SUBSTITUTED XANTHINE DERIVATIVES

RELATED APPLICATIONS

5 This application claims the benefit of U.S. Provisional Application No.:

61/239,336, filed on September 2, 2009, the entire teachings of which are incorporated herein.

BACKGROUND OF THE INVENTION

10 Many current medicines suffer from poor absorption, distribution,

metabolism and/or excretion (ADME) properties that prevent their wider use. Poor ADME properties are also a major reason for the failure of drug candidates in clinical trials. While formulation technologies and prodrug strategies can be employed in some cases to improve certain ADME properties, these approaches

15 have failed to overcome the inherent ADME problems that exist for many drugs and drug candidates. One inherent problem is the rapid metabolism that causes a number of drugs, which otherwise would be highly effective in treating a disease, to be cleared too rapidly from the body. A possible solution to rapid drug clearance is frequent or high dosing to attain a sufficiently high plasma level of drug. This,

20 however, introduces a number of potential treatment problems, such as poor patient compliance with the dosing regimen, side effects that become more acute with higher doses, and increased cost of treatment.

In some select cases, a metabolic inhibitor will be co-administered with an important drug that is rapidly cleared. Such is the case with the protease inhibitor

25 class of drugs that are used to treat HIV infection. These drugs are typically co- dosed with ritonavir, an inhibitor of cytochrome P450 enzyme CYP3A4, the enzyme responsible for their metabolism. Ritonavir itself has side effects and it adds to the pill burden for HIV patients who must already take a combination of different drugs. Similarly, dextromethorphan which undergoes rapid CYP2D6 metabolism is being

30 tested in combination with the CYP2D6 inhibitor quinidine for the treatment of pseudobulbar disease.

BOST 1635628.1 In general, combining drugs with cytochrome P450 inhibitors is not a satisfactory strategy for decreasing drug clearance. The inhibition of a CYP enzyme activity can affect the metabolism and clearance of other drugs metabolized by that same enzyme. This can cause those other drugs to accumulate in the body to toxic 5 levels.

A potentially attractive strategy, if it works, for improving a drug's metabolic properties is deuterium modification. In this approach, one attempts to slow the CYP-mediated metabolism of a drug by replacing one or more hydrogen atoms with deuterium atoms. Deuterium is a safe, stable, non-radioactive isotope of hydrogen.

10 Deuterium forms stronger bonds with carbon than hydrogen does. In select cases, the increased bond strength imparted by deuterium can positively impact the ADME properties of a drug, creating the potential for improved drug efficacy, safety, and tolerability. At the same time, because the size and shape of deuterium are essentially identical to hydrogen, replacement of hydrogen by deuterium would not

15 be expected to affect the biochemical potency and selectivity of the drug as

compared to the original chemical entity that contains only hydrogen.

Over the past 35 years, the effects of deuterium substitution on the rate of metabolism have been reported for a very small percentage of approved drugs (see, e.g., Blake, MI et al, J Pharm Sci, 1975, 64:367-91; Foster, AB, Adv Drug Res

20 1985, 14: 1-40 ("Foster"); Kushner, DJ et al, Can J Physiol Pharmacol 1999, 79-88;

Fisher, MB et al, Curr Opin Drug Discov Devel, 2006, 9: 101-09 ("Fisher")). The results have been variable and unpredictable. For some compounds deuteration caused decreased metabolic clearance in vivo. For others, there was no change in metabolism. Still others demonstrated decreased metabolic clearance. The

25 variability in deuterium effects has also led experts to question or dismiss deuterium modification as a viable drug design strategy for inhibiting adverse metabolism. (See Foster at p. 35 and Fisher at p. 101).

The effects of deuterium modification on a drug's metabolic properties are not predictable even when deuterium atoms are incorporated at known sites of 30 metabolism. Only by actually preparing and testing a deuterated drug can one

determine if and how the rate of metabolism will differ from that of its undeuterated counterpart. Many drugs have multiple sites where metabolism is possible. The

BOST 1635628.1 . 9 . site(s) where deuterium substitution is required and the extent of deuteration necessary to see an effect on metabolism, if any, will be different for each drug.

