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Crystal engineering of flavonoids

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Title:
Crystal engineering of flavonoids
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Kavuru, Padmini
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University of South Florida
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Polyphenols
Pharmaceutical cocrystals
Hydrogen bond
French paradox
Bioavailability
Dissertations, Academic -- Chemistry -- Masters -- USF   ( lcsh )
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non-fiction   ( marcgt )

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Abstract:
ABSTRACT: Crystal engineering is attracting attention in the pharmaceutical industry because the design of new crystal form of drugs can improve their stability, bioavailability and other relevant physical characteristic properties. Therefore, crystal engineering of nutraceuticals such as flavonoids by exploring their hydrogen bonding interactions can generate novel compounds such as pharmaceutical cocrystals. Flavonoids are polyphenolic secondary plant metabolites that are present in varying levels in fruits, vegetables and beverages. The "French paradox", low cardiovascular mortality rate in spite of high intake of saturated fat among the Mediterranean populations made flavonoids an appropriate target for therapeutic researchers. The work herein deals with the crystal engineering of two flavonoids, quercetin and hesperetin, which are already known to exhibit antioxidant properties and reduce cardiovascular effects in humans. However, they have limited bioavailability and poor water solubility. Several new forms of quercetin and hesperetin in the form of solvates and cocrystals were synthesized. These new crystal forms were characterized by various techniques: FT-IR, DSC (Differential Scanning Calorimetry), single X-ray diffraction, powder X-ray diffraction, TGA (Thermal Gravimetric Analysis) and melting point. The new compounds were also studied via dissolution studies performed in 1:1 ethanol/water (V/V%). Thus, crystal engineering proves to be effective way to enhance the solubility and bioavailability of the target flavonoid molecules.
Thesis:
Thesis (M.S.)--University of South Florida, 2008.
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Includes bibliographical references.
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by Padmini Kavuru.
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Document formatted into pages; contains 87 pages.

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Crystal Engineering of Flavonoids by Padmini Kavuru A thesis submitted in partial fulfillment Of the requirements for the degree of Master of Science Department of Chemistry College of Arts and Sciences University of South Florida Major Professor: Michael J. Zaworotko, Ph.D. Julie P. Harmon, Ph.D., Abdul Malik, Ph.D. Date of Approval: April 11, 2008 Keywords: Polyphenols, Pharmaceutical Cocrystals, Hydrogen bond, French Paradox, Bioavailability Copyright 2008, Padmini Kavuru

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Dedication To my husband, parents and in-laws

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Acknowledgements I would like to thank m y advisor, Prof essor Michael J. Zaworotko, for the opportunity to conduct re search under his supervision, and for his advice and guidance throughout the Graduate Program. I would al so like to thank Dr. Julie P. Harm on and Dr. Abdul Malik, my committee members, for their helpful comments and encouragements. In addition, I would like to acknowledge all m embers of my research group, as well as Faculty and Staff of the Chem istry Department of University of South Florida, for their friendly accommodation. At last I would like to express my deepest thanks to m y husband, parents, in-laws, other family members and friends who constantly supported me throughout the period of studies.

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i TABLE OF CONTENTS LIST OF TABLES iv LIST OF FIGURES iv ABSTRACT ix 1. INTRODUCTION 1 1.1 Nutraceuticals 1 1.2 Flavonoids 1 1.3 Structure and classification 3 1.4 Bioactivity and bioavailability 6 1.5 Crystal Engineering 9 1.5.1 Supramolecular Chemistry 10 1.5.2 Supramolecular synthons 11 1.5.3 Cocrystals 12 1.5.4 Pharmaceutical cocrystals 14 2. CSD ANALYSIS 18 2.1 Cambridge Structural Database 18 2.2 CSD statistics for Phenols and Polyphenols 19 2.3 Interactions of phenolic-OH with other functional groups 21 2.4 Discussions 25 3. QUERCETIN 26

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ii 3.1 Description 26 3.2 Strategy for Crystal E ngineering of Quercetin 27 3.3 Solvates of Quercetin 29 3.3.1 Form I and Form II (Pyrid ine solvates of Quercetin) 29 3.3.2 Form III (THF solvate of Quercetin) 32 3.3.3 Form III ( Quercetin-Acetone solvate) 33 3.3.4 Discussions 35 3.4 Cocrystals of Quercetin 36 3.4.1 QuercetinCaffeineMeth anol cocrystal solvate 36 3.4.2 Quercetin-Isonicotinamide 39 3.4.4 Discussions 42 4. HESPERETIN 43 4.1 Description 43 4.2 Strategy for Crystal Engineering of Hesperetin 44 4.2.1 Hesperetin-Isonicotinamid e cocrystal (Cocrystal 3) 45 4.2.2 Hesperetin-Nicotinic acid zwitterion cocrystals 49 4.2.4 Discussions 54 5. DISSOLUTION STUDIES OF THE COCRYSTALS 55 5.1 Dissolution experiments of quercetin and hesperetin cocrystals 57 5.2 Dissolution study of Quercetin:Ca ffeine cocrystal (Cocrystal 1) 60 5.2 Dissolution study of Quercetin-I sonicotinamide (Cocrystal 2) 61 5.3 Dissolution study of Hesperetin -Isonicotinamide (Cocrystal 3) 62 5.4 Dissolution study of Hesperetin-NA (Cocrystal 4) 63

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iii 5.5 Discussions 64 6. CONCLUSIONS AND FUTURE DIRECTIONS 66 REFERENCES 67 APPENDICIES 76 Appendix A: 77 Appendix B 85

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iv LIST OF TABLES Table 1.1 Structures of different of Flavonoids 5 Table 1.2 Classification Flavonoids and their co mmon food sources 6 Table 2.1 CSD statistics for Phenols and Polyphenols 20 Table 3.1 Solvates of Quercetin 27

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v LIST OF FIGURES Figure 1.1 Skeleton of Flavonoid 3 Figure1.2 Structure of Flavonoid with the chromane ring 3 Figure1.3 Aurones (2-benzyl-coumarone) 4 Figure1.4 Example of Glycoside, Rutin (quercetin-3-rutinoside) 4 Figure1.5 Hypothesis of the links between the working mechanisms of flavonoids a nd their effects on disease. NO, nitrous oxide 7 Figure 1.6 Supramolecular synthons: (a) Carboxylic acid homosynthon, 12 (b) Carboxylic acidpyridine heterosynthon Figure 1.7 Crystal structure of the triclinic form of quinhydrone 13 Figure 1.8 The Hoogsteen base-pairing in the structure of 9-methyladenine 13 Figure 1.9 Occurrence of the term cocrysta l in papers published between 1991 and 2007 (SciFinder search 2/2008). 14 Figure 1.10 Different forms of drugs in which they are administered. 15 Figure 1.11 Crystal packing of 1:1 co-crystals of 5, 5-diethylbarbituric acid 16 Figure 1.12 (a) Itraconazolesuccinic acid cocrystal, (b) Carbamazepinesaccharin cocrystal 16 Figure 2.1 (a) Growth of the CSD since 1972, (b) Predicted growth of the CSD during the decade 2001 2010 19 Figure 2.2 Structure of polyphenol for the CSD search 19 Figure 2.3 Supramolecular phenolic OH OH homosynthon (Synthon I) 22 Figure 2.4 Histogram for synthon I 23 Figure 2.5 Supramolecular phenolic OHOH heterosynthon (Syn II) 21

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vi Figure 2.6 Histogram for synthon II 21 Figure 2.7 Supramolecular phenolic OHNarom heterosynthon (Syn III) 22 Figure 2.8 Histogram for synthon II 24 Figure 2.9 Supramolecular phenolic OHO heterosynthon (Syn IV) 25 Figure 2.10 Histogram for synthon IV 24 Figure 2.11 (a) Supramolecular phenolic OHCO heterosynthon (Syn V) (b) Supramolecular phenolic OHNH heterosynthon (Syn VI) 24 Figure 2.12 Histograms for synthon V and synthon VI 25 Figure 3.1 Structure of Quercetin 26 Figure 3.2 Representation of different rings in Quercetin 27 Figure 3.3 Hydrogen bonding in Quercetin dihydrate 28 Figure 3.4 Illustration of dihedral angl e between the planes. (Red plane contains ring s A and C and the blue plane contains ring B) 29 Figure 3.5 Single crystal of quercetin-pyridine solvate 30 Figure 3.6 (a) Hydrogen bonding in Form I (b) Hydrogen bonding in Form II 31 Figure 3.7 The dihedral angle in From I (a) and Form II (b) 31 Figure 3.8 Single crystal of quercetin-THF solvate (Form II) 32 Figure 3.9 Comparison of hydrogen bonding in Fo rm III (left) with that of Form II (right) 33 Figure 3.10 Crystals of Quercetin-Acetone solvate (Form III) 34 Figure 3.11 Hydrogen bonding in Form III (Acetone molecules colored red) 34 Figure 3.12 Representation of crisscross grid network in Quercetin-Acetone 35 solvate Figure 3.13 The asymmetric unit of 1:1:1 cocrystal solvate of quercetin, caffeine and methanol 36

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vii Figure 3.14 Single crystal of que rcetin:caffeine:methanol cocrystal solvate (Cocrystal I) 37 Figure 3.15 Intermolecular inte ractions in the 1:1:1 co crystal solvate of querc etin, caffeine and methanol (Green). 38 Figure 3.16 Illustration of sheets ge nerated in the cocrystal 1. 39 Figure 3.17 The asymmetric unit of the1:1 quercetin isonicotinamide cocrystal 39 Figure 3.18 Single crystal of querc etin-isonicotinamide cocrystal 39 Figure 3.19 Intermolecular inte ractions in the 1:1 Quercetin-Isonicotinamide cocrystal. 40 Figure 3.20 Illustration of R4 4 (18) graph set in quercetin and isonicotinamide cocrystal. 41 Figure 4.1 Structure of hesperetin (hydrogen are not included) 43 Figure 4.2 Illustration of rings in Hesperetin 44 Figure 4.3 Hydrogen bonding in (a) Hesp eretin (b) Hesperetin monohydrate 44 Figure 4.4 Illustration of th e dihedral angles 45 Figure 4.5 The asymmetric unit of hesper etin:isonicotinamide 1:1 cocrystal 45 Figure 4.6 Single crystal of hespere tin-isonicotinamide cocrystal 46 Figure 4.7 Overall hydrogen bonding in the Hesperetin-Isonicotinamide cocrystal 47 Figure 4.8 Illustration supramolecular synthons in the cocrystal 3. 47 Figure 4.9 The cavity formed in hesperetin:isonicotinamide cocrystal. 48 Figure 4.10 Illustration of 8-fold interp enetrated network in Cocrystal 3 48 Figure 4.11 Illustration of dihe dral angle in hesperetin molecules (red plane contains rings A and C; blue contains ring C) 49 Figure 4.12 Hesperetin Nicotinic acid 1:1 zwitterion cocrystal (Form I) 49 Figure 4.13 Single crystals of hesperetin-NA cocrystals. 50

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viii Figure 4.14 The hydrogen bonding in Form I cocrystal.(Blue: R-Hesperetin ) 51 Figure 4.15 Illustration of hydrogen bonds in NA: a) NA in pure form b) NA in the cocrystal, hesperetin molecules are deleted for clarity. 51 Figure 4.16 Representation of dihedral a ngle between the rings. (red plane contains rings A and C, blue pl ane contains ring B) 52 Figure 4.17 The hydrogen bonding in Form II cocrystal (yellow and maroon: NA molecu les, (+) hesperetin: blue, (-) hesperetin) 53 Figure 4.18 The dihedral angle formed betw een the planes of rings A, C and ring B in Form II 53 Figure 5.1 Powder dissolution profiles for (1), (2), (3), and (4) in water at 20 C. 56 Figure 5.2 Dissolution profiles into 0.1 N HCl at 25 C for Sporanox beads and the cocrystals 56 Figure 5.3 Average plasma time curves of carbamazepine concentrations (SEM)from a cross-ove r experiment in fasted beagle dogs (n = 4)given oral doses of 200 mg of the active drug as Tegretol tablets and co-crystal 57 Figure 5.4 UV spectrums of Quercetin and Hesperetin in 1:1 Ethanol/Water 59 Figure 5.5 Dissolution profiles of quercetin dihydrate, solvated and the desolvated cocrystal in 1:1 Ethanol/water 61 Figure 5.6 Dissolution profiles of quercetin dihydrate and quercetinisonicotinamide cocrystal in 1:1 EtOH/Water (V/V%) 61 Figure 5.7 Dissolution profiles of hesperetin and hesperetin-isonicotinamide (1:1) cocrystal in 1:1 EtOH/Water (V/V%) 62 Figure 5.8 Dissolution profiles of hesperetin a nd its cocrystals 63

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ix CRYSTAL ENGINEERING OF FLAVONOIDS PADMINI KAVURU ABSTRACT Crystal engineering is attrac ting attention in the pharmaceu tical industry because the design of new crystal form of drugs can improve their stability, bioavailability and other relevant physical characteristic propertie s. Therefore, crystal engineering of nutraceuticals such as fla vonoids by exploring their hydrog en bonding interactions can generate novel compounds such as pha rmaceutical cocrystals. Flavonoids are polyphenolic secondary plant metabolites that are present in varyi ng levels in fruits, vegetables and beverages. The French paradox, low cardiovascular mortality rate in spite of high intake of saturated fa t among the Mediterran ean populations made flavonoids an appropriate target for therapeutic researchers. The work herein deals with the crystal e ngineering of two flavonoids, quercetin and hesperetin, which are already known to exhibit antioxidant properties and reduce cardiovascular effects in humans. Howe ver, they have limited bioavailability and poor water solubility. Several new forms of quercetin and hesperetin in the form of solvates and cocrystals were synthesized. These new crystal forms were characterized by various techniques: FT-IR, DSC (Differen tial Scanning Calorimetry), single X-ray diffraction, powder X-ray diffraction, TGA (Thermal Gravimetric Analysis) and

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x melting point. The new compounds were also studi ed via dissolution studies performed in 1:1 ethanol/water (V/V%). Thus crystal engineering proves to be effective way to enhance the solubility and bioavailability of the target flavonoid molecules.

