NITRATE ESTERS OF STEROLS/STANOLS AND USE THEREOF IN TREATING OR PREVENTING CARDIOVASCULAR DISEASE, ITS UNDERLYING CONDITIONS AND OTHER DISORDERS

FIELD OF THE INVENTION

This present invention relates to the field of sterol and stanol derivatives, pharmaceutical compositions comprising these derivatives and various uses of these derivatives in a wide variety of therapeutic areas.

BACKGROUND OF THE INVENTION

While recent advances in science and technology are helping to improve quality and add years to human life, the prevention of atherosclerosis, an underlying cause of cardiovascular disease ("CVD") has not been sufficiently addressed. Atherosclerosis is a degenerative process resulting from interplays of inherited (genetic) factors and environmental factors such as diet and lifestyle. Research to date suggests that cholesterol may play a role in atherosclerosis by forming atherosclerotic plaques in blood vessels, ultimately restricting blood supply to the heart muscle or alternatively to the brain or limbs, depending on the location and stability of the plaque in the arterial tree (1 ,2). Overviews have indicated that a 1% reduction in a person's total serum cholesterol yields a 2% reduction in risk of a coronary artery event (3). Statistically, a 10% decrease in average serum cholesterol (e.g. from 6.0 mmol/L to 5.3 mmol/L) may result in the prevention of 100,000 deaths in the United States annually (4).

Sterols are naturally occurring compounds that perform many critical cellular functions. Phytosterols such as campesterol, stigmasterol and beta-sitosterol in plants, ergosterol in fungi and cholesterol in animals are each primary components of cellular and sub-cellular membranes in their respective cell types. The dietary source of phytosterols in humans comes from plant materials i.e. vegetables and plant oils. The estimated daily phytosterol content in the conventional western-type diet is approximately 60-80 milligrams in contrast

to a vegetarian diet which would provide about 500 milligrams per day.

Phytosterols have received a great deal of attention (particularly over the past ten years) due to their ability to decrease serum cholesterol levels when fed to a number of mammalian species, including humans. While the precise mechanism of action remains largely unknown, the relationship between cholesterol and phytosterols is apparently due in part to the similarities between the respective chemical structures (the differences occurring in the side chains of the molecules). It is assumed that phytosterols displace cholesterol from the micellar phase and thereby reduce its absorption or possibly compete with receptor and/or carrier sites in the cholesterol absorption process.

This history of phytosterols and use in cholesterol lowering goes back much further than this past decade of resurgence. Over forty years ago, EIi Lilly marketed a sterol preparation from tall oil and later from soybean oil called Cytellin™ which was found to lower serum cholesterol by about 9% according to one report (5). Various subsequent researchers have explored the effects of sitosterol preparations on plasma lipid and lipoprotein concentrations (6) and the effects of sitosterol and campesterol from soybean and tall oil sources on serum cholesterols (7). A composition of phytosterols which has been found to be highly effective in lowering serum cholesterol is disclosed in US Patent Serial No. 5,770,749 to Kutney et al. and comprises no more than 70% by weight beta- sitosterol, at least 10% by weight campesterol and stigmastanol (beta-sitostanol). It is noted in this patent that there is some form of synergy between the constituent phytosterols, affording even better cholesterol-lowering results than had been previously achieved.

Despite the obvious and now well recorded advantages of phytosterols, not only in the treatment of CVD and its underlying conditions such as hypercholesterolemia, hyperlipidemia, atherosclerosis, hypertension, thrombosis but in the treatment of other diseases such as Type Il diabetes, dementia cancer and aging, the administration of phytosterols and the incorporation thereof into foods, pharmaceuticals and other delivery

vehicles was complicated by the fact that they are highly hydrophobic (i.e. they have poor water solubility). This problem was finally addressed by the creation of ascorbic acid derivatives of phytosterols and phytostanols, as described in WO 01/00653, such derivatives being highly water soluble and capable of being readily incorporated into various pharmaceutical delivery matrices. Compounds within this ascorbyl derivative family have been developed by Forbes Medi-Tech Inc. under the reference name VP4.

Phytosterols and phytostanols remain compounds of great interest in the cardiovascular therapeutics field. It is an object of the present invention to enhance the therapeutic efficacy of phytosterols and phytostanols in manners heretofore unconsidered.

SUMMARY OF THE INVENTION

The present invention provides in one aspect, sterols and stanols linked to an NO releasing moiety. There are a number of means by which this linkage can be accomplished and the present invention is not intended to be limited to any one compound so formed. The crux of the present invention is the linkage of sterols and stanols to NO releasing moieties.

In one aspect, the present invention provides novel sterol and stanol nitroderivatives, including pharmaceutically acceptable salts or stereoisomers thereof, represented by the general formulae:

1) M-T-Y _A -NO ₂ ; and

2) M-T-Y _A -R ₁

wherein:

M is a sterol or stanol, or residue thereof; T= -PO ₃ R ₁ - (Ri being selected from the groups

as provided below, or H or an alkyl group with from 1-10 carbons or an aryl group with from 1-10 carbons) -SO ₂ NH-, -SO ₂ NR-, -(CO)-,-O-, -S-, -NH-, -N(SO ₂ R)-, (R being alkyl with 1-10 carbon atoms, preferably methyl);

YA = ascorbyl moiety (optionally substituted at one or more of the -OH groups with, interchangeably, R ₁ , other -NO releasing agents, C1-10 alkyl or C 1 -10 aryl groups or H) -(B)bo-( ^c )co- wherein: bO and cO are the integers 1 or 0, with the proviso that bO and cO cannot be simultaneously 0,

B = -TB-X ₂ -TBI-, in which:

TB = CO or X, wherein X = O, S, NH, NR, and R is as defined above, T _B is CO when T is -SO ₂ NH-, -SO ₂ NR-, -O-, -S-, -NH-, -N(SO ₂ R)-, T _B is X when T is -CO-;

TBI = CO or X, in which X is as defined above;

X ₂ is a divalent radical and is selected from the following compounds: a)

wherein: n1 and n2 are integers O or 1 ; R ² and R ³ are independently selected from H or

CH ₃ ;

b)

wherein: n2 and R ² are as above defined;