SUMMARY OF THE INVENTION

5 This invention relates to novel compounds that are substituted xanthine derivatives and pharmaceutically acceptable salts thereof. For example, this invention relates to novel substituted xanthine derivatives that are structurally related to pentoxifylline. This invention also provides compositions comprising one or more compounds of this invention and a carrier and the use of the disclosed 10 compounds and compositions in methods of treating diseases and conditions for which pentoxifylline and related compounds are beneficial.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 A and IB depict the serum levels of a compound of this invention, 15 pentoxifylline and certain of their respective metabolites in four individual dogs following oral administration of a combination of pentoxifylline and that compound of this invention.

FIG. 2 depicts the time course of the production of the specific metabolites measured in FIG. 3 following incubation of various compounds of this invention, 20 pentoxifylline, (S)-M1 and (R)-Ml with rat whole blood.

FIG. 3 depicts the relative amount of specific metabolites produced following incubation of various compounds of this invention, pentoxifylline, (5)-Ml and ( ?)-Ml with rat whole blood.

FIG. 4 depicts the time course of the production of the specific metabolites 25 measured in FIG. 5 following incubation of various compounds of this invention, pentoxifylline, (5)-Ml and ( ?)-Ml with human liver microsomes.

FIG. 5 depicts the relative amount of specific metabolites produced following incubation of various compounds of this invention, pentoxifylline, (5)-Ml and ( ?)-Ml with human liver microsomes

30

DETAILED DESCRIPTION OF THE INVENTION

The terms "ameliorate" and "treat" are used interchangeably and include both therapeutic and prophylactic treatment. Both terms mean decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease

BOST 1635628.1 (e.g., a disease or disorder delineated herein), lessen the severity of the disease or improve the symptoms associated with the disease.

"Disease" means any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

5 It will be recognized that some variation of natural isotopic abundance

occurs in a synthesized compound depending upon the origin of chemical materials used in the synthesis. Thus, a preparation of pentoxifylline will inherently contain small amounts of deuterated isotopologues. The concentration of naturally abundant stable hydrogen and carbon isotopes, notwithstanding this variation, is small and

10 immaterial as compared to the degree of stable isotopic substitution of compounds of this invention. See, for instance, Wada E et al, Seikagaku, 1994, 66: 15; Gannes LZ et al, Comp Biochem Physiol Mol Integr Physiol, 1998, 119: 725. In a compound of this invention, when a particular position is designated as having deuterium, it is understood that the abundance of deuterium at that position is

15 substantially greater than the natural abundance of deuterium, which is 0.015%. A position designated as having deuterium typically has a minimum isotopic enrichment factor of at least 3340 (50.1% deuterium incorporation) at each atom designated as deuterium in said compound.

The term "isotopic enrichment factor" as used herein means the ratio

20 between the isotopic abundance and the natural abundance of a specified isotope.

In other embodiments, a compound of this invention has an isotopic enrichment factor for each designated deuterium atom of at least 3500 (52.5% deuterium incorporation at each designated deuterium atom), at least 4000 (60% deuterium incorporation), at least 4500 (67.5% deuterium incorporation), at least

25 5000 (75%o deuterium), at least 5500 (82.5% deuterium incorporation), at least 6000 (90%) deuterium incorporation), at least 6333.3 (95%> deuterium incorporation), at least 6466.7 (97% deuterium incorporation), at least 6600 (99% deuterium incorporation), or at least 6633.3 (99.5% deuterium incorporation).

In the compounds of this invention any atom not specifically designated as a

30 particular isotope is meant to represent any stable isotope of that atom. Unless

otherwise stated, when a position is designated specifically as "H" or "hydrogen", the position is understood to have hydrogen at its natural abundance isotopic composition. Also unless otherwise stated, when a position is designated specifically as "D" or "deuterium", the position is understood to have deuterium at

BOST 1635628.1 . A . an abundance that is at least 3340 times greater than the natural abundance of deuterium, which is 0.015% (i.e., at least 50.1% incorporation of deuterium).

The term "isotopologue" refers to a species that differs from a specific compound of this invention only in the isotopic composition thereof.