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1 1. INTRODUCTION 1.1 Nutraceuticals The term Nutraceutical is an intermediate of nutrition and pharmaceutical. 1 It is used to describe a medicinal or nutritional compone nt of food, plant or naturally occurring material in purified or concentrated form claimed to have a medicinal effect on human health. Such foods are also called functiona l foods. It can also refer to individual chemical present in common foods. The pos ition of nutraceuticals on the legislative grounds is marginal between pharmaceutics and food. The term nutraceu tical was coined by Dr. St ephen De Felice (founder and chairman of Foundation of I nnovation in Medicine) in 1976. 1 He defined it as: Nutraceuticals are food, or parts of food, th at provide medical or health benefits, including the prevention and treatment of disease . Nutraceuticals include a wide range of products, such as polyphenols (Flavonoids), vitamins, oils from fish and flax seed, glucosamine, chondroitin, resveratrol, calcium-fortif ied juices etc. 1 1.2 Flavonoids Flavonoids are naturally occu rring polyphenolic compounds that are found in fruit, vegetables, grains, bark, roots, stems, flowers, tea, and wine. 2 These natural products were known for their beneficial effects on health long before flavonoids were isolated as

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2 the effective compounds. There are more than 4000 varieties of flavonoids which have been identified till now and are considered responsible for the attractive colors of flowers, fruit, and leaves. 3, 4 These are responsible for many of the plant colors that dazzle us with their brilliant shades of yellow, red and orange. Many of the fl avonoids are dietary antioxidants and constituents of medicinal herbs. These compounds were discovered in by a Hungarian scientist, Dr Albert Szent-Gyorgyi in 1938, who discovered Vitamin C (Nobel laureate) by mistake. Rusznyk and Szent-Gyrgi (1936) suggested that flavonoids be known as vitamin P or vitamin C2. 5-8 However, by the 1950s the vitamin claim had been abandoned due to a lack of substantive evidence. Flavonoids are also known as plant secondary metabolites which does not effect the normal growth, development or reproduction of organisms like the primary metabolites neither their absence result in immediate death. 9 But, these compounds are used as defenses against predators, parasites and diseases, for interspecies competition, and to facilitate the reproductive processes (coloring agents, attractive smells, etc) and hence they are called as the First Line of Defense . 10 The research on flavonoids took a rapid momentum with the di scovery of the French para dox, i.e., low cardiovascular mortality rate observed in Mediterranean popul ations with red wine consumption and a high saturated fat intake. 22, 23 The flavonoids in red wine are responsible for this effect. In addition, epidemiologic studies suggest a protective role of dietary flavonoids against coronary heart disease. Afte r a number of subsequent stud ies on animals it was found that flavonoid intake is inversely correlated with mortality due to coronary heart disease. 11, 12 These potential benefits are be ing used to promote the consumption of flavonoid-rich foods, beverages and dietary supplements.

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1.3 Structure and classification The basic structure of flavonoids is based on the 15 carbon skeleton, i.e. C 6 C 3 C 6. The two C6 represents the number of carbon atoms of the two phenyl groups and the C3 represents the number of carbon atoms that bridge the two phenyl rings by a linear three carbon chain. 7-10 Figure 1.1 explains the above desc ription in most convenient way. Figure 1.1 Skeleton of Flavonoid In some of the flavonoids, the three carbon linear chain is replaced by a chromane ring and the position of ring could be at 2, 3 or at 4 th carbon atom. The overall structure now consists of three rings A, B and C. The posit ion of the ring B varies from one class of flavonoid to the other. Figure 1. 2 Structure of Flavon oid with the chromane ring In some of the flavonoids, the six-membered he terocyclic ring C occurs in an isomeric open form or is replaced by a five me mbered ring. The oxygen bridge involving the central carbon atom (C 2 ) of the three carbon chain occurs in a limited number of cases resulting in a heterocyclic ri ng which is of the furan type. 3

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Figure 1.3 Aurones (2-benzyl-coumarone) Most of the flavonoids occur as glucosides in which the C6-C3-C6 aglycone part of the molecule is linked with different number of sugars. If the linkage of the sugar to the flavonoid aglycone is through an OH group then these are called as O-glycosylflavonoids and if the linkage is through C-C bond then these are called as C-glycosylflavonoids. 17 The type of sugar and the position of sugars vary for different glyc osides. These have a more complex structure than the phenolic skeleton materials. Glycoside = Aglycone + Glycone (sugar) Figure 1.4 Example of Glycoside, Rutin (quercetin-3-rutinoside) The most common citrus flavanone-glycosides are rutin, natrirutin, naringin, hesperedin and neohesperidin. Naringin (in grapefruits) and hesperidin (in oranges) are the two major flavonoid-glycosides present in the citrus fruits, and are primar ily concentrated in the peel and the tissue of the fruit. 18 The table below represents the structures of various flavonoids. 4

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Table 1.1 Structures of different of Flavonoids Anthocyanidins Flavanols Flavonols Flavonones Flavones Isoflavones Flavonoids are divided into va rious classes on the basis of their molecular structure. 19 The six main groups of flavonoids are listed in Tabl e 1.2, which includes the known members of 5

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6 each group and the food sources in which they are present. Table 1.2 Classification Flavonoids and their common food source Flavonoid Subclass Dietary Flavonoids Some Common Food Sources Anthocyanid ins Cyanidin, Delphinidin, Malvidin, Pelargonidin, Peonidin, Petunidin Red, blue and purple berries, red and purple grapes, red wine Flavanols Monomers (Catechins) : Catechin, Epicatechin, Epigallocatechin Epicatechin gallate, Epigallocatechin gallate Dimers and Polymers : Theaflavins, Thearubigins, Proanthocyanidins Catechins : Teas (particularly green and white), chocolate, grapes, berries, apples Theaflavins, Thearubigins : Teas (particularly black and oolong) Proanthocyanidins : Chocolate, apples, berries, red grapes, red wine Flavonones Hesperetin, Naringenin, Eriodictyol Citrus fruits and juices, e.g., oranges, grapefruits, lemons Flavonols Quercetin, Kaempferol, Myricetin, Isorhamnetin Widely distributed: yellow onions, scallions, kale, broccoli, apples, berries, teas. Flavones Apigenin, Luteolin Parsley, thyme, celery, hot peppers, Isoflavones Daidzein, Genistein, Glycitein Soybeans, soy foods, legumes 1.4 Bioactivity an d bioavailability Flavonoids are widely known for their antioxidant activity. Important effect of flavonoids is the scavenging of oxygen-derived free radicals. In vitro experimental systems showed that flavonoids possess anti-inflammatory, anti-all ergic, antiviral, and anti-carcinogenic properties. 20 An overview of the hypothetical links between the working mechanisms and clinical effects of flavonoi ds is given in Figure 1.5. 21

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Figure 1.5 Hypothesis of the links between the working mechanisms of flavonoids and th eir effects on disease. NO, nitrous oxide One clue to the health benefits of flavonoids comes from studies of the "French paradox.22 French eat almost four times more butter and three times more lard-and have higher cholesterol levels and blood pressures-than do Americans and yet the French are 2.5 times less likely than Americans to die of coronary heart disease. Many peopl e have suggested that the liberal French consumption of red wine protects against coronary heart disease, apparently by lowering cholesterol levels or pr eventing abnormal blood clot s. In fact, at least eight medical studies have found that a glass or two of wine daily protects against heart disease. But some studies have reported that red wine is better than white wine, suggesting that some of the benefits might be unrelate d to the alcohol. The antioxidant property of flavonoids can be explained in the followi ng way: Flavonoids are oxidized by radicals, resulting in a more stable, less-reactive radi cal. In other words, flavonoids stabilize the reactive oxygen species by reacting with the reactive compo und of the radical. Because of 7

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8 the high reactivity of the hydroxyl group of th e flavonoids, radicals are made inactive, according to the following equation: 22 Flavonoid (OH) + R Flavonoid (O) + RH ( 1) where, R is a free radical and O is an oxygen free radica l. Selected flavonoids can directly scavenge super oxides; whereas other flavonoids can scavenge the highly re active oxygen derived radical called peroxynitrite. Epicatechin and rutin are also powerful radical scavengers. The scavenging ability of rutin may be due to it s inhibitory activity on the enzyme xanthine oxidase. By scavenging radi cals, flavonoids can inhib it LDL oxidation in vitro. 24 This action protects the LDL particles a nd, theoretically, flavonoids may have preventive action against atherosclerosis. With the exception of fl avanols (catechins and proa nthocyanidins), flavonoids occur in plants and most foods as glycosides. Even after cooking, most flavonoid glycosides reach the small intestine intact. Only flavonoi d aglycones and flavonoid glucosides (bound to glucose) are absorbed in the small intestine, where they are rapidly metabolized to form methylated, glucuronidated or sulfated metabolites. 25 Hollman and Katan suggested that the glycosylated forms of quercetin are absorbed more readily than are the aglycone forms; however, this has been questioned by other rese archers. The role of flavonoid glycosylation in facilitating absorption is questioned by the fact that catechi n, which is not glycosylated in nature, is absorbed rela tively efficiently. As most of the flavonoids are poorly absorbed by the body and the major percentage is excreted out which, limits its bioavailability. Even though numerous strategies exist fo r enhancing the bioavailability of drugs with low aqueous solubility, the success of these approaches is not yet able to be guaranteed and is greatly dependent on the physical and chemical nature of the molecules being developed. Crystal

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9 engineering offers a means to improved solubi lity and dissolution rate and stability, which can be adopted through an in-depth knowledge of crystallization processes and the molecular properties of active pharmaceutical ingredients. 26 1.5 Crystal Engineering Crystal engineering is descri bed as the exploita tion of noncovalent interactions between molecular or ionic components for the rational design of solid-state structures that might exhibit interesting electrical, magne tic, and optical properties. It is also recognized that it is becoming increasingly evident that the specifi city, directionality, a nd predictability of intermolecular hydrogen bonds can be utilized to assemble supramolecula r structures of, at the very least, controlled dimensionality. 27 The noncovalent interactions which have been exploited in this fi eld are hydrogen bonding, dipole interactions, ionic bonds, hydrophobic interactions, London dispersion forces etc. The term crystal engineering was coined by Pepinsky in 1955, but it was popularized by the work of Schmidt in solid state photo chemistry and this is considered as the beginning era of crystal engineering. 28 By his work it was clear that the ch emical and physical properties of the crystalline solids are dependent on the arrangement of the molecules in the crystal lattice. Gautam Desiraju, in this context define crystal engi neering as below: Crystal engineering is the understanding of intermolecu lar interactions in the context of crystal packing and in the utilization of such understanding in the de sign of new solids with desired physical and chemical properties. Crystal engineering can be used to manipulate th e solubility and dissoluti on rate of the active pharmaceutical ingredients (API) in the crystalline state w ithout compromising with its