Y ¹ is -CH ₂ -CH ₂ - or -CH=CH-(CH ₂ ) _n2 - wherein n2' is an integer O or

1 ;

wherein: n4 is an integer from 1 to 20 and n5 is an integer from 0 to 20, R ⁴ and R ⁴ R ⁵ and R ^5' are independently selected from H, CH ₃ , OH ₁ NH ₂ , NHCOCH ₃ , COOH; when the bond between the C ^A and C ^B carbons is a double bond R ⁴ and R ⁵ or R ⁴ and R ⁵ are absent;

C is the bivalent radical -T ₀ -Y-, wherein:

T ₀ = CO, X wherein X is as defined above, or -(CH2)n ₆ OC(O)- wherein n6 is an integer from 1 to 20, preferably n6 is 1 ;

Y is a bivalent radical having the following meanings:

d) -R ¹ O-, in which R ¹ is:

- straight or branched CrC ₂ o-alkylene eventually containing one or more heteroatoms selected from oxygen, nitrogen, sulphur, or one or more groups - O(CO)-, -NH(CO)-, -S(CO)-, eventually substituted with one or more of the following groups -OH, -SH, - NH ₂ , -NHCOR ⁶ , in which R ⁶ is straight or branched CrCio-alkyl, preferably CH ₃ ;

- cycloalkylene containing from 5 to 7 carbon atoms into cycloalkylene ring, wherein one or more carbon atoms can be replaced by heteroatoms selected from nitrogen, oxygen or sulphur, and the ring can be substituted with side chains R ⁶ , R ⁶ being as defined above;

e)

f)

wherein n7 is an integer from 0 to 20, and n7" is an integer from 1 to 20;

9)

wherein m is an integer from 1 to 6, preferably from 1 to 4, Rf is a hydrogen atom or CH ₃ ;

h)

wherein:

nlX is an integer from 0 to 10, preferably from 1 to 5; nllX is an integer from 1 to 10, preferably from 1 to 5;

RTIX, RTIX', RTIIX, RTIIX', are the same or different, and are H or straight or branched CrC ₄ -alkyl, preferably R _T ιχ, RTIX ¹ , RTIIX, RTIIX ¹ are H;

Y ³ is an heterocyclic saturated, unsaturated or aromatic 5 or 6 members ring, containing one or more heteroatoms selected from nitrogen, oxygen, sulphur, and selected from

with the proviso that:

when b0 = 0, cO = 1 and T = -SO ₂ NH-, -SO ₂ NR-, -O-, -S-, -NH-, -N(SO ₂ R)- wherein R is

as defined above, then T ₀ = (CO) or -(CH ₂ ) _n6 O(CO)-; when bO = 0, cO = 1 and T = CO then T ₀ = X wherein X is as defined above; when bO = 1 and T = -SO ₂ NH-, -SO ₂ NR-, -0-,-S-, -NH-, -N(SO ₂ R)- wherein R is as defined above, then T _B = CO; when bO = 1 and T = CO then TB = X wherein X is as defined above; when bO = 1 , cO = 1 and TBI = CO then T ₀ = X wherein X is as above defined; when bO = 1 , cO = 1 and TBI = X, wherein X is as above defined, then T ₀ = (CO); when bO = 1 , cO = O the TBI has only the meaning of -O-.

Preferred compounds of formula (I) are those wherein bO = O, cO = 1 , T and T _c are as defined in claim 1 , Y is a straight CrC ₆ alkylene or

wherein n7 is O or 1 , and n7' is 1 or 2, or

wherein m is 2, Rf is hydrogen.

Particularly preferred compounds of formula 1 ) are those wherein bO = O, cO = 1 , T = -N(SO ₂ R)-, T ₀ = CO or -(CH ₂ ) _n6 O(CO)- wherein n ₆ = 1 and R = CH ₃ and those wherein bO = O, cO = 1 , T = -SO ₂ NH- and T _c = CO or-(CH ₂ )neO(CO)- wherein n ₆ = 1.

In another aspect, the present invention provides novel phytosterol and phytostanol nitroderivatives, including pharmaceutically acceptable salts or stereoisomers thereof, represented by the general formulae:

wherein in the foregoing formulae, R is selected from CH ₃ and CH2CH3

and Ri is selected from the group consisting of:

wherein n is an integer from 1 to 20.

The present invention further provides novel phytosterol and phytostanol nitroderivatives, including pharmaceutically acceptable salts or stereoisomers thereof, represented by the general formulae

wherein in the foregoing formulae, R is selected from CH ₃ and CH ₂ CH ₃ and Ri and R2 is independently selected from the group consisting of:

and wherein n is an integer from 1 to 20.

The present invention further provides novel phytosterol and phytostanol nitroderivatives, including pharmaceutically acceptable salts or stereoisomers thereof, represented by the general formulae:

wherein R is either CH ₃ or CH2CH3; and a) one of R-i, R ₂ , R ₃ or R ₄ is selected from the group consisting of:

wherein n is an integer from 1 to 20 and the remaining three groups of Ri, R2, R ₃ or R ₄ are OH; or b) one of R ₁ , R2, R ₃ or R ₄ is selected from the group consisting of:

wherein n is an integer from 1 to 20 and the remaining three groups of R-i, R ₂ , R3 or R ₄ are OH.

wherein R is either CH ₃ or CH ₂ CH ₃ ; and a) any two of R-i, R2, R3 or R ₄ is selected from the group consisting of:

wherein n is an integer from 1 to 20 and the remaining three groups of R-i, R ₂ , R ₃ or R ₄ are OH; or

b) any two of R-i, R2, R3 or R ₄ is selected from the group consisting of:

wherein n is an integer from 1 to 20 and the remaining three groups of Ri, R. ₂ , R ₃ or R ₄ are OH.

The present invention further provides novel phytosterol and phytostanol nitroderivatives, including pharmaceutically acceptable salts or stereoisomers thereof, represented by the general formulae:

wherein R is either CH ₃ or CH ₂ CH ₃ and a) Ri and R2 are independently selected from the following:

Or b) ) Ri is H and R2 is selected from one of the three compounds noted below:

and wherein n is an integer from 1 to 20.

The present invention further comprises pharmaceutical compositions for treating or preventing or ameliorating CVD and its underlying conditions including atherosclerosis, hypercholesterolemia, hyperlipidemia, hypertension, thrombosis, and related diseases such as Type Il diabetes, as well as the diseases forming metabolic syndrome , as well as diseases that are increasingly recognized as having oxidative injury and/or NO-

insufficiency components as part of their etiology such as dementia, Alzheimer's disease, aging, and cancer, as well as use in wound healing, for general microbial and viral infections, for bronchopulmonary diseases such as asthma and obstructive pulmonary syndromes, ulcerative and non-ulcerative Gl inflammatory diseases; for dermatitis, eczema and other inflammatory cutaneous syndromes which comprises one or more derivatives as described herein, with the above noted formulae, and a pharmaceutically acceptable carrier therefor.

The present invention further provides a method for treating or preventing or ameliorating all of the above mentioned diseases, conditions and syndromes by administering to an animal a therapeutically effective amount of one or more derivatives as described herein along with a pharmaceutically acceptable carrier therefore.

The phytosterol and phytostanol nitroderivatives of the present invention represent an unexpected advantage over the known phytosterols, in free or derivatized form. Contrary to any expectation, the derivatives of the present invention are characterized by the fact that they show an improved therapeutic profile and high activity across a variety of therapeutic areas.

The derivatives of the present invention can be prepared and used as such or they can be easily incorporated into a wide selection of pharmaceutical preparations.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is illustrated by way the following non-limiting drawings in which:

Figure 1 is a flow chart describing the synthesis of one nitroderivative in accordance with the present invention.

PREFERRED EMBODIMENTS OF THE INVENTION

The following detailed description is provided to aid those skilled in the art in practising the invention. However this detailed description should not be construed so as to unduly limit the scope of the present invention. Modifications and variations to the embodiments discussed herein may be made by those with ordinary skill in the art without departing from the spirit or scope of the present invention.

According to the present invention, there are provided novel nitroderivatives synthesized by ester linkage of an NO-releasing moiety to a sterol or stanol compound. These derivatives are suitable for use per se in treating or preventing CVD and its underlying conditions, such as atherosclerosis, hypercholesterolemia, hyperlipidemia, hypertension, thrombosis, and related diseases such as Type Il diabetes, as well as in treating and preventing other diseases that include oxidative damage as part of the underlying disease process such as dementia, Alzheimer's disease, aging, and cancer.

It should be noted that, throughout this disclosure, the terms "derivative", "structure" and "analogue" are used interchangeably to describe the novel unitary compounds which are covered by formulae noted herein.

Nitric oxide (NO) is a highly reactive, diffusible, and unstable radical which is a unique messenger molecule involved in the regulation of diverse physiological processes including smooth muscle contractility, platelet reactivity and central and peripheral neurotransmission. In the immune system NO has been recognized as an important effector molecule for macrophages as well as a homeostatic regulator. Depending on the type and phase of the inflammatory reaction and the individual vascular or cellular reponses studied, NO can exert both proinflammatory and antiinflammatory actions. The term "NO" or nitric oxide", as used herein, encompasses all charged and uncharged nitrogen oxide species, particularly including uncharged nitrogen monoxide

radical (NO) and charged nitrogen monoxide species, particularly including nitrosonium cation (NO+) and nitroxyl (HNO/NO-).

Unlike most other neurotransmitters that only transmit information from a presynaptic to a postsynaptic neuron, the small NO molecule can diffuse widely and can thereby act on several nearby neurons, even on those not connected by a synapse. It is conjectured that this process may be involved in memory through the maintenance of long-term potentiation. In addition, NO is an important non-adrenergic, non-cholinergic (NANC) neurotransmitter in various parts of the gastrointestinal tract. It causes relaxation of the gastrointestinal smooth muscle. In the stomach it increases the capacity of the fundus to store food/fluids.

In the classic signal-transduction paradigm for blood-flow modulation by NO, nitric oxide synthase (NOS) in the vascular endothelium (inner cell lining) of blood vessels synthesizes NO that diffuses to the underlying vascular smooth muscle. NO-dependent soluble guanylyl cyclase stimulation within the vascular smooth-muscle cell relaxes the muscle, thereby dilating the artery and increasing regional blood flow through it. Although this mechanism is not the only one by which nitrogen oxides influence vascular tone, it is thought to contribute to the therapeutic action of organic nitrate vasodilators (including nitroglycerin, isosorbide dinitrate, and amyl nitrate) in the treatment of coronary heart disease. In vivo, the organic nitrate vasodilators are ultimately biotransformed to NO and a variety of other nitrogen oxides/NO-derived metabolites by a complex, incompletely understood process involving both enzymatic and nonenzymatic metabolic routes (Janero et al., Proc. NatL Acad-Sci. USA 101:16958; 2004).

The term "NO releasing agent (or compound)", as used herein includes any and all compounds that, in a living system: a) provide, deliver, release and/or transfer any

chemical form of bioactive nitrogen oxide, particularly, including charged and uncharged nitrogen monoxide species, to the intended site of action, in a form active for their intended purpose and/or b) stimulate or otherwise alter or modulate the endogenous production of NO and/or endothelium-derived relaxing factor (EDRF) refers to all aliphatic and aromatic NO releasing compounds described herein.

As used herein, the term "sterol" includes all sterols without limitation, for example: (from any source and in any form: α, β and Y) sitosterol, campesterol, stigmasterol, brassicasterol (including dihydrobrassicasterol), desmosterol, chalinosterol, poriferasterol, clionasterol, ergosterol, coprosterol, codisterol, isofucosterol, fucosterol, clerosterol, nervisterol, lathosterol, stellasterol, spinasterol, chondrillasterol, peposterol, avenasterol, isoavenasterol, fecosterol, pollinastasterol, cholesterol and all natural or synthesized forms and derivatives thereof, including isomers.