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10 bioactivity. There are many possible ways that may be achieved from recent developments in the study of molecular solids and reviews topical issues such as habit modification, polymorphism, solvation, co-crystal form ation and surface modification. Therefore, particular attention is paid to the area of co-crystallization, which is an emerging area of strategic importance to the pharmaceutical sector. 27 1.5.1 Supramolecular Chemistry Supramolecular chemistry is a relatively new field of chemistry which focuses literally on going "beyond" molecular chemistry. 29 The importance of supramolecular chemistry was recognized by Donald J. Cram, Jean-Marie Lehn, and Charles J. Pedersen and for their work in this area they were awarded Nobel Prize for Chemistry in 1987. The important breakthrough that allowed the elucidation of the double helica l structure of DNA occurred when it was realized that ther e were two separate strands of nucleotides connected through hydrogen bonds. The utilization of noncovalent bonds is essential to re plicate because they allow the strands to be separated and us ed to template new double stranded DNA. The development of selective "host-guest" complexes, in which a host molecule recognizes and selectively binds a certain guest was cited as an important co ntribution. Unlike, the organic synthesis that involves the making and break ing of covalent bonds to synthesize a desired molecule, supramolecular chemistry utilizes far weaker and reversible noncovalent interactions, such as hydrogen bonding, meta l coordination, hydrophobi c forces, van der Waals forces, pi-pi interactions, sometimes electrostatic effects to assemble molecules into multimolecular complexes. Various fields that are classified under supramolecular chemistry

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11 include molecular self-assembly, molecular r ecognition, host-guest ch emistry, mechanicallyinterlocked molecular architectures and dynamic covalent chemistry. 30 It is important for the development of new pharmaceutical therapies by understanding the interactions at a drug binding site. In addition, supramolecular system s have been designed to disrupt proteinprotein interactions that are important to cellu lar function. It also ha s application in green chemistry where reactions have been develope d where the reactions ta ke place in the solid state directed by non-covalent bonding. Such pr ocedures are highly de sirable since they reduce the need for solvents duri ng the production of chemicals. 1.5.2 Supramolecular synthons Supramolecular synthons are the smallest struct ural units within which is encoded all the information inherent in the mutual rec ognition of molecules to yield solid state supermolecules, that is, crystals. A key aspect of crystal engineer ing is therefore the dissection of a target networ k into supramolecular synthons not the molecular synthons which connect the supramolecular synthons. 31 Such a dissection simplifies the analysis of a target network and is important in crys tal engineering because it recognizes the interchangeability of supramolecular s ynthons in a family of structures. 32, 33 Supramolecular synthons are classified into two categories: supramolecular homosynthons and supramolecular heterosynthons 34 The supramolecular homosynthons are formed between the same, self-complementary functional groups and supramolecular heterosynthons are formed between different but complementary fu nctional groups. Examples of supramolecular homosynthons include the dimers formed between carboxylic acids 35, 36 and amides 37 whereas supramolecular heterosynth ons include carboxylic acidamide, 38-42

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hydroxylpyridine, 43-45 and carboxylic acidpyridine.46-49 (a) (b) Figure 1.6 Supramolecular synthons: (a) Carboxylic acid homosynthon, (b) Carboxylic acidpyridine heterosynthon The supramolecular homosynthons usually exis ts in structures of single-component compounds, for example the carboxylic acids ex ists as dimers. Where as, if multiple functional groups are present, a supramolecular hete rosynthon is more likely to be formed as the formation of heterosynthons wins over the homosynthons when ever these two are competing with each other. When a supramol ecular heterosynthon is formed between two functional groups that are located on different molecules, a multicomponent is formed. 1.5.3 Cocrystals Cocrystals belong to the class of compounds which are long known but little studied E arlier, cocrystals were known with different names like; molecular compounds,50 organic molecular compounds,51 addition compounds,52 molecular complexes,53 solid-state complexes,54 or heteromolecular crystals.55 In1844, the first cocrystal of benzoquinone and hydroquinone was synthesized by Wohler, and then followed by studies of halogen derivatives of quinhydrone.50, 56 Until 1960 the structural information for the cocrystal was absent. The elucidation of DNA structure 57, 58 through X-ray analysis inspired many people to come out with numerous nucleobase complexes in 1950s and 60s 59-62 and then the term cocrystal 12

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was first coined in this context 63 later it was subsequently popularized by Etter. 64 During this time, Hoogstein came up with the new base pair called Hoogstein base pair a cocrystal formed between 9-methyladenine and 1-methylthymine. 61 Figure 1.7 Crystal structure of the triclinic form of quinhydrone Figure 1.8 The Hoogsteen base-pair The term cocrystal sounds very simple but the definition is often a topic of debate. 65, 66 The Zaworotko research group defines cocrystal as: A cocrystal is a multiple component crystal in which all components (molecu le or ion and a molecular cocrystal former) are solid under ambient conditions when in their pure form . All components are solid under ambient conditions has important practical consideratio ns, because synthesis of cocrystals can be achieved via the solid-state. 67 Aakeroy and co-workers, 68 they defined cocrystals as (i) compounds constructed from neutral molecules; (i i) made from reactants that are solids at ambient conditions; and (iii) structurally homogeneous crystalline materials that contains at 13

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least two neutral buildin g blocks with a well-defined stoich iometry. A broad definition of a cocrystal given by Dunitz: a crystal cont aining two or more components together 69 would include molecular adducts, salts, solvates/hydrates, inclusion compounds, etc. What ever way people define the term cocrystal, the field of co crystal is attaining the heights of excellence, and Figure 1.9 reveals the growing interest in the subject. Therefore, it is evident that cocrystals play a vital role in pharmaceutical science and elsewhere. 14 199119921993199419951996199719981999200020012002200320042005200620070 50 100 150 200 250 300 Number of hits containing the term "Cocrystal"Year Figure 1.9 Occurrence of the term cocrystal in papers published between 1991 and 2007 (SciFinder search 2/2008). 1.5.4 Pharmaceutical cocrystals Most of the active pharmaceutical ingredients (API s) are administered in the solid state as part of an approved dosage type li ke tablets, capsules, etc as it is the convenient and compact format to store a drug.

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Figure 1.10 Different forms of drug s in which they are administered. APIs can exist in a variety of distinct so lid forms, where each form may display unique physicochemical properties such as hygrosc opicity, morphology, and (most importantly) solubility. The solid form of the drug dictates the properties like stab ility, hygroscopicity, dissolution rate, solubility and bioavailability. Figure 1.10 repres ents the different ways in which drugs are administered. 70 This makes the pharmaceutics to look for a crystal form with the best properties for therapeutic use and ma nufacturability. Therefore, whatever form of drug is selected, it must be amenable to ha ndling and processing and as a drug, it must be effective and safe. Sometimes, the pharmaceutic developers come across a problem, that the most common polymorphic form of the drug is l east stable. Therefore the current approaches to change the properties of APIs include the utilization of ionic salts, solvates, hydrates, and polymorphs. 71 In this context pharmaceutical cocrysta llization is potentially attractive for improving the material properties while leaving an API unaltered. The intellectual property implications of creating co-crystals are also highly relevant. A pharmace utical co-crystal can described as a multiple component crystal in which at 20 least one component is molecular and a solid at room temperature (the co-cryst al former) and forms a supramolecular synthon with a molecular or ionic API 72, 73 The first application of crysta l engineering to generate of 15

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pharmaceutical co-crystals was a series of st udies by Whitesides et al. with the use of substituted barbituric acid, including barbital and melamine deriva tives and generated a supramolecular linear tape, crinkled tape, and rosette motifs.74-80 (a) (b) Figure 1.11 Crystal packing of 1:1 co-crystals of 5, 5-diethylbarbituric acid (barbital) To date, many pharmaceutical cocrystals have b een repo rted in the scientific and patent literature and most of the cocrys tals have been found to exhib it improved material properties like solubility, stability, bioa vailability etc over the pure API. A few examples pharmaceutical cocrystals are: carbamazepin e (CBZ), aspirin, profens, piracetam, caffeine, loracarbef, cephalexin, cefaclor, con azo les, topiramate, modafinil, phenytoin, olanzapine, nabumetone, fluoxetine, theophyllin e, sulfadim idine, trimethroprim, and paracetamol.81-97 (a) (b) Figure 1.12 (a) Itraconazolesuccinic acid co crystal, (b) Carbamazepinesaccharin cocrystal 16

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17 Therefore, the applications of concepts of supramolecular synthesis and crystal engineering to the development of pharmaceutical cocrys tals offer many opport unities for the drug development and delivery. So it would not be exaggeration in saying that sooner or later pharmaceutical cocrystals will gain a br oader foothold in drug formulation.

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18 2. CSD ANALYSIS 2.1 Cambridge Structural Database The Cambridge Structural Database (CSD) is an important product of the Cambridge Crystallographic Data Centre (CCDC), which was establ ished by Olga Kennard at Cambridge University in 1965. 98 It comprises software for database access, structure visualization and data analysis, and structural knowledge. It has a collection of bibliographic, chemical and crystallographic information for organic molecules and metal-organic compounds whose 3D structures have been determined using X-ray diffraction or neutron diffraction and the information is stored in form of a cif file ( .cif ). The collection also includes the results of single crystal studies powder diffraction studies which yield 3D atomic coordinate data for at leas t all non-H atoms. The crystal structure data arising from publications in the open literatur e and private communications vi a direct data deposition are also assimilated in the CSD. The basic softwares that CSD relies on are: ConQuest (search and information retrieval), Mercury (structure visualization), Vista (numerical analysis) and PreQuest (database creation). The CSD helps to compare ex isting data with that obtained from crystals grown in their laboratories. It is evident from Figure 2.1 that there is a rapid growth of the CSD since 1970, and it is predicted growth w ould increase exponentially during the decade 2001-2010. Thus CSD is proves to be an inexha ustible source for cr ystallographers and interpretation of existing crystal structures with complete knowledge on interplay between

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supramolecular synthons would help in designing new multicomponent crystals. (a) (b) (b) Figure 2.1 (a) Growth of the CSD since 1972, (b) Predicted growth of the CSD during the decade 2001 2010 2.2 CSD statistics for Phenols and Polyphenols Flavonoids are known as polyphenolic compounds a nd to design new crystal forms (solvates, cocrystals etc) of these compounds it is neces sary to know about the statistics of existing synthons of hydroxyl groups with other functio nal groups present in the CSD. Although phenols and alcohols have the hydroxyl group comm on in their structures as they differ in acidity. Phenols have relatively higher acidities due to the aromatic ring tightly coupling with the oxygen and a relatively loose bond between the oxygen and hydrogen. The acidity of the hydroxyl group in phenols is intermediate betwee n that of aliphatic alcohols and carboxylic acids (their pK a is between 10 and 12). The polyphenols for the CSD search were recognized by the presence of more than one hydroxyl grou p per molecule as represented in the Figure 2.2. Figure 2.2 Structure of polyphenol for the CSD search 19

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20 All the searches were done by considering the constraints: no ions, only organics, R factor: 7.5% and structures with 3D coordinates. Th e statistics for the o ccurrence of various interactions of phenols and polyphenols with ot her functionalities in the CSD are tabulated below. Table 2.1 CSD statistics for Phenols and Polyphenols (January 2008 update) Moieties present in a structure No. Struc (Phenols) Structures with synthon No. Struc (Polyphenols) Structures with synthon *OH (Phenols only) 6932 O-H OH (synthon I) 1320/ 6932 (19.0%) 1196 / 6932 (19.2%) O-H OH 492 / 1196 (41.1%) *OH & CO (ket & ald) 960 O-H CO (synthon II) 569 / 960 (67.0%) & O-H OH (phenolic) 202 homosynthons (20.0%) 446 O-H CO 186 / 446 (41.7%) & O-H OH (phenolic) 163 homosynthons (36.6%) *OH & N arom 566 O-H N arom (synthon III) 315 / 566 (59.3%) & O-H OH (phenolic) 88 homosynthons (15.5%) 151 O-H N arom 128 / 151 (84.8%) & O-H OH (phenolic) 46 homosynthons (30.5%) *OH & O (ether) 2391 O-H O (synthon IV) 278 / 2391 (11.6%) & O-H OH (phenolic) 383 homosynthons (16.0%) 330 O-H O 66 / 330 (23.5%) & O-H OH (phenolic) 131 homosynthons (39.7%) *OH & CONH 2 (1 o amides) 85 O-H O=C (synthon V) 16 / 85 (%) OH NH 2 (synthon VI) 14 / 85, (16.5%) & O-H OH (phenolic) 6 homosynthons (7.1%) 10 O-H O=C 3 / 10 (30%) OH NH 2 8 / 10 (80%) & O-H OH (phenolic) 2 homosynthons (20%) Constrains: no ions, only organics, R factor: 7.5% and structures with 3D coordinates represents phenolic OH group