The term "stanol" refers to, for example: (from any source and in any form: α, β and v) saturated or hydrogenated sterols including all natural or synthesized forms and derivatives thereof, and isomers, including sitostanol, campestanol, stigmastanol, brassicastanol (including dihydrobrassicastanol), desmostanol, chalinostanol, poriferastanol, clionastanol, ergostanol, coprostanol, codistanol, isofucostanol, fucostanol, clerostanol, nervistanol, lathostanol, stellastanol, spinastanol, chondrillastanol, pepostanol, avenastanol, isoavenastanol, fecostanol, and pollinastastanol.

It is to be understood that modifications to the sterols and stands i.e. to include side chains also falls within the purview of this invention. It is also to be understood that, when in doubt throughout the specification, and unless otherwise specified, the term "sterol" encompasses both sterol and stanol. The terms "phytosterol" and "phytostanol" may also be used and refer to all p/a/rt-derived sterols or stanols respectively.

The sterols and stanols for use in forming derivatives in accordance with this invention may be procured from a variety of natural sources or they may be artificially synthesized.

For example, they may be obtained from the processing of plant oils (including aquatic plants) such as corn oil and other vegetable oils, wheat germ oil, soy extract, rice extract, rice bran, rapeseed oil, sunflower oil, sesame oil and fish (and other marine-source) oils. They may also be derived from yeasts and fungi, for example ergosterol. Accordingly, the present invention is not to be limited to any one source of sterols. US Patent Serial No. 4,420,427 teaches the preparation of sterols from vegetable oil sludge using solvents such as methanol. Alternatively, phytosterols and phytostanols may be obtained from tall oil pitch or soap, by-products of forestry practises as described in US Patent Serial No.5, 770, 749, incorporated herein by reference. A further method of extracting sterols and stands from tall oil pitch is described in Canadian Patent Application Serial No. 2,230,373 which was filed on February 20, 1998 (corresponding to PCT/CA99/00150 which was filed on February 19, 1999) and US Patent Application Serial No 10/060,022 which was filed on January 28, 2002 the contents of all of which are incorporated herein by reference.

Accordingly, it is to be understood that the widest possible definition is to be accorded to the terms "sterol" and "stanol" as used herein, including, but not limited to: free sterols and stanols, esterified sterols and stanols with aliphatic or aromatic acids (thereby forming aliphatic or aromatic esters, respectively), phenolic acid esters, cinnamate esters, ferulate esters, phytosterol and phytostanol glycosides and acylated glycosides or acylglycosides. Thus, the terms "sterols" and "stanols" encompasses all analogues, which may further have a double bond at the 5-position in the cyclic unit as in most natural sterols, or one or more double bonds at other positions in the rings (for example, 6, 7, 8(9), 8(14), 14 5/7) or no double bonds in the cyclic unit as in stanols. Further, there may be additional methyl groups as, for example, in άi-sitosterol.

Sterols and/or stanols once isolated from their source are generally formed into a solid powder through precipitation, filtration and drying, spray drying, lyophilization or by other conventional work-up techniques.

In one preferred form, the nitroderivative of the present invention is formed of naturally- derived or synthesized beta-sitosterol, campestanol, sitostanol and campesterol. Some of the preferred forms of nitroderivatives of the present invention include the following: sitosteryl ascorbyl phosphate monoesters; sitostanyl ascorbyl phosphate monesters, campesteryl ascorbyl phosphate monoesters; campestanyl ascorbyl phosphate monesters, sitosteryl ascorbyl phosphate diesters; sitostanyl ascorbyl phosphate diesters, campesteryl ascorbyl phosphate diesters; campestanyl ascorbyl phosphate diesters, sitosteryl phosphate monesters, sitostanyl phosphate monesters, campesteryl phosphate monesters, campestanyl phosphate monoesters, sitosteryl phosphate diesters, sitostanyl phosphate diesters, campesteryl phosphate diesters, campestanyl phosphate diesters, and sterodienyl phosphate esters.

Within the scope of the present invention, an NO releasing agent is linked in a number of possible ways to sterol and/or stanol based compounds. In one embodiment, represented by formulae I to IV, an NO releasing agent is attached to either a phosphate "linker" between an ascorbyl moiety and a sterol/stanol moiety or to the ascorbyl moiety itself. In another embodiment, represented by formulae V-X, representing diesters, two NO releasing agents, which may be the same or different are attached to positions on the ascorbyl moiety which is joined to the sterol or stanol via a phosphate "linker" and/or are joined to the phosphate linker. In yet another embodiment, represented by formulae Xl- XII, various monoester and diesters are provided in which the NO releasing agents may be attached different positions on either the ascorbyl moiety or the phosphate linker. In yet another embodiment, the NO releasing agent is attached to a phosphate "linker" which is itself attached to a sterol/stanol moiety (and wherein there is no ascorbyl moiety).

It is to be clearly understood, as seen from the entirety of the structures provided herein, that more than one NO releasing agent may be attached to either or both of the ascorbyl moiety and the phosphate linker. For example, in structure V, an NO releasing compound is attached to both position 3 on the ascorbyl moiety and to the phosphate linker. In structure Vl, an NO releasing compound is attached to both position 5 on the ascorbyl

moiety and to the phosphate linker, substituting one hydroxyl group. In structure VII, an NO releasing compound is attached to both position 6 on the ascorbyl moiety and to the phosphate linker, substituting one hydroxyl group. In structure VIII, an NO releasing compound is attached to positions 3 and 5 on the ascorbyl moiety. In structure IX, an NO releasing compound is attached to positions 3 and 6 on the ascorbyl moiety. In structure X, an NO releasing compound is attached to positions 5 and 6 on the ascorbyl moiety. In structure Xl, an NO releasing compound is attached to any one of positions 3, 5 and 6 on the ascorbyl moiety or to phosphate linker, substituting one hydroxyl group therein. In structure XII, an NO releasing compound is attached to any two of positions 3, 5 and 6 on the ascorbyl moiety or to phosphate linker, substituting one hydroxyl group therein. In structure XIII ₁ an NO releasing compound is attached to one or both of the positions on the phosphate linker substituting two hydroxyl groups, wherein the sterol to which the phosphate is joined is saturated i.e. a stanol. In structure XIV, an NO releasing compound is attached to one or both of the positions on the phosphate linker substituting two hydroxyl groups, wherein the sterol to which the phosphate is joined is unsaturated. In structure XV, an NO releasing compound is attached to one or both of the positions on the phosphate linker substituting two hydroxyl groups, thereby forming a sterodienyl phosphate ester.

Some of the compounds of the present invention include a phosphate linker, at least one NO molecule and a sterol or stanol moiety, and additionally comprise an ascorbyl moiety. It has been previously found by Forbes Medi-Tech Inc. that there are significant benefits in chemically linking sterols or stanols to ascorbic acid. The union benefits and enhances both parts of this structure. The sterol moiety, formerly poorly soluble, becomes, as part of the derivative, much more readily soluble in aqueous and non-aqueous media. Accordingly, administration of the sterol becomes possible without any further enhancements to modify its delivery.

The methods of preparation of sterol/stanol chemically joined to ascorbic acid via a phosphate linker is now well known and documented and can be found in WO 01/00653,

the entire contents of which are incorporated herein by reference.

There are many processes by which the structures comprising phytosterols and/or phytostanols linked to ascorbic acid can be formed. In general, the selected phytosterol or stanol (or halophosphate, halocarbonate or halo-oxalate derivatives thereof) and ascorbic acid are mixed together under reaction conditions to permit condensation of the "acid" moiety with the "alcohol" (phytosterol). These conditions are the same as those used in other common esterification reactions such as the Fisher esterification process in which the acid component and the alcohol component are allowed to react directly or in the presence of a suitable acid catalyst such as mineral acid, sulfuric acid, phosphoric acid, p-toluenesulfonic acid. The organic solvents generally employed in such esterification reactions are ethers such as diethyl ether, tetrahydrofuran, or benzene, toluene or similar aromatic solvents and the temperatures can vary from room to elevated temperatures depending on the reactivity of the reactants undergoing the reaction.

In a preferred mode, the process to form the ester derivative comprises "protecting" the hydroxyl groups of the ascorbic acid or derivatives thereof as esters (for example, as acetate esters) or ethers (for example, methyl ethers) and then condensing the protected ascorbic acid with the phytosterol/phytostanol halophospahte, halocarbonate or halo- oxalate under suitable reaction conditions. In general, such condensation reactions are conducted in an organic solvent such as diethyl ether, tetrahydrofuran, or benzene, toluene or similar aromatic solvents. Depending on the nature and reactivity of the reactants, the reaction temperatures may vary from low (-15°C) to elevated temperatures. The result of this process is a compound in which an ascorbyl moiety is chemically joined indirectly to phytosterols or phytostanols via a phosphate linker.

Included within the scope of the present invention are various new compounds in which

NO releasing agents are chemically joined to either or both of:

1) a phosphate linker attached to phytosterols and phytostanols; and/or

2) an ascorbyl moiety chemically joined indirectly to phytosterols or phytostanols via a phosphate linker.

In both embodiments, the common elements, at a minimum, are a phytosterol/phytostanol moiety, a phosphate linker and at least one NO releasing agent. In both embodiments, the NO releasing agent replaces one or more hydroxyl groups on the ascorbyl moiety and/or on the particular phosphate linker, as described and shown herein.

The reaction by which this may be achieved is simple and utilizes the fact that carboxylic acids (of which of the NO releasing agents within the scope of the present invention all are) react with alcohols (OH groups are inherent on both the ascorbyl moiety and the phosphate linker) in the presence of an acid catalyst to produce esters. Once such reaction is called "Fischer esterification", and is well known and practised in the art. As shown by way of example in Figure 1 , attachment of the phosphate linker to a sterol/stanol and subsequently attaching an NO releasing agent to the phosphate can be achieved by a series of simple reactions. The process is further described above in regards to the ascorbyl derivatives. It is to be understood that while Figure 1 shows the NO releasing agent as an aliphatic compound, the process is equally applicable to the selected aromatic NO releasing agents described herein, for example, the various nitromethyl benzoic acids.

The compounds of the present invention, which have one or more asymmetric carbon atoms, can exist as optically pure enantiomers, pure diastereomers, enantiomers mixtures, diastereomers mixtures, enantiomers racemic mixtures, racemates or racemates mixtures. Within the scope of the present invention are all possible isomers, stereoisomers and their mixtures of the compounds having structures I through XVI.

Salts

The present invention encompasses not only the parent structures, the nitrosated

sterol/stanol phosphate esters, but also the salts thereof. These salts are even more water soluble than the corresponding parent compounds and therefore their efficacy in vivo is enhanced. It is possible to form such salts in the particular compounds possessing a free "P-OH" (structures II, III, IV, VIII, IX, and X)

In such cases, salt formation of the derivatives of the present invention can be readily performed by treatment of the parent compound with a series of bases (for example, sodium methoxide or other metal alkoxides) to produce the corresponding alkali metal salts. Other metal salts of calcium, magnesium, manganese, copper, zinc, and the like can be generated by reacting the parent with suitable metal alkoxides. Thus hydrogen presents the parent compound and any metal, alkali earth metal, or alkali metal represents the corresponding salt.

Nitration

Nitration methods which may a suitable for use within the scope of the present invention are practised in the art. The classic synthesis of an NO -releasing agent relies upon a nitration reaction with silver nitrate and precipitation of the nitro product. An alternative nitration method such as described in Ranatunge et al. J. Med. Chem. 47:2180; 2004 may be successfully used.

Other Methods of Forming Nitrate Esters

Methods of forming nitrate esters are described in WO2004/000781 , WO2004/105754, WO2005/050224, and WO03/080568, all to Nicox SA, the contents of which are fully incorporated herein by reference.

Combination of Derivatives with HMG-Coenzyme A Reductase Inhibitors and Other Compounds

In a further embodiment, the derivatives of the present invention may be combined, prior to administration or concurrently, with one or more agents or compounds which treat cardiovascular disease. For example, they may be combined with compounds that

inhibit cholesterol synthesis. These compounds include, but are not limited to 3-hydroxy-3-methyl glutaryl coenzyme-A (HMG-CoA) reductase inhibitors, nitrosated 3-hydroxy-3-methyl glutaryl coenzyme-A (HMG-CoA) reductase inhibitors, nitrosylated 3-hydroxy-3-methyl glutaryl coenzyme-A (HMG-CoA) reductase inhibitors and nitrated 3-hydroxy-3-methyl glutaryl coenzyme-A (HMG-CoA) reductase inhibitors . The combination of these cholesterol synthesis-limiting compounds and the phytosterol derivatives of the present invention is synergistic and initiates and perpetuates both "systemic or enteric" and "extrinsic" effects.