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2.3 Interactions of phenolic -OH gr oup with other functional groups The CSD search reveals that there are 6232 hits for phenols and 1196 polyphenols (constraints: no ions, onl y organics, R factor: 7.5% and structures with 3D coordinates). H O O H Figure 2.3 Supramolecular phenolic OH OH homosynthon (Synthon I) Out of 6232 phenols the OHOH phenolic supram olecular homosynthon was found in 19.0% structures and out of 1196 polyphenols it was 41.1%. The bond range for synthon I was determined by the histogram generated by Vista software and it was found to be 2.5-3.07 (cut off range). This implies that the remaining structures form supramolecular heterosynthons in the presence of other comp eting groups and the Figure 2.4 represents the cut off range for synthon I. Figure 2.4 Histogram for synthon I 21

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The search for the entries w ith phenolic OH group and the carbonyl group (aldehydes and ketones only) revealed that out of 960 hits 67.0% were found to have phenolic OHCO supramolecular heterosynthon (synthon II) and only 20.0% have OHOH phenolic supramolecular homosynthon (synthon I) O H OC Figure 2.5 Supramolecular phenolic OH CO heterosynthon (Synthon II) In case of polyphenols the presence of synt hon II was only 41.7% and 36.6% have synthon I out of 446 entries with polyphenols and car bonyl compounds. The cut off range for synthon II was determ ined as 2.5-3.10 as shown in Figure 2.6. Figure 2.6 Histogram for synthon II Another search for the occurrence of phenolic OHNarom supramolecular heterosynthon (synthon III) among the structur es containing aromatic n itrogen with phenols and polyphenols was made. Figure 2.7 Supramolecular phenolic OH Narom heterosynthon (Synthon III) 22

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The search results in 566 hits for compounds containing aromatic ni trogen and phenols. Out of the 566 hits 59.3% were found to have s ynthon III and 15.5% synthon I. For polyphenols the percentage for the occurrence of synthon III is much higher than that of phenols. Out of 151 total structures containing polyphenols and aromatic nitrogen, 84.8% were found to have synthon III and 30.5% have synthon I. The dist ance range for synthon III was found to be 2.5-3.125 as shown in Figure 2.8. Figure 2.8 Histogram for synthon III The search for the phenolic OHO supramolecular heterosynthon (synthon IV) for ether with phenols and polyphenols shows th at ethers are not good hydrog en bond acceptors as other functional groups. Even though the occurrence ethers with phenols and polyphenols is high when compared to other functional groups, out of 2391 hits only 11.6% of the total structures are capable of forming synthon IV and 16.0% form synthon I. OH O Figure 2.9 Supramolecular phenolic OH O heterosynthon (Synthon IV) 23

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For polyphenols out of 330 hits 23.5% were f ound to have the synthon IV and 39.7% form synthon I. The distance range for the occurren ce of synthon IV was found to be 2.55-3.07 Figure 2.10 Histogram for synthon IV The similar kind of search was made for the presence of am ides with phenols and polyphenols was made for the occurrence of phenolic OHCO (synthon V) and phenolic OHNH (synthon VI) supramolecular heterosynthons. Out of 85 hits containing phenols and amides only 18.8% hits form the synthon V and 16.5% form synthon VI and only 7.1% of the total hits form synthon I. O H O NH2 O H O N H H (a) (b) Figure 2.11 (a) Supramolecular phenolic OH CO; (b) Supramolecular phenolic OH NH2 heter osynthons synthon There were only 10 hits for am ides and polyphenols and out of which only 3 hits were found to form synthon V and 8 hits form synthon VI and only 2 hits were found to form synthon I. 24

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The bond ranges for the synthons V and VI were found to be 2.6-3.0 2.75-3.175 and respectively as shown in Figure 2.12. (a) (b) Figure 2.12 Histogram for (a) Synthon V; (b) Synthon VI 2.4 Discussions Upon reviewing the statistics obtained from CSD searches, it is evident that aromatic nitrogen seems to be more prominent a nd competitive functional group to hydrogen bond with phenols and polyphenols. Even the carbonyls are good competitors for hydrogen bonding with the phenols and polyphenols. The data obtained from CSD sta tistics is useful in selecting the cocrystal formers (CCFs) that contains the co mpetitive functional groups and are capable of forming hydrogen bond to the target nutraceuticals. Compounds containing aromatic nitrogen, carbonyl and amide func tional groups are good candidates as CCFs for polyphenolic compounds such as flavonoids. T hus CSD proves to be very good tool for crystal engineering and would provi de a valuable insight for crys tal engineering strategies in generation of new multicomponent materials. 25

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3. QUERCETIN Quercetin (3, 3, 4, 5-7-pentahydroxyflavone) is a bio flavonoid which is widely distributed in the plant kingdom. 99-102 It is the most abunda nt of all the flavonoid molecules found in many often consumed foods including apples, c itrus fruits, onions, tea, berries and vegetables, as well as ma ny seeds, nuts, flowers, barks and leaves. 103 It is also present in medicinal botanicals li ke Ginkgo biloba, Hypericum perforatum, Sambuscus Canadensis and many others. 104 Figure 3.1 Structure of Quercetin 3.1 Description Quercetin (Mol Wt = 302.2) is a bioflavonoi d (pigment), found in almost all herbs, fruits, and vegetables. Bioflavonoids provi de the body with anti-inflammatory and antioxidant protection, and querc etin is one of the most pow erful and effective herbal anti-inflammatory, and antioxidant supplements in the market today. 105, 106 Quercetin has been shown to prevent the development of a variety of conditions related to inflammation by direct inhibition of several initial processes of inflamma tion and free-radical damage, including arthritis, allergies, macular dege neration, heart disease and various forms of 26

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cancer. 107 Quercetin also shows anti-tumor properties. 108 The estimated average daily dietary consumption of quercetin is about 30 mg/day. However, the manufacturers recommended daily dose of over-the-counter quercetin supplements ranges from 400 1200 mg/day. 109 There is no recommended standard dose for quercetin but there have been some occasional reports of nausea wh en taken as high doses in supplements. 106 Quercetin exhibits poor solubil ity in water, it is practically insoluble in water but it is soluble in 100% sodium hydroxide and methanol but in its glycoside form (with a sugar attached), which is the common form of its occurrence in fruits and vegetables, is more soluble in the body. 110 Some of the glycoside forms of quercetin are rutin and quercitrin. 3.2 Strategy for Crystal Engineering of Quercetin The quercetin molecule consists of three ri ngs designated A, B and C respectively and is illustrated in Figure 3.2. The five hydroxyl groups, labeled as a-e can act as hydrogen bond acceptors and/or donors and the ether and carbonyl moieties are capable of serving as hydrogen bond acceptors. O OHd OHeOHbOHaO B C A OHc Figure 3.2 Representation of diff erent rings in Quercetin Based on the CSD search as discussed in Chapter 2 for the occurrence of supramolecular 27

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synthons between polyphenols and other function alities, aromatic nitrogen, carbonyls and amides look more promising competitors for hydrogen bonding. To date, the crystal structure of pure quercetin is not known but there are two entries for quercetin dihydrate with ref codes FEFBEX 111 and FEFBEX01 112 and a formamide solvate (EVIJUO) 113 and other hydrates (LORKEI) 114 in the CSD. Figure 3.3 repr esents the packing in the quercetin dihydrate. The hydrogen bonding for th e dihydrates of quercetin with the ref codes FEFBEX and FEFBEX01 are same but the R factor for FEFBEX01 (R fac 4.8) is better than FEFBEX (R fac 9.5). Figure 3.3 Hydrogen bonding in Quercetin dihydrate In the dihydrate form quercetin crystallizes with two water molecules that participate in extended hydrogen bonding network through the crystal lattice. In the crystal structure quercetin exists as a dimer which is formed by the OH b and the adjacent carbonyl moiety and these are also involved in the intramol ecular hydrogen bonding in the dimer (circled in Fig 3.3). All the hydroxyl a nd carbonyl groups and the wate r molecules participate in hydrogen bonding. All the three ring s of quercetin molecule in the crystal lattice are not 28

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planar. The rings A and C lie in the same pl ane whereas the ring B is not. The dihedral angle between the plane containing rings A and C and the plane containing ring B is 8.10 o as shown in Figure 3.4. The overall hydr ogen bonding leads to the stacking of infinite 2 D sheets. Figure 3.4 Illustration of dihedral angle between th e planes. (Red plane cont ains rings A, C and the blue plane contains ring B) 3.3 Solvates of Quercetin Three solvates of quercetin with pyridine, tetrahydrofuran (THF) and acetone namely I, II, III, were obtained by differe nt methods. All the chemical s used during the preparation were purchased from Sigma Al drich and used as received. Form I and II Quercetin-Pyridine solvates Form III Quercetin-THF solvate Form IV Quercetin-Acetone solvate Table 3.1 Solvates of Quercetin 3.3.1 Form I and Form II (Pyr idine solvates of Quercetin) Synthesis: 34 mg of quercetin dihydrate (purchased from Sigma Aldrich and used as received) was dissolved in 5ml of Pyridine; th is solution was layered with 4 mL of water 29

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and refrigerated. After 4 hours golden yellow crystals were produced (yield25mg). The crystallization experiment was conducted in an unmodified atmosphere and the solvents were dried by standard methods prior to use. mp-314 C. Figure 3.5 Single crystal of quercetin-pyridine solvate The attempts to get pure crystals of querc etin using pyridine solvate resulted in two different forms of quercetin-pyr idine solvate namely Form I and Form II. The Form I in due course of time transforms to Form II. The crystal structure of Form I reveals that two molecules of pyridine hydrogen bond to each molecule of quercetin. The quercetin molecules dont exists as a dimer as found in its dihydrate form (FEFBEX01). But the quercetin molecules forms a cyclic pattern, described by Etters 115 graph set R 2 2 (8). The cyclic pattern includes heterosynthons such as OH CO (D = 2.724 ) and OH OH (D = 2.914 ). Where as the crystal structure Form II reveals that quercetin molecules exists as dimer as found in quercetin dihydrate (FEFBE X01). But the only difference being that in FEFBEX01 the dimer includes OH b and the carbonyl moiety whereas in Form II the dimer includes OH c and the carbonyl moiety (D = 2.749 ) and the OH b is just involved in the intramolecular hydr ogen bonding (D = 2.608(3) Ao). Figure 3.6 represents the hydrogen bonding in Form I and Form II. The R 2 2 (8) graph set in Form I and the quercetin dimer in Form II are circled in the figure. 30

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(a) (b) Figure 3.6 (a) Hydrogen bonding in Form I (b) Hydrogen bonding in Form II The dihedral angle between the planes cont aining the rings A, C and ring B for for m I and Form II are 21.11o and 23.71o respectively. These angles ar e higher than that is found in FEFBEX01 (8.10o). This indicates that quercetin molecules in Form I and II are not that planar as in FEFBEX 01 (quercetin dihydrate). (a) (b) Figure 3.7 The dihedral angles in From I (a) and Form II (b) The presence of quercetin-pyridine solvate in tw o different forms indicates that quercetin exhibits polymorphism.65 The CSD search has no entries for the crystal structure of polymorphic quercetin but the literature sear ches reveal that quercetin exhibits polymorphism.116-119 The Form II could be most stable than Form I because according to Ostwalds step rule134, 135 which states: In general it is not the most stable but the least stable polymorph that crystallizes first. In both the forms the pyridine molecules hydrogen bond to OHa and OHe of quercetin molecules. The hydrogen bond distances for the OHa Narom and OHe Narom in From I are 2.705 and 2.677 respectively whereas 31

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for Form II it is 2.765 and 2.64 The dist ances are in accordance with the distance ranges found in CSD as discussed in Ch apter 2. Overall supramolecular hydrogen bonding in Form I results in 2D tape and in Form II it results in a 2D sheet. Desolvation occurs when the crystals are heated at 150 o C for 10 min and they become amorphous. The amorphous powder could be pure quercetin as it decomposes at 316 o C which coincides with the literature value. 120 3.3.2 Form III (THF solvate of Quercetin) Synthesis: 34 mg of quercetin dihydrate (purchased from Sigma Aldrich and used as received) was dissolved in 5ml of THF; this solution was layered by 4 mL of hexane and refrigerated. After 4 days golden yellow crys tals in the form of plates were produced (yield-20.5mg). The crystallization expe riment was conducted in an unmodified atmosphere and the solvents were dried by st andard methods prior to use. mp 316 C. Figure 3.8 Single crystal of quercetin-THF solvate (Form III) The crystal structure reveals that there are three THF molecules in the asymmetric unit. Two of the THF molecules are involved in the formation of supramolecular network with the quercetin molecules and the third distorte d THF molecule just act as a guest. All the hydroxyl and the carbonyl groups of quercetin and the THF molecules are involved in hydrogen bonding. In Form III also quercetin exis ts as a dimer (circle in Fig 3.9) with a 32

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bond distance of 2.749(3) Ao similar to that found in Form II (quercetin-pyridine solvate) with hydrogen bond distance of 2.749 (OH CO). Figure 3.9 compares the hydrogen bonding in Form II and III. The dimer in Form III can also be described as R2 2 (10) graph set.115 Figure 3.9 Comparison of hydrogen bonding in Form III (left) with that of Form II (right) Desolvation of Form III crystals occurs after 15 -20 minutes when they are taken out from the mother liquor and leaves the system amorphous and the decomposition temperature of the powder coincides with that of quercetin.120 3.3.3 Form III (Quercetin-Acetone solvate) Synthesis: 34 m g of quercetin dihydra te and 13.3 mg of caprol actam were dissolved in 5ml of acetone and the refrigerated for 3 days. Golden yellow crystals of solvate III were obtained wherein quercetin and acetone were in 1:1 stoichiometric ratio (yield-20mg). All the chemicals used during the preparation were purchased from Sigma Aldrich and used as received. The crystallization experiment was conducted in an unmodified atmosphere and the solvents were dried by stan dard methods prior to use. mp 316oC. 33

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Figure 3.10 Crystals of Que rcetin-Acetone solvate (Form III) The crystals of solvate III were obtained dur ing an attempt to prepare the cocrystal of quercetin and caprolactam by using acetone as a solvent. In Form III also quercetin exists as a dimer, but this dimer is formed by OH d and OH e of quercetin molecule form strong with hydrogen bond distance of 2.754 which fa lls well with in the range for alcoholalcohol supramolecular homosynthon as discu ssed in Chapter 2. Figure 3.11 represents the dimer that is formed in Form III. All hydroxyls and the carbonyl groups of quercetin participate in the hydrogen bonding. Further, OH e of both quercetin molecule hydrogen bonds with OH d of adjacent quercetin molecule with OH OH interaction (D = 2.765 ) and the OH d hydrogen bonds to the carbonyl gro up of another adjacent quercetin molecule and forms a strong OH O hydrogen bond (D = 2.682 ). Figure 3.11 Hydrogen bonding in Form III (Acetone molecules colored red) 34

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The OHa of each quercetin molecule is bifu rcated in hydrogen bonding with acetone molecule forming OHO hydrogen bond (D = 2.596 ) and OHOH supramolecular homosynthon with adjacent OHa quercetin molecules (D = 2.750 ). All the hydrogen bond distances mentioned above fall well with in the range of distan ces found in CSD as discussed in Chapter 2. The overall hydrogen bon ding results in a cri sscross grid network as represented in Figure 3.12. Figure 3.12 Representation of crisscross grid network in Quercetin-Acetone solvate (Space fill model) 3.3.4 Discussions The attempts to obtain the pure crystals of quercetin from quercetin dihydrate resulted in the form ation of new forms namely, Form I, Form II, Form III and Form IV. The Form I and Form II are the polymorphs of quercetin whereas, the Form III and IV are the solvates of THF and acetone respectively. The quercetin molecule exists as dimer in Form II and III similar to that of quercetin dihydrate (FEFBEX01)90 where as such kind of dimer is absent in Form I. Hydrogen bonding in Form I and II results in the formation of linear tapes where as the Form III results in cross grid network. Desolvation occurs for Form II after 5-10 minutes when the crystals are taken out of the mother liquor and an amorphous powder is obtained where as, the Form I and III are pretty st able in absence of mother liquor. In order to get rid of the solvent molecules in Form I and III, the crystals were heated at 150 and 55 oC respectively. The amorphous powders from all the three 35

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forms were analyzed by DSC and melt temp se parately and it was found that in all the three cases the powder decomposes at 328 o C, that exactly coincides with the decomposition temperature of quercetin. The amorphous form obtained could be pure quercetin, but this could not be confirmed as the crystals of pure quercetin were not produced by other means. 3.4 Cocrystals of Quercetin Crystal engineering of quercetin lead to th e generation of two novel cocrystals namely Cocrystal I and II with caffeine and isonicoti namide were obtained by slow evaporation. The functional groups that are more likely to undergo hydrogen boning in quercetin are the hydroxyls and the carbonyl. These functiona l groups were utilized to target the various functional groups in caffeine and isoni cotinamide such as the nitrogen in the imidazole ring and the imide groups in caffeine and the aromatic nitrogen and the amide groups in isonicotinamide. The CSD searches (C hapter 2) reveal th at the polyphenols are more likely to form supramolecular synthons with functional gr oups like carbonyl and aromatic nitrogen. The percentages obtained for OH N arom and OH CO supramolecular heterosynthons in the CSD search es are 84.8% and 74.4% respectively. 3.4.1 QuercetinCaffeineMethanol cocrystal solvate 36 Figure 3.13 The asymmetric unit of 1:1:1 cocr ystal solvate of quercetin, caffeine and methanol

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37 Synthesis: 68 mg of quercetin dihydrate and 38 mg of caffeine were dissolved in approximately 5 mL of methanol and heated until a clear solution was obtained. Slow evaporation of this solution in refrigerator resulted in 1:1 crystals after 3 days (yield30 mg). All the chemicals used during the prep aration were purchased from Sigma Aldrich and used as received. All cr ystallization experiments were conducted in an unmodified atmosphere and the solvents were dried by standard methods prior to use. mp: 246C. Figure 3.14 Single crystal of quercetin-caffei ne-methanol cocrystal solvate (Cocrystal I) he crystal structure reveals that the imide group and the aromatic nitrogen of caffeine are the functional group that interacts with the hydroxyl groups of quercetin. Caffeine molecules interact with quercetin molecules via the formation of OHcNarom and OHaCO supramolecular heterosynthons. The fo rmer supramolecular heterosynthon is formed by the interaction of OHc quercetin and the aromatic nitrogen of the imidazole ring in caffeine (D = 2.821(3) A ) and the latter results due to the hydrogen bonding between OHa of quercetin and the CO moiety of the imide group of caffeine (D= 2.716(3) A ). The carbonyl in the caffeine molecule hydrogen bonds to the methanol molecule (D=2.712(3) A). All the above distances are in accordance with the OHO synthon and OHcNarom distance ranges found in CSD as discussed in Chapter 2. T o o o

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Figure 3.15 Intermolecular interactions in the 1:1:1 cocrystal solvate of quercetin, caffeine and methanol (colored green) Adjacent quercetin molecules interact by utilizing some of the remaining hydrogen bonding sites as follows: trifurcation of the OH b moiety of quercetin molecules through supramolecular homosynthon and OH CO supramolecular heterosynthon. The OH OH supramolecular homosynthon is formed between the OH b of quercetin molecules and OH d and OH e of adjacent quercetin molecules (D = 2.774(3) A o 2.847(3) A o respectively). OH b is also engaged in OH CO intramolecular heterosynthon (, D = 2.602(3) A o ). The bond distances fall in the range found in the CSD searches for the respective synthons as discussed in Chapter 2. These hydrogen bond interactions afford a supramolecular sheet that stacks up. Thes e sheets are furthe r connected via the interaction of methanol molecules with OH e moieties of quercetin molecules through OH OH supramolecular homosynthons (D = 2.633(3) A o ). The overall effect of the hydrogen bonding results in the formation of a network where the methanol could be described as a guest although as shown in Figure 3.16. 38

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Figure 3.16 Illustration of sheets generated in the cocrystal 1. 3.4.2 Quercetin-Isonicotinamide Figure 3.17 The asymmetric unit of the1:1 qu ercetin isonicotinamide cocrystal Synthesis: 67.6 mg of Quercetin di hydrate and 24.6 mg of isonicotinamide were dissolved in approximately 5 mL of metha nol and heated until a clear solution was obtained. Slow evaporation of this solution in refrigerator resulted in 1:1 crystals after 2 days (yield-31.5mg). All the chemicals used during the preparati on were purchased from Sigma Aldrich and used as re ceived. The crystallization e xperiment was conducted in an unmodified atmosphere and the solvents were dried by standard methods prior to use. mp: 256-260C. Figure 3.18 Single crystal of quercetin-isonicotinamide cocrystal (Cocrystal 2) 39

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The aromatic nitrogen and the carbonyl mo iety of the amide group and the carbonyl group in quercetin acts as hydrogen bond accepto rs where as all the hydroxyl groups of quercetin can act as hydrogen bond acceptors/don ors. Isonicotinamide molecules interact with quercetin molecules via, CO OH and NH OH supramolecular heterosynthons and OH OH supramolecular homosynthons. One of the two hydrogen atoms in the amino moiety of the isonicotinamide hydrogen bonds to the carbonyl group of adjacent quercetin molecules and the other hydrogen atom interacts with OH d of a different quercetin molecule giving rise to NH CO (D = 3.018(4) A o ) and NH OH (D = 3.028(4) A o ) supramolecular heterosynthons, respectively. These distances are in accordance with the distance ranges found in the CSD search fo r amides in Chapter 2. The carbonyl of the amide moiety hydrogen bonds to OH e quercetin molecules whereas the N arom atom of the isonicotinamide molecule interacts with OH a of quercetin molecules and thereby generates CO OH (D = 2.607(3) A o ) and N arom OH (D = 2.684(3) A o ) supramolecular heterosynthons respectively. The distances fall within the ra nge found in CSD as discussed in Chapter 2. Figure 3.19 Intermolecular inte ractions in the 1:1 Quercetin-Isonicotinamide cocrystal. 40

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The OHd & OHe moieties of two quercetin molecules and the amide moieties and the syn hydrogen atoms of the amino group of two isonicotinamide molecules form a four component assembly composed of two R 2 2 (8) and one R 2 2 (10) supramolecular synthons (Etters graph set). 115 Both the patterns result in R 4 4 (18) graph set as represented in Figure 3.6. Figure 3.20 Illustration of R 4 4 (18) graph set in quercetin and isonicotinamide cocrystal. The R 2 2 (8) graph set is formed by two querce tin molecules and one isonicotinamide molecules which includes OH CO supramolecular heterosynthon formed between OHe moieties and the carbonyl groups of isoni cotinamide molecules (D = 2.607(3) A o The R 2 2 (10) supramolecular synthon occurs betw een two quercetin molecules in which OH OH supramolecular homosynthons are formed between OH d & OH e groups of two quercetin (D= 2.765(3) A o ). The dimer [R 2 2 (10)] is formed between two quercetin molecules remains intact a nd the exterior hydrogen bonding sites are utilized in the hydrogen bonding with N-H of isonicotinamide (D 3.028(4) A o ). The bond distances found in the supramolecule are in accordan ce with the distances found in CSD for the respective synthons as discussed in Chapter 2. 41

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42 3.4.4 Discussions Crystal engineering has lead to the generati on novel cocrystals of quercetin and thus confirms that flavonoids are capable of gene rating novel cocrystals with pharmaceutically acceptable molecules such as caffeine and isonicotinamide. As caffeine and isonicotinamide have no solubility problems whereas caffeine has problem with physical stability against hydration. The CSD reveals that there are 17 entries fo r the cocrystals of caffeine. There are also evidences which indi cate that cocrystallization of caffeine with suitable cocrystal formers (CCF) overcome the hydration problem. 121 Even isonicotinamide 122 seems to be a good CCF as there are cocrystals of it in the literature. The CSD statistics as discussed in Chapter 2 reveals that aromatic nitrogen and carbonyl functional groups are good acceptors to hydrogen bond with the hydroxyls of polyphenolic compounds. Therefore caffe ine and isonicotinamide (isomer of nicotinamide, known as niacin ) 123 stand as suitable CCFs fo r cocrystallization and are pharmaceutically acceptable molecules. Thus cr ystal engineering allows one to choose complementary components for polyphenolic compounds for designing supramolecules.