Furthermore, the derivatives of the present invention may be combined with ACE inhibitors, angiotensin Il receptor antogonists, beta-andrenergic blockers, calcium channel blockers, and anti-thrombotics (such as aspirin) along with all chemical forms of NO-donor containing ACE inhibitors, angiotensin Il receptor antogonists, beta- andrenergic blockers, calcium channel blockers, and anti-thrombotics, in nitrosylated, nitrosated and nitrated forms.

Although the mechanism of action of the derivatives of the present invention is not certain, it is believed that cholesterol levels are lowered through a process called "biliary diversion", the uncoupling of communication between the biliary enterocytes and the hepatocytes. Enteric cholesterol production may increase but cholesterol is released in the bile and not reabsorbed through the enterocytes. Any cholesterol synthesis limiting compound would work in concert with these derivatives as the mechanism of the mechanism of the former is the decrease of enteric cholesterol synthesis and bile cholesterol secretion. As cholesterol synthesis is decreased, the phytosterol/cholesterol intestinal ratio increases which decreases cholesterol absorption and increases phytosterol transport via the enterocyte shuttle mechanism.

The finding of this synergistic co-effect between the phytosterol derivatives of the present invention and the compounds which limit cholesterol synthesis, such as statins, is critically important as the dosage of these latter compounds may be significantly

reduced when administered in conjunction with the derivatives described herein. It has recently been discovered that there are some critical side-effects to statins such as Lovastatin, so the dosage reduction afforded by the synergy with the derivatives described herein is particularly compelling. Examples of other statins with which the derivative of the present invention may be combined include: atorvastatin (Lipitor™), superstatin, simvastatin (Zocor™), pravastatin (Pravachol™) and rosuvastatin (Crestor™).

In the alternative, the compounds of the present invention may be combined, prior to administration or concurrently, with one or more compounds selected from the group consisting of: ACE inhibitors, angiotensin Il receptor antogonists, beta-andrenergic blockers, calcium channel blockers, anti-thrombotics such as aspririn, nitrosated ACE inhibitors, nitrosated angiotensin Il receptor antogonists, nitrosated beta-andrenergic blockers and nitrosated aspirin.

Thus, the present invention further comprises pharmaceutical kits comprising one or more containers filled with one or more of the compounds and/or compositions of the present invention and one or more "other" compounds used to treat cardiovascular disease, such as statins, as noted above.

Examples of Preferred Compounds

Potential Advantages of Novel Phytosterol Analogues

The novel derivatives of the present invention, containing at least one nitrogen releasing atom, a sterol/stanol moiety and a phosphate linker affords many therapeutic advantages. The compounds of formulae I through IVX are prodrugs for sterols/stanols and NO. Under physiological conditions, NO will be released from the sterol "carrier" molecule revealing broader and more pronounced pharmacological activity that either NO or sterols alone.

Methods of Use

The sterol/stanol nitroderivatives of the present invention may be administered to animals, in particular humans, directly and without further modification or may be treated to enhance further the solubility and/or dispersability. Alternatively, the derivatives may be incorporated into various vehicles as described further below in order to treat and/or prevent CVD ₁ its underlying conditions such as hypercholesterolemia, hyperlipidemia, arteriosclerosis, hypertension, thrombosis, related diseases such as Type Il diabetes, as well as other diseases that include oxidative damage as part of the underlying disease process such as dementia, aging, and cancer. In populations, which are considered "high-risk" for CVD or any of the oxidation related disorders, it is contemplated that the derivatives of the present invention be used in primary, secondary and tertiary treatment programs.

Without limiting the generality of the foregoing, the derivatives of the present invention may be admixed with various carriers, excipients or adjuvants to assist in direct administration or to assist in the incorporation of the composition into pharmaceuticals. In order to appreciate the various possible vehicles of the delivery of the derivatives, the list below is provided. The doses of the derivatives will vary depending upon, among other factors, the mode of delivery, the patient size and condition, the result to be achieved, as well as other factors known to those skilled in the art of medicinal agents.

Pharmaceutical Dosage Forms:

It is contemplated within the scope of the present invention that the derivatives of the present invention may be incorporated into various conventional pharmaceutical preparations and dosage forms such as tablets (plain and coated) for use orally, bucally or lingually, capsules (hard and soft, gelatin, with or without additional coatings) powders, granules (including effervescent granules), pellets, microparticulates, solutions (such as micellar, syrups, elixirs and drops), lozenges, pastilles, ampoules, emulsions, microemulsions, ointments, creams, suppositories, slurries, gels, transdermal patches and modified release dosage forms together with customary excipients and/or diluents and stabilizers.

The compounds of the present invention can be administered to a patient either by themselves, or in pharmaceutical compositions where they are mixed with suitable carriers, adjuvants or excipients.

Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention. With proper choice of carrier and suitable manufacturing practice, the compounds of the present invention, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection.

Pharmaceutical compositions, comprising one or more of the compounds of the present invention, include compositions wherein the active ingredients are contained in an effective amount to achieve their intended purpose. Determination of the effective amounts within such compositions is well within the capability of those skilled in the art, especially in light of the disclosure provided herein.

The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving,

granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

Oral liquid preparations may be in the form of, for example, emulsions, syrups, or elixirs, or may be presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, for example sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcelluiose, carboxymethylcellulose, aluminium stearate gel, hydrogenated edible fats; emulsifying agents, for example lecithin, sorbitan monooleate, or acacia; nonaqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters such as esters of glycerine, propylene glycol, or ethyl alcohol; preservatives, for example methyl or propyl p-hydroxybenzoate or sorbic acid; and if desired conventional flavouring or colouring agents.