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4. HESPERETIN Hesperetin (RS-2,3-dihydro-5,7-dihy droxy-2-(3-hydroxy-4-methoxyphenyl)-4H-1benzopyran-4-one) is a naturally occurring flava none which is unique among the others as it is the only one which is faintly sweet while most of the flavonoids ar e tasteless or bitter. 124 Hesperetin is the aglycone form of hesperedin which is abundantly present in citrus fruits. Animal studies have shown that hesperetin has very good anti-inflammatory properties. 125, 111 Figure 4.1 Structure of hesperetin (hydrogen are not included) 4.1 Description Hesperetin (Mol.Wt 302.27 g/mol) is a bioflavon oid that is present in the plants in its glycoside form (hesperedin). We have discusse d earlier in Chapter 1, how glycoside forms of flavonoids are more soluble that the aglycone form. Hesperedin is more soluble than hesperetin because of the sugar ring attached to it. 126 It is a phenolic antioxidant 127 antiallergic 128 and antimutagenic 129 It may scavenge the reactive oxygen species as superoxide anions and may pr otect against peroxidation. Stud ies on rats have shown that hesperetin helps in reducing the cholestero l by the inhibition of 3-hydroxy-3-ethylglutaryl coenzyme A. 130 In vitro studies have also shown that hesp eretin has some anti cancer activity too. 131 43

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4.2 Strategy for Crystal Engineering of Hesperetin Hesperetin has similar structure to that of que rcetin, the difference being rings B and C. In ring B there is a m ethoxy group instead of a hydroxyl and saturated ring C. Figure 4.2 represents the structure of he speretin with its 3 rings A, B, C. The hydroxyl groups are labeled a, b and c. The hesperetin molecu le has two hydroxyl groups on ring A and one on ring B act as hydrogen bond acceptors and/or donor s. Hesperetin has also has an ether and carbonyl (ketone) moieties on ring C th at can act as hydrogen bond acceptors. O OCH3 OHcOHbOHaO B C A Figure 4.2 Illustration of rings in Hesperetin The data available in the CSD reveals that there are two crys tal structures of hesperetin discovered so far, one is th e pure h esperetin (YEHROS) 132 and the other is the monohydrate form (FOYTOC).133 (a) (b) Figure 4.3 Hydrogen bonding in (a) Hesperetin (b) Hesperetin monohydrate Figure 4.3 represents the hydrogen bonding in both the available form s of hesperetin as a 44

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racemate and monohydrate. Hesperetin molecule in YEHROS is not a planar molecule as represented in Figure 4.4 (a). (a) YEHROS (b) FOYTOC Figure 4.4 Illustration of the dihedral angles (blue plane contains rings A and C; red plane contains the ring C). The two rings A and C lie in the same plane a nd the ring B lies in the other plane with a dihedral angle of 53.59 o Whereas, in FOYTOC hespere tin adopts almost a planar conformation while the conformatio n in YEHROS seems to be more favorable as there is less repulsive steric hindrance between th e rings with a dihedral angle of 2.91 o as represented in Figure 4.4 (b). 133 4.2.1 Hesperetin-Isonicotinamide cocrystal (Cocrystal 3) Figure 4.5 The asymmetric unit of hesperetin:isonicotinamide 1:1 cocrystal 45

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Synthesis: 60 mg of Hesperetin and 24.6 mg were dissolved in approximately 5 mL of ethanol and heated until a clear solution was obt ained. Slow evaporation of this solution in refrigerator resulted in 1:1 crystals after 5 days. All crystallization experiments were conducted in an unmodified atmosphere and th e solvents were dried by standard methods prior to use. All chemicals were purchased from Aldrich and used as obtained. m.p 172176 o C. Figure 4.6 Single crystal of hesp eretin-isonicotinamide cocrystal Crystallization of hesperetin with isonicotin amide resulted in a 1:1 cocrystal. In the isonicotinamide molecule, the aromatic nitrogen and the carbonyl moiety of the amide group act as hydrogen bond acceptors. The two hydrogen atoms of the amino group of the amide moiety act as the hydrogen bond donors. The X-ray crystal structure reveals the existence of a supramolecular homosynthon (amide dimer) formed by the interaction of two isonicotinamide molecules. This results in a graph set notation 115 R 2 2 (8) with O---N arom interaction as represented in Fi gure 4.8 with bond distance of 2.868(3) A o that fall well with the range found in CSD in Chapter 2. The s upramolecular synthon form ed in the cocrystal includes OH---N hydrogen bond between the nitrogen atom of isonicotinamide and the OH a of the adjacent hesperetin molecule with an O---N bond distance of 2.623(2) A o OH a is further bifurcated as it forms a hydrogen bond with the anti N-H of the isonicotinamide 46

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dimer with an N---O bond distance of 3.031(3) A o The amide dimer remains intact and as observed in the crystal struct ure of pure isonicotinamide. Figure 4.7 Overall hydrogen bonding in the Hesperetin-Isonicotinamide 1:1 cocrystal The hesperetin molecules lie opposite to each other around the crystallographic inversion center as represented in Figure 4.8. The adjacen t hesperetin molecule s interact via OH--CO utilizing the hydroxyl moieties on ring B with a bond distance of 2.720(2) A o There is also intramolecular hydrogen bonding between the carbonyl (ketone) group on ring C and the OH c with a bond distance of 2.584(2) A o All the hydrogen bond distances found in hesperetin and isonicotinamide falls well with in the expected as discussed in Chapter 2. Figure 4.8 Illustration supramolecula r synthons in the cocrystal 3. Figure 4.7 represents the how the hesperet in and isonicotinamide molecules arrange 47

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themselves in the crystal la ttice. The hydrogen bonding in th e cocrystal 3, the hesperetin molecules form a one dimensional tape along Z-axis and these tape s are connected through centrosymmetric amide dimers formed by isonicotinamide molecules forming a two dimensional sheets with cavities (31 x 10 2 ). Figure 4.9 The cavity formed in hesp eretin:isonicotinami de cocrystal. The cavities are filled by similar sheets and its eight fold interpenetrated. The overall hydrogen-bonding pattern is that of a zigzag 2-D sheet with interpenetra tion and it means that all hydrogen bond donors and acceptors in both mol ecules are satisfied as shown in Figure 4.10. Figure 4.10 Illustration of 8-fold interpenetrated network formed in Cocrystal 3 48

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The hesperetin molecules maintain the angular conformation similar to what is observed in pure hesperetin crystal structure (YEHROS). The dihedral angle observed for hesperetin molecules in the cocrystal between the planes containing rings A, C and ring B is greater by 24.2 o than found YEHROS. The hesperetin mol ecules are more stable in angular conformation as the steric hi ndrance is reduced. Figure 4.11 represents the dihedral angle between the planes of the rings in hesperetin molecule. Figure 4.11 Illustration of dihedral angle in hesperetin molecules (red plane contains rings A and C; blue contains ring C) 4.2.2 Hesperetin-Nicotinic acid cocrystals Figure 4.12 Hesperetin Nicotinic acid 1:1 zwitterion cocrystal (Form I) Synthesis: 60 mg of Hesperetin and 24.6 mg of nicotinic acid were dissolved in approximately 5 mL of methanol and heated until a clear solution was obtained. Slow 49

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evaporation of this so lution in refrigerator resulted in 1:1 crystals after 5 days. All crystallization experiments were conducted in an unmodified atmosphere and the solvents were dried by standard methods prior to use. All chemicals were purchased from Aldrich and used as obtained. m.p 198-204 o C. Figure 4.13 Single crystals of hesperetin-NA cocrystals. Crystallization of hesperetin with nicotinic aci d results in two1:1 cocrystals in which the nicotinic acid exists as a zwitterionic state, Form I contains only one of the enantiomer of R-hesperetin (Cahn-Ingo ld-Prelog priority rules) and conve rts to Form II which contains both forms of (+ ) hesperetin (Cahn-Ingold-Prelog priority rules) crystalliz es with nicotinic zwitterion. As Form I contains only one of th e enantiomer of hesperetin in the crystal structure it is called as the racemic conglomerat e and Form II is the racemate of hesperetin. During the crystallization process both the cocrystals were genera ted in the same vial and the reproduction of the Form I cocr ystal was not achieved. This indicates that Form I is kinetically more favored and Form II is th ermodynamically. Figure 4.13 represents the overall hydrogen bonding in cocrystal Form I. The crystal structure of Form I reveals the existence of nicotinic acid (NA) as a zwitterion ic species in the cocrystal, which is not an amino acid. The zwitterion of NA can be confirmed by the following structural information: a) The C-O bond distances in the carboxylate ion were found to be 1.241 7& 1.250 and 50

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the corresponding C-N-C (protonated pyridine) bond angle equal to 121.54 o Where as, the C-O & C=O bond distances and the C-N-C bond angle in NA are 1.217 &1.289 and 117.65 o respectively. Figure 4.14 Overall hydrogen bonding in the hesperetin Form I cocrystal. (Blue colorR-Hesperetin) Pure NA forms a linear tape where they are hydrogen bonded to each other in a head to tail fashion with a bond distance of 2.666 Similar kind of tape is also present in Form I cocrystal with hydrogen bond distance of 2.604 Figure 4. 15 represents the tapes that are formed in NA in pure NA (a) and in Form I cocrystal (b) respectively. (a) (b) Figure 4.15 Illustration of hydrogen bonds in NA: a) NA in pure form b) NA in the cocrystal, hesperetin molecules are deleted for clarity. 51

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The CSD search for the cocrystals of NA rev eals that there are only two entries with ref codes AWIDEB 136 and SESLIM. 137 The two cocrystals were made by using 3,5dinitrobenzoic and 4 amino benzoic acid resp ectively. The crystal structure of SESLIM reveals that NA acid form a molecular tape similar to that found in pure NA and Form I cocrystal. The other modes by which the remaining hydrogen bonding sites are exploited includes O---O (D = 2.504 ) interaction between NA and the OH a of hesperetin molecule. The hesperetin molecules ar e interconnected via OHOH supramolecular hydrogen bonding (D = 2.867 ) and forms linear tapes that sandw ich the NA molecular ta pes. There is weak hydrogen bonding between the OHb of hesperetin molecule with the ether group of the other hesperetin molecules with a bond distance of 3.001 All the hydrogen bond distances fall in the ranges found in the CSD as discussed in Chapter 2. In Form I, the hesperetin changes in conformation adopts almost a planar c onformation similar to that in hesperetin monohydrate (FOYTOC) with a dihedral angle of 4.68 o between the planes containing the rings A, B and C. Figure 4.16 represents the dihedral angle. Figure 4.16 Representation of dihedral angle between the rings. (red plane contains rings A and C, blue plane contains ring B) The crystal structure of the Form II reveals (C ahn, Inngold and Prelog rule s) that hesperetin crystallizes out as a racema te, is a result of thermodynamically favored product. The NA zwitterions hydrogen bond with each other in a h ead-to-tail fashion and thereby form chains 52

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that are sandwiched by chains of hesperetin mol ecules. The crystal is still polar as the headto-tail chains of NA zwitterions are parallel throughout the crysta l structure. The three chains are linked by lateral hydrogen bonds and therefore form a network that could be described as a supramolecular tape as a represented in Fi gure 4.17. The molecular structure of hesperetin as it exists in Form II rev eals that one of the three hydroxyl groups engages in an intramolecular hydrogen bond with the carbonyl moiety. All the hydrogen bonds formed in both the forms have their distances that fall well with in the bond range s obtained in the CSD searches as discussed in Chapter 2. Figure 4.17 The hydrogen bonding in the hesperet in Form II cocrystal (yellow and maroon: NA molecules, (+) hesperetin: blue, (-) hesperetin) green) In Form II, the hesperetin maintains the almost planar conformation as found in Form I but it is slightly greater. In Form I it is 4.69 o and in Form II it is around 11.38 o Figure 4.18 The dihedral angle formed between the planes of rings A, C and ring B in Form II 53

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54 4.2.4 Discussions Crystal engineering has lead to the generation novel cocrys tals of hesperetin with pharmaceutically acceptable molecules such as isonicotinamide and nicotinic acid (NA). For the first time crystal engineering has b een applied to generate cocrystals of Flavonoids. As we discussed earlier in Ch apter 2 that compounds containing aromatic nitrogen are more likely to form hydrogen bond with phenolic OH groups. Both isonicotinamide and NA have aromatic nitrogen and make them good cocrystal formers for the cocrystallization of hesper etin. In addition to aromatic nitrogen isonicotinamide and NA have amide and acid groups respectively which are capable of forming hydrogen bonds with hesperetin. The CS D search reveals that there are almost 26 cocrystals of isonicotinamide. Among the 26 cocrystals three are with polyphenols such as phloroglucinol, 138 hydroquinone, 138 and resorcinol. 138 NA has two cocrystals dinitrobenzoic 136 acid and aminobenzoic acid. 137 Thus isonicotinamide and NA seem to be good cocrystal former (CCF) for cocrystallization.

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55 5. DISSOLUTION STUDIES OF COCRYSTALS Most of the active pharmaceutical ingredients (API s) are administered in the solid state as part of an approved dosage type like tablets, capsules, etc as these are the convenient and compact format to store a drug. 70 The absorption of all the forms of a drug depends on its physical properties. 139 There are examples of drugs that are commercially available that suffer problems related to solubility, dissoluti on rate and absorption in the neutral form. These entire problems thus restrict the bioa vailability of drugs. In this context the application of crystal engineer ing to the field of pharmaceuti cs provides a solution. It has intensified the research in the pharmaceutical in dustries as it offers a means to alter the solubilities of drugs by the discovery of new crystal forms there by improving the bioavailability. 140, 141 The new forms include polymorphs solvates, salts, hydrates and cocrystals. There are evidences where in it is proved that drugs that are commercially available, such as fluoxitine HCl (Prozac*) 86 Itraconazole (Sporanox*) 75 with low solubility show a drastic impr ovement in the solub ilities via cocrystall ization. But not all cocrystals show improved properties, and they are not a panacea for all problems. For example the cocrystals 67 forms of Fluoxetine hydrochloride, an anti depressant drug, with a series of acids, has shown variations in its solubility when compared to the pure API. For fluoxetine HC l: succinic acid cocrysta l an approximate 3fold increase in the dissolution of the AP I relative to fluoxetine HC l, the cocrystal of fluoxetine HCl: benzoic acid dissolves at a rate that is approxim ately half of the rate for the API alone, while the di ssolution rate for fluoxetine HC l: fumaric acid cocrystal is

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roughly similar to that of fluoxetine HCl. Thes e results reveal that, by cocrystallizing an API with different guest molecules, it is possible to increase or decrease the dissolution rate of the API or to leave the effective di ssolution rate essentially unchanged. The Figure 5.1 represents the resu lts of dissolution studies carried out for various cocrystals of fluoxetine HCl. Figure 5.1 Powder dissolution profiles for (1), (2), (3), and (4) in water at 20 C. Another example of API that forms cocrys tals is Itraconazole (Sporanox), a low solubility drug that is available com merc ially in the amorphous form. The dissolution studies of the cocrystals of Itraconazole dr ug with carboxylic acids s how a wide range of solubilities ranging from 420 fold than that of the crystalline form of API. Figure 5.2 Dissolution profiles into 0.1 N HCl at 25 C for Sporanox beads and the cocrystals 56

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The carbamazepine (CBZ): saccharin cocrystal is also an appropriate example in this context. CBZ, commercially available as Tegretol, is another example of low soluble drug. 14 2 The CZB:saccharin cocrystal has bette r solubility when compared to CBZ dihydrate and pure CBZ. The cocrystal even offe rs more stability (cocrystal is more stable than pure CBZ for more than 24hrs) to CBZ in the cocrystal. Figure 5.3 compares the plasma concentration of CBZ and the cocrystal in fated beagle dogs. Figure 5.3 Average plasma time curves of carbamazepine concentrat ions (SEM) from a cross-over experiment in fasted beagle dogs (n = 4) given oral doses of 200 mg of the active drug as Tegretol tablets and co-crystal 5.1 Dissolution experiments of quer cetin and hesperetin cocrystals Dissolution is a characterization test comm only used by the pharmaceutical industry to guide formulation design and control product quality. 143 It is also the only test that measures the rate of in vitro drug release as a function of time, wh ich can reflect either reproducibility of the product manufacturing pr ocess or, in limited cases, in vivo drug release. The common analytical methods used for quantifying drug release in dissolution tests are: spectrophotometric, chroma tographic, mass spectrometric, and 57

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58 potentiometric. 144 The UV/VIS spectrophotometric method has been the traditional analytical method for dissolution testing. A compound will exhibit absorption in the UV region if it contains one or more chromophores, such as aromatic nitro, azoxy, nitroso, carbonyl, or azo groups. For quant itation of any given drug, the desired absorption (A) should be 0.3.0. In the UV absorption spectr a of flavonoid, there are generally two main absorption bands, band I (300n m) and band II (240 nm) which are associated with the absorption of the cinnamoyl system and the absorption of the benzoyl moiety in the molecules respectively. 145, 146 The two absorption bands are more sensitive to the environment of pH due to the weak acidity of th e phenolic hydroxyl groups of flavonoid. In the UV absorption spectra of hesperetin in ethanol, the main absorption peak is assigned as band II, centered in about 290 nm, and there is only a weak broad shoulder peak in about 330 nm. 145, 147 The reason for this is because the B ring of hesperetin is not conjugated with the carbonyl group in ri ng C. Whereas quercetin has two bands at 255 nm and 374 nm respectively. The band II for quercetin is broader than band I and this is due to the conj ugation of ring B with ring C. As quercetin and hesperetin did not show any UV absorbance spectrum in water raised a situation to look fo r a suitable solvent in which these compounds are soluble and suitable for the dissolution studies. Most of the flavonoids are soluble in alcohols; therefore a mixture of solvent system c onsisting of 1:1 ethanol/water (V/V%) was adopted in order to carry out the dissol ution experiments. Figure 5.4 represents UV spectrum of quercetin and hesper etin in 1:1 ethanol/water (V/V%) solvent system.

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Figure 5.4 UV spectrums of Quercetin and He speretin in 1:1 Etha nol/Water (V/V%) 59 tandard For cocrystals 2, 3, 4 the dissolu tion were carried out in 50 % EtOH/Water (v/v) mixture. In order to avoid the interference from methanol in the quercetin:caffeine:methanol cocrystal solvate, methanol/water so lvent system was employed. A 3:2 methanol/water (V/V%) solvent system was used for the dissolution of cocrystal 1. The dissolution study for cocrystal 1 was carried at 400nm as caffeine di d not absorb at this wavelength and did not interfere with the determination of concentr ation of quercetin. In the similar way the wavelengths for the dissolution of cocrystals 2, 3, 4 (cocryst al 2 = 360nm, cocrystal 3 & 4 = 300nm) were selected in such way that the interf erence of the cocrystal formers was not observed while determining the concentrati on of respective flavonoids. The standard calibration curves all the cocrystals were plot ted by preparing the standard solutions of the cocrystals in their respective so lvent systems. For the bulk po wder dissolution of cocrystals 1-4 the powder was sieved using ASTM s sieve in order to ge t the particle sizes

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60 was ranging between 53 75 m. All the experiments were carried at room temperature in 3 sets for each cocrystal in order to have reliabilit y. Approximately 900mg of the sample was added to the beakers containing 80mL of solvent mi xture and the resulting slurry was stirred at 200 rpm with the help of spinning vane on a stir plat e. At regular intervals of time an aliquot drawn out from the beaker and it was filtered using a 0.25 m nylon filter. Appropriate amount of the resulting solution was diluted with the solvent mixture and then the UV absorption was recorded. 5.2 Dissolution study of Quercetin:Ca ffeine cocrystal (Cocrystal 1) The dissolution studies of cocrystal 1 (solvated and desolvated forms) in water did not show any improvements in the solubility of querce tin. Therefore, the disso lutions of methanol solvate of quercetin-caffeine cocrystal (KP05) and desolvated cocrystal were carried out in 3:2 methanol/water (V/ V %) solvent system. The Figure 5.4 represents the comparison of the dissolution of quercetin dihydrate with the methanol solv ate and desolvated cocrystals. It is evident from the Fig 5.5 that there is an appr oximate 6-fold improvement in the solubility of quercetin in the methanol solvate cocrystal and 21-fold improvement in the desolvated cocrystal in 3:2 methanol/water (V/ V %) so lvent system when compared to quercetin dihydrate. The solubility of que rcetin in the desolvated cocr ystal (amorphous form) has much better solubility than the solvat ed cocrystal in the first hour. But, later (after 1 hour) the desolvated cocrystal converts to the solvat ed cocrystal as the so lubility goes down and coincides with the solvated cocrystal. The dissolution experiment demonstrates that the pure cocrystal has better solubility than the solvated cocrystal. Thus, cocrys tallization can tailor the solubility of a compound and could afford to improve the bioavailability.

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0 1 2 3 4 5 6 7 8 0 50 100 150 200 250 Time (Minutes)Concentration (mg/mL) Quercetin dihydrate KP05 Desolvated cocrysta Figure 5.5 Comparison dissolution profiles of quercetin methanol solvated cocrys tal and the desolvated cocrystal in 3:2 MeOH/Wat er (V/V%) solvent system 5.2 Dissolution study of Quercetin-Isonicotinamide (Cocrystal 2) The dissolution quercetin-isonicotinamide co crystal (KP10) was carried out in 1:1 ethanol/water (V/V%) so lvent system as ther e was no improvement in the solubility of quercetin. It is clear from the Figure 5.5 that the solubility of quercetin in the cocrystal reaches the maximum value within the first five minutes and then the solubility goes down and gets almost close to that of quercetin di hydrate. The solubility of pure quercetin through out the experiment is almost constant this i ndicates that the solubility of pure quercetin reaches the saturation limit within five minutes The dissolution of quercetin cocrystal with isonicotinamide reveals that the solubility of quercetin has been improved 6-folds in 1:1 ethanol/water (V/V%) solvent system. 61

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0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 50 100 150 200 250 Time (minutes)Concentration (mg/mL) Quercetin dihydrate KP10 Figure 5.6 Dissolution profiles of quercetin and qu ercetin-isonicotinamide (1 :1) cocrystal in 1:1 EtOH/Water (V/V%) solvent mixture 5.3 Dissolution study of Hesperetin -Isonicotinamide (Cocrystal 3) The dissolution of cocrystal 3 (KP15) was al so carried out in 1: 1 ethanol/water (V/V%) solvent system as it did not showed any improvement the solubility in water. The solubility of pure hesperetin remains constant through out the time. But the solubility of hesperetin in the cocrystal increases slowly reaches a maximum value after 40 min and later it deteriorates down, if extrapolated probably could reach to the saturated limit similar to that pure hesperetin. Figure 5.6 represents the comparison of the concentrations of pure hesperetin and hesperetin in the cocrystal plotted against time. Th is indicates that the sol ubility of hesperetin has been improved 10-12 fold via cocrystall ization in 1:1 ethanol/water (V/V%) solvent system. 62

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0 1 2 3 4 5 6 7 8 9 0 50 100 150 200 250 Time (Minutes)Concentration (mg/mL) Hesperetin KP15 Figure 5.7 Dissolution profiles of hesperetin and he speretin-isonicotinamide (1:1) cocrystal in50 % EtOH/Water (v/v) solvent system 5.4 Dissolution study of Hesperetin-NA (Cocrystal 4) The dissolution experiment of cocr ystal 4 (KP22) reveals that the solubility of hesperetin in cocrystal increases tremendously from zero to 3.5 mg/mL of 1:1 ethanol/water (V/V%) solvent system within five minut es of the experiment. In the due course of time the solubility of hesperetin (cocrystal) decreases reaches ar ound 2 mg/mL after 3 hrs was carried out in 1:1 ethanol/water (V/V%) solvent system. The Fi gure 5.7 represents the comparison of the dissolution of hesperetin with its cocrystals with isonicotinamide and nicotinic acid. The NA cocrystal shows an approximate 5-fold increase in the solubility of hesperetin. 63

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0 1 2 3 4 5 6 7 8 9 0 50 100150200250 Time (Minutes)Concentration (mg/mL) KP22 Hesperetin KP15 Figure 5.8 Comparison of dissolution profiles of hesperetin with its cocrystals 5.5 Discussions The application of crystal engineering to the field of nutraceuticals has been demonstrated by synthesizing the cocrystals with pharmaceutically acceptable molecules. Incorporating pharmaceutically acceptable molecules with the desired target nutraceuticals provides an opportunity to tailor the solubi lities and thereby generating more chances to improve the bioavailability. Crystal engineeri ng plays a vital role in setting up the strategies for selecting appropriate nutraceutical:guest combinations that could form complementary hydrogen bonds and producing the novel cocrystals. In order to check wh ether the cocrystallization ha s altered the solubility of hesperetin and quercetin it was important do the dissolution experiments for the pure flavonoids as well as the cocrystals. But owi ng to the poor solubility of hesperetin and quercetin in water has forced to adopt a different solvent for th e dissolution of the cocrystals. 64

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65 As most of the flavonoids show good solubility in alcohols, 1:1 ethanol -water solvent system was adopted. It is evident from the dissoluti on experiments that al l the cocrystals of hesperetin and quercetin showed an improved solubility. Thus conf irming the fact that cocrystallization offers a potential means to tailor the physical prope rties of the target molecules in the solid state. As the solubi lity and bioavailability are interdependent, cocrystallization could be a better alternat ive to tailor the solub ility, in return the bioavailability.