It is contemplated within the scope of the present invention that the compositions of the present invention may be incorporated into various conventional pharmaceutical preparations and dosage forms such as tablets (plain and coated) for use orally, bucally or lingually, capsules (hard and soft, gelatin, with or without additional coatings) powders, granules (including effervescent granules), pellets, microparticulates, solutions (such as micellar, syrups, elixirs and drops), lozenges, pastilles, ampoules, emulsions, microemulsions, ointments, creams, suppositories, gels, transdermal patches and modified release dosage forms together with customary excipients and/or diluents and stabilizers.

The compositions of the present invention, adapted into the appropriate dosage form as described above may be administered to animals, including humans, orally, by injection (intravenously, subcutaneously, intra-peritoneally, intra-dermally or intra-muscularly), topically or in other ways.

Preferably, the compositions of the present invention are delivered in the form of phospholipid systems such as liposomes and other hydrated lipid phases, by physical inclusion. This inclusion refers to the entrapment of molecules without forming a covalent bond and is widely used to improve the solubility and subsequent dissolution of active ingredients.

Hydrated lipid systems, including liposomes, can be prepared using a variety of lipid and lipid mixtures, including phospholipids such as phosphatidylcholine (lecithin), phosphodiglyceride and sphingolipids, glycolipids, and the like. The lipids may preferably be used in combination with a charge bearing substances such as charge-bearing phospholipids, fatty acids, and potassium and sodium salts thereof in order to stabilize the resultant lipid systems. A typical process of forming liposomes is as follows:

1) dispersion of lipid or lipids and the composition of the present invention in an organic solvent (such as chloroform, dichloromethane, ether, ethanol or other alcohol, or a combination thereof). A charged species may be added to reduce subsequent aggregation during liposome formation. Antioxidants (such as ascorbyl palmitate, alpha- tocopherol, butylated hydroxytoluene and butylated hydroxyanisole) may also be added to protect any unsaturated lipids, if present;

2) filtration of the mixture to remove minor insoluble components;

3) removal of solvents under conditions (pressure, temperature) to ensure no phase separation of the components occur;

4) hydration of the "dry" lipid mixture by exposure to an aqueous medium containing dissolved solutes, including buffer salts, chelating agents, cryoprotectorants and the like; and

5) reduction of liposome particle size and modification of the state of lamellarity by means

of suitable techniques such as homogenization, extrusion etc..

Any procedure for generating and loading hydrated lipid with active ingredients, known to those skilled in the art, may be employed within the scope of this invention. For example, suitable processes for the preparation of liposomes are described in references 16 and 17, both of which are incorporated herein by reference. Variations on these processes are described in US Patent Serial No. 5,096,629 which is also incorporated herein by reference.

US Patent Serial No. 4,508,703 (also incorporated herein by reference) describes a method of preparing liposomes by dissolving the amphiphillic lipidic constituent and the hydrophobic constituent to form a solution and thereafter atomizing the solution in a flow of gas to produce a pulverent mixture.

In another preferred form, the compositions of the present invention can be administered as cyclodextrin complexes. Cyclodextrins are a class of cyclic oligosaccharide molecules comprising glucopyranose sub-units and having a toroidal cylindrical spatial configuration. Commonly available members of this group comprise molecules containing six (alpha- cyclodextrin), seven (beta-cyclodextrin) and eight (gamma-cylcodextrin) glucopyranose molcules, with the polar (hydrophilic) hydroxyl groups oriented to the outside of the structure and the apolar (lipophilic) skeletal carbons and ethereal oxygens lining the interior cavity of the toroid. This cavity is capable of accomodating (hosting) the lipophilic moiety of an active ingredient (the guest molecule, here the composition of the present invention ) by bonding in a non-covalent manner to form an inclusion complex.

The external hydroxyl substituents of the cyclodextrin molecule may be modified to form derivatives having improved solubility in aqueous media along with other desired enhancements, such as lowered toxicity, etc.. Examples of such derivatives are: alkylated derivatives such as 2,6-dimethyl-beta-cyclodextrin; hydroxyalkylated derivatives such as hydroxypropyl-beta-cyclodextrin; branched derivatives such as diglucosly-beta-

cyclodextrin; sulfoalkyl derivatives such as sulfobutylether-beta-cyclodextrin; and carboxymethylated derivatives such as carboxymethyl-beta-cylcodextrin. Other types of chemical modifications, known to those in the art, are also included within the scope of this invention.

The cyclodextrin complex often confers properties of improved solubility, dispersability, stability (chemical, physical and microbiological), bioavailability and decreased toxicity on the guest molecule.

There are a number of ways known in the art to produce a cyclodextrin complex. Complexes may be produced, for example, by using the following basic methods: stirring the anthocyanin composition into an aqueous or mixed aqueous-organic solution of the cyclodextrin, with or without heating; kneading, slurrying or mixing the cyclodextrin and the present composition in a suitable device with the addition of an appropriate quantity of aqueous, organic or mixed aqueous-organic liquid, with or without heating; or by physical admixture the cylcodextrin and the composition of the present invention using a suitable mixing device. Isolation of the inclusion complex so formed may be achieved by co- precipitation, filtration and drying; extrusion/ spheronisation and drying; subdivision of the moist mass and drying; spray drying; lyophilization or by other suitable techniques depending on the process used to form the cyclodextrin complex. A further optional step of mechanically grinding the isolated solid complex may be employed.

These cyclodextrin complexes further enhance the solubility and dissolution rate and increase the stability of the present compositions.

The precise modes of delivery of the composition of the present invention in each case will depend upon the objectives of the administration protocol i.e. whether it be preventing, reducing, eliminating or ameliorating a dyslipidemic condition or disorder. In the case of existing conditions and disorders, it will depend upon the severity of the disorder, and as discussed above, the age, size and gender of the individual.

Determining appropriate dosages and administration schedules is well within the purview of one skilled in this field.

EXAMPLES

The present invention is described by the following examples which are provided for illustrative purposes and are not intended, in any way, to limit the scope of the present invention:

EXAMPLE 1- Synthesis of 4-(nitromethyl) benzoic acid — one example of a NO releasing reagent

The commercially available 4-(hydroxymethyl) benzoic acid (10 g) was reacted with 3 equivalents of silver nitrate in acetonitrile as solvent. The reaction was allowed to proceed at room temperature for several hours and the resulting 4-(nitromethyl) benzoic acid precipitated from the reaction mixture upon addition of water. The product ( 8 g) obtained as a white powder, after drying, was utilized in the reaction below, without further purification.