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66 6. CONCLUSIONS AND FUTURE DIRECTIONS The study of crystal engineering remained as an understudied subject until Schmidts work on topochemical reactions 15 popularized it. Later it expa nded to larger areas like Supramolecular Chemistry. 16 In the field of pharmaceutics cr ystal engineering has excelled greatly as it offers a wide range of application to the drug discover y. It has lead to the discovery of new forms of drugs as solvates, hydrates, polymorphs 58 and cocrystals 54 which allows handling problems pertaining to the phys iochemical properties of drugs such as hygroscopicity, solubility and bioavailability. In case most of the APIs and nutraceuticals th ese properties can be tailored by cocrystallizing pharmaceutically acceptable mol ecules. In this regard, crystal engineering proves to be an efficient tool. It directs the one to select appropriate cocrystal formers to generate cocrystals by explor ing the non-covalent interact ions such as hydrogen bonding. The flavonoids have many benefits to the human health; the problem lies with their solubility and bioavailabi lity that limits their usage. 2 Therefore, flavonoids have been selected for the research work. The results here in demonstrate the wide range application of crystal engineering to generate new forms of quercetin and he speretin. The utilization of crystal engineering to synthesi ze cocrystals has been demonstr ated using the cocrystals of quercetin and hesperetin with pharmaceutically acceptable molecules like caffeine, nicotinic acid and isonicotinamide. The work also de veloped new solvent systems for dissolutions where, the dissolutions studies in water are not possible. The applica tion of such solvent

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67 systems may not be allowed pharmaceutically but could act as templates for other cocrystals as the results indicate increase in the sol ubility of the quercetin and hesperetin via cocrystallization. These results could be the subject of future contributions.

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68 REFERNCES: 1. Brian Lockwood, Pharmaceutical Press: London, UK, 2007. 2. Middleton, E.; Kandaswami, C. Biochem Pharmacol. 1992; 43(6):1167-1179 1992 3. Groot, H. Rauen, U. Fundament als of Clinical Pharmacology 1998; 12:249. 4. Jitka, Psotova1.; Iarka, ChlopCikova1.; Petra, Miketova1.; Jan, Hrbac.; Vilim, imanek. Phytother. Res. 2004; 18, 516. 5. Divi, R. L.; Chang, H. C.; Doerge, Dr. Biochem Pharmacol 1997; 54(10):10871096 1997. 6. Gil, M. I.; Ferreres, F.; Tomas, Barberan. F. A. J Agric Food Chem 1999 ; 47(6): 2213-7 7. Groff, J. L.; Gropper, S. S.; Hunt, S. M. Advanced Nutrition and Human Metabolism. West Pu blishing Company, New York, 1995. 8. Ioku, K.; Aoyama, Y.; Tokuno, A. et al. J Nutr Sci Vitaminol (Tokyo) 2001; 41(7): 78-83 2001. 9. Ververidis, Filippos.; Trantas, Emmanou il.; Douglas, Carl.; Vollmer, Guenter.; Kretzschmar, Georg.; Panopoulos, Nickolas. Jou. Biotech 2007. 2:10, 1235. 10. Adebamowo, C. A.; Cho, E.; Sampson, L. et al, Int J Cancer 2005; 114:628-633. 11. Formica, J. V.; Regelson, W.; Food Chem Toxicol, 1995 ; 33:1061. 12. Huxley1, C. C.; Neil, Haw.. Eur. J. Cli Nutr. 2003; 57, 904 13. Knekt, P.; Jarvinen, R.; Reunanen, A.; Maatela, J. BMJ. 1996; 312:478. 14. Corbett, J. R, Academic Press, 1974. 15. Matsumura, F. Plenum Press New York, 1985. 16. O'Brien, R. D Academic Press, 1967. 17. Shimkim, M. B.; Anderson, N. N. Acute toxi cities of rotenone and mixed pyrethrins in mammals. Proc. Soc. Exp. Biol. Med. 1936; 34: 135-138.

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76 APPENDICIES

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77 Appendix A: 1. Crystallographic data and Experimental data for the new crystal forms of Quercetin Form I Form II Form III Form IV Cocrystal 1 Cocrystal 2 Formula C25H20N2O7 C25H20N2O7 C27H26O10 C165H10O875 C24H24N4O10 C21H16N2O8 m.p. (C) 316 (decomp) 316 (decomp) 318 (decomp) 316 (deco mp) 246 256-260 Mol. Wt. 460.43 460.43 510.48 351.25 528.47 424.36 Crystal System Monoclinic Triclinic Triclinic Monoclinic Monoclinic Triclinic Space Group P2(1)/c P-1 P-1 C2/c P2(1)c P-1 A () 16.727(7) 10.188(2) 9.0830(3) 38.192 10.315(2) 4.9780(10) B () 17.564(8) 10.224(2) 10.858(3) 3.6344 14.853(4) 12.636(3) C () 6.919(3) 12.082(3) 13.063(3) 21.227 15.229(4) 15.571(3) () 90 104.167(4) 102.344(5) 90 90 110.53(3) () 91.456(12) 109.081(3) 93.516(4) 104.57(3) 100.667 97.63(3) () 90 107.775 111.149(4) 90 90 99.39(3) Volume () 2032.2(14) 1046.3(4) 1255.5(5) 2851.7(10) 2292.9(4) 885.7(3) calc density (mg/cm3) 1.505 1.461 1.350 1.636 1.531 1.591 Solvent Pyridine Pyridine THF Acetone Methanol Methanol

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1.1 Experimental data for Form I and Form II DSC thermogram, FT-IR spectrum and X-ray powder diffraction patterns of bulk sample (black ) calculated from the single crystal st ructure of Form I (green) and From II (red) and TGA. 78 6 9 603 24 821.54 864.36 941.66 1014.34 1261.14 131741 1381.26 1521.21 1561.0 5 1609.03 1664.71 3277.13 3411.55qdh 70 75 80 85 90 95 1 00 %T 1000 2000 3000 4000 Wavenumbers (cm-1) 510152025303540 0 2000 4000 6000 8000 10000 12000 14000 Expt Form II Form IRelative intensity2 degree

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1.2 Experimental data for Form III DSC thermogram, FT-IR spectrum and X-ray powder diffraction patterns of bulk sample (red) and calculated from the single crystal structur e (black) and TGA. 79

PAGE 93

6 0 796.47 876.89 929.88 1016.48 108891 1159.21 1439.67 1594.91 1620.27 1654.29 3261.41qdh-thf 50 60 70 80 90 100 %T 1000 2000 3000 4000 Wavenumbers (cm-1) 510152025303540 2000 4000 6000 8000 10000 12000 Calculated (100K) Experimental (293K)Relative intensity2 degree 80

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1.3 Experimental data for Form IV DSC thermogram, FT-IR spectrum and X-ray powder diffraction patterns of bulk sample (red) and calculated from the single crystal structur e (black) and TGA. 704.44 754.32 788.05 821.60 879.19 931.68 998.64 1009.72 1041.10 1092.75 1140.01 1163.99 1208.14 1244.46 1287.20 1316.12 1353.90 1451.46 1510.73 1554.60 1608.45 1657.25 3226.27 76 78 80 82 84 86 88 90 92 94 96 98 1000 2000 3000 Wavenumbers (cm-1) 81 510152025303540 0 2000 4000 6000 8000 10000 kp08 CalculatedRelative intensity2 degree

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1.4 Experimental data for Cocrystal 1 DSC thermogram, FT-IR spectrum and X-ray powder diffraction patterns of bulk sample (red) and calculated from the single crystal structur e (black) and TGA. 82

PAGE 96

1157.83 1278.00 1358.69 1434.37 1460.43 1509.70 1554.21 1633.26 1687.16 3307.02 72 74 76 78 80 82 84 86 88 90 92 94 96 98 100 1000 2000 3000 4000 Wavenumbers (cm-1) 510152025303540 0 1000 2000 3000 4000 5000 6000 Calculated kpo5Relative intensity2 degree 83

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1.5 Experimental data for Cocrystal 2 DSC thermogram, FT-IR spectrum and X-ray powder diffraction patterns of bulk sample (red) and calculated from the si ngle crystal structure (black). 84 510152025303540 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 Experimental CalculatedRelative intensity2 degree

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85 Appendix B 2. Crystallographic data and Experimental data of the new crystal forms of Hesperetin Cocrystal 3 Cocrystal 4 (Form I) Cocrystal 4 (Form II) Formula C 22 H 20 N 2 O 7 C 21 H 16 N 2 O 8 C 21 H 16 N 2 O 8 m.p. (C) 172-176 198-204 256-260 Mol. Wt. 424.40 424.36 424.36 Crystal System Monoclinic Orthorhombic Triclinic Space Group P21/c P2 1 2 1 2 1 P-1 a () 25.653(10) 6.411(3) 6.7055(15) b () 5.129(2) 12.340(6) 11.494(3) c () 14.880(6) 23.418(3) 12.402(3) () 90 o 90 o 89.635(4) o () 103.384(7) o 90) o 85.483(4) o () 90 o 90 o 86.623(4) o Volume () 1904.7(13) 1852.6(15) 951.3(4) calc density (mg/cm3) 1.480 1.525 1.485 Solvent Methanol Methanol Methanol

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2.1 Experimental data of Cocrystal 3 DSC thermogram, FT-IR spectrum and X-ray powder diffraction patterns of bulk sample (red) and calculated from the si ngle crystal structure (black). 638.5 730.61 759.06 801.96 816.09 858.85 959.58 1012.67 1058.37 1082.52 1127.5 1167.75 1218.97 1262.1 13060 1335.52 1399.01 137.65 172.12 1595.9 1629. 1696.99 2962.21 3108.22 3156.07 322.5 76 78 80 82 8 86 88 90 92 9 96 98 100 1000 2000 3000 000 Wavenumbers (cm-1) 510152025303540 0 2000 4000 6000 8000 10000 kp15 CalculatedRelative intensity2 degree 86

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.2 Experimental data of Cocrystal 4 (Form I and Form II) DSC thermogram, FT-IR spectrum and X-ray powder diffraction patterns of bulk sample 2 (red), calculated from the single crystal of Form I (black) and calculated from the single crystal of Form II (Green). 87 719.80 741.61 756.98 799.60 837.11 872.02 946.70 971.74 1012.98 1046.94 1072.72 1085.65 1152.85 1178.04 1192.33 1234.85 1274.53 1320.93 1368.45 1397.16 1438.54 1452.92 1508.66 1580.62 1628.35 3394.36 72 74 76 78 80 82 84 86 88 90 92 94 96 98 100 3000 2000 1000 Wavenumbers (cm-1) 510152025303540 0 2000 4000 6000 8000 10000 12000 Form I (P1) Form II (P212121) ExptRelative intensity2 degree


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Crystal engineering of flavonoids
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ABSTRACT: Crystal engineering is attracting attention in the pharmaceutical industry because the design of new crystal form of drugs can improve their stability, bioavailability and other relevant physical characteristic properties. Therefore, crystal engineering of nutraceuticals such as flavonoids by exploring their hydrogen bonding interactions can generate novel compounds such as pharmaceutical cocrystals. Flavonoids are polyphenolic secondary plant metabolites that are present in varying levels in fruits, vegetables and beverages. The "French paradox", low cardiovascular mortality rate in spite of high intake of saturated fat among the Mediterranean populations made flavonoids an appropriate target for therapeutic researchers. The work herein deals with the crystal engineering of two flavonoids, quercetin and hesperetin, which are already known to exhibit antioxidant properties and reduce cardiovascular effects in humans. However, they have limited bioavailability and poor water solubility. Several new forms of quercetin and hesperetin in the form of solvates and cocrystals were synthesized. These new crystal forms were characterized by various techniques: FT-IR, DSC (Differential Scanning Calorimetry), single X-ray diffraction, powder X-ray diffraction, TGA (Thermal Gravimetric Analysis) and melting point. The new compounds were also studied via dissolution studies performed in 1:1 ethanol/water (V/V%). Thus, crystal engineering proves to be effective way to enhance the solubility and bioavailability of the target flavonoid molecules.
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