EXAMPLE 2 — Preparation of compound having ascorbyl moiety joined to phytosterol or phytostanol via a phosphate linker

a) Protection of ascorbic Acid

Oleum (24%, 8.3g) was added dropwise to acetone (50ml). Ascorbic acid (12g) was introduced to the mixture at 0°C and the reaction mixture was stirred at O ⁰ C for 6 hours. The obtained crystals were filtered off under suction, the filtered cake was pressed to dryness and then washed with acetone (30ml). The product, 5,6-isopropylidene ascorbic acid (14g) was obtained.

b) Attachment to Phytostanols (an identical procedure was used to attach to phytosterols)

A solution of phytostanol mixture (24g) (campestanol: 36.4%; sitostanol: 62.3%) in toluene (500ml) and pyridine (25ml) was added dropwise to a mixture of phosphorous oxychloride (9ml) in toluene (200ml) at 0°C. The mixture was stirred at room temperature for 3 hours. The pyridine hydrochloride was filtered off and the mother liquor was concentrated to recover the toluene. The residue was dissolved in dry THF (100ml) and a solution of the above prepared protected ascorbic acid (14g) in dry THF (400ml) was added dropwise at OºC. The stirring at room temperature was maintained for 1 hr. The solution was concentrated to remove the solvent. Ethanol (400ml) and 3N HCI (200ml) were added, the mixture was heated to 5O ⁰ C for 30 min and concentrated. Ethyl acetate (600ml) was added, the resultant solution was washed with water (3X300ml), dried over sodium sulfate, concentrated and the product (phytostanol-phosphate-ascorbate) was obtained as a white powder 22g.

EXAMPLE 3 Synthesis of Compound Vl

Phytostanol phosphate ascorbate (herein referred to as "VP4") and prepared in accordance with Example 2, in an amount of 0.5 g, was reacted with 4 equivalents of 1 ,3-dicyclohexyl carbodiimide (DCC) in methylene chloride and 4 equivalents of 4- (nitromethyl) benzoic acid. The mixture was allowed to react at 35 C for several hours until completion (TLC monitoring). Addition of water to the mixture was followed by separation of layers and removal of the organic layer. Evaporation of solvent provided the crude product. Extensive column chromatography on silical gel, utilizing a mixture of isopropyl alcohol, chloroform and water did afford some purification of the mixture. Finally, further precipitation utilizing a mixture of acetonitrile and hexane, afforded product Vl (0.4 g) with a purity greater then 95%.

By varying the equivalents of 4-(nitromethyl) benzoic acid from 1-4 equivalents, and careful control of the above reaction by TLC monitoring, it is possible to produce minor amounts of the products I and III.

Examples 4-7 refer to Figure 1 and the various steps that may be used in preparing compounds wherein phytosterols and phytostanois are linked to one or more NO- releasing agents via a phosphate linker.

EXAMPLE 4 — Synthesis of Compound 2 (Figure 1)

Phytosterols (11.8 g), 5% palladium on alumina (0.32 g), and isopropanol (100 g) were charged into a pressure tube. The tube was degassed and filled with hydrogen. This procedure was repeated twice. The reaction mixture was stirred at 70 ⁰ C under the pressure of hydrogen (60 psi) for 24 h. THF (80 ml) was added and solids were dissolved. Reaction mixture was filtered through Celite at 60 ⁰ C to remove catalyst. The filtrate was concentrated to dryness under reduced pressure. The product was dried under oil pump vacuo overnight and used for the next reaction without further purification.

EXAMPLE 5— Synthesis of Compound 3 (Figure 1 )

A solution of compound 1 (6.0 g) and dry pyridine (2 ml_) in 40 ml_ of anhydrous THF was slowly added to a solution of POCI ₃ (1.31 ml_, 14.2 mmol) in anhydrous THF(20 ml_) at 0 ⁰ C (ice-bath) under argon via syringe. Reaction mixture was stirred at 0 ⁰ C for 1 h, and then stirred at room temperature for 3 h. The mixture was directly used for the next reaction.

EXAMPLE 6— Synthesis of Compound 4 (Figure 1)

The above reaction mixture was cooled to 0 ⁰ C and 3-bromopropanol (3.2 mL, 9.75 mmol) was added via syringe under argon. Then cold bath was removed and reaction mixture was stirred at room temperature for 3 h. Ethyl acetate (100 mL) and water (50 mL) were added to reaction mixture. The aqueous layer was extracted twice with EtOAc (2 x 50 mL). The combined organic layers were dried over anhydrous MgSO ₄ and solvent was removed under reduced pressure. The residue was purified by chromatography on silica gel, eluted with a mixture of EtOAc and MeOH (85:15). Compound 4 was obtained as a colorless syrup (7.03 g).

EXAMPLE 7 — Synthesis of Compound 5 (Figure 1)

A mixture of compound 4 (7.0 g, 9.66 mmol) and AgNO ₃ (4.92 g, 28.98 mmol) in CH ₃ CN (60 mL) was stirred at room temperature overnight. The resulting solid was removed by filtration and filtrate was concentrated to dryness under reduced pressure. The residue was purified by chromatography on silica gel, eluted with a mixture of hexane and EtOAc (70:30). Compound 5 was obtained as white sticky solid (5.1 g).

EXAMPLE 8 — Synthesis of Compound having structure XIII

The reaction mixture containing compound 3 (Figure 1 ) was prepared as described in Example 5, was cooled to 0°C and 4(nitormethyl)benzylalcohol (9g) was added to the mixture. The cold bath was removed and the reaction mixture was stirred at room temperature for 3 hours. Ethyl acetate (100ml) and water (50ml) were added to the reaction mixture. The aqueous layer was extracted twice with ethyl acetate (2x50ml) and twice with methylene chloride (2x 50ml). The combined organic layers were dried over anhydrous MgSO ₄ and the solvent removed under reduced pressure. The residue was purified by chromatography on silica gel, eluted with a mixture of ethyl acetate:methanol (85:15). Compound XIII was obtained as a white solid (7g).

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