5 The term "compound," when referring to a compound of this invention, refers to a collection of molecules having an identical chemical structure, except that there may be isotopic variation among the constituent atoms of the molecules. Thus, it will be clear to those of skill in the art that a compound represented by a particular chemical structure containing indicated deuterium atoms, will also contain lesser

10 amounts of isotopologues having hydrogen atoms at one or more of the designated deuterium positions in that structure. The relative amount of such isotopologues in a compound of this invention will depend upon a number of factors including the isotopic purity of deuterated reagents used to make the compound and the efficiency of incorporation of deuterium in the various synthesis steps used to prepare the

15 compound. However, as set forth above, the relative amount of such isotopologues in toto will be less than 49.9% of the compound.

The invention also provides salts of the compounds of the invention. A salt of a compound of this invention is formed between an acid and a basic group of the compound, such as an amino functional group, or a base and an acidic group of the

20 compound, such as a carboxyl functional group. According to another embodiment, the compound is a pharmaceutically acceptable acid addition salt.

The term "pharmaceutically acceptable," as used herein, refers to a component that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and other mammals without undue toxicity,

25 irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. A "pharmaceutically acceptable salt" means any non-toxic salt that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this invention. A "pharmaceutically acceptable counterion" is an ionic portion of a salt that is not toxic when released from the salt

30 upon administration to a recipient.

Acids commonly employed to form pharmaceutically acceptable salts include inorganic acids such as hydrogen sulfide, hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid and phosphoric acid, as well as organic acids such as para-toluenesulfonic acid, salicylic acid, tartaric acid, bitartaric acid,

BOST 1635628.1 _ _ ascorbic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucuronic acid, formic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid,

benzenesulfonic acid, lactic acid, oxalic acid, para-bromophenylsulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid and acetic acid, as well as 5 related inorganic and organic acids. Such pharmaceutically acceptable salts thus include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate,

monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate,

10 suberate, sebacate, fumarate, maleate, butyne-l,4-dioate, hexyne-l,6-dioate,

benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, terephthalate, sulfonate, xylene sulfonate,

phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate, maleate, tartrate, methanesulfonate, propanesulfonate, naphthalene- 1-

15 sulfonate, naphthalene-2-sulfonate, mandelate and other salts. In one embodiment, pharmaceutically acceptable acid addition salts include those formed with mineral acids such as hydrochloric acid and hydrobromic acid, and especially those formed with organic acids such as maleic acid.

The invention also includes solvates and hydrates of the compound of the

20 invention. As used herein, the term "hydrate" means a compound which further includes a stoichiometric or non-stoichiometric amount of water bound by non- covalent intermolecular forces. As used herein, the term "solvate" means a compound which further includes a stoichiometric or non-stoichiometric amount of solvent such as water, acetone, ethanol, methanol, dichloromethane, 2-propanol, or

25 the like, bound by non-covalent intermolecular forces.

1 2

It is understood that the carbon atom that bears substituents Y and Y in

1 2 3

Formulae A, Al, I and B can be chiral in some instances (when Y , Y and R are different from one another) and in other instances it can be achiral (when at least two

1 2 3 1 ofY ¹ Y" and R ^J are the same). This carbon atom (i.e., the carbon atom bearing Y 30 and Y ) is indicated by an "*" in Formulae A, Al, I and B. As such, chiral

compounds of this invention can exist as either individual enantiomers, or as racemic or scalemic mixtures of enantiomers. Accordingly, a compound of the present invention will include racemic and scalemic enantiomeric mixtures, as well as individual respective stereoisomers that are substantially free from another

BOST 1635628.1 - f - possible stereoisomer. The term "substantially free of other stereoisomers" as used herein means less than 25% of other stereoisomers, preferably less than 10% of other stereoisomers, more preferably less than 5% of other stereoisomers and most preferably less than 2% of other stereoisomers, or less than "X"% of other

5 stereoisomers (wherein X is a number between 0 and 100, inclusive) are present.

Methods of obtaining or synthesizing an individual enantiomer for a given compound are well known in the art and may be applied as practicable to final compounds or to starting material or intermediates.

Unless otherwise indicated, when a disclosed compound is named or 10 depicted by a structure without specifying the stereochemistry and has one or more chiral centers, it is understood to represent all possible stereoisomers of the compound.

The term "stable compounds," as used herein, refers to compounds which possess stability sufficient to allow for their manufacture and which maintain the 15 integrity of the compound for a sufficient period of time to be useful for the

purposes detailed herein (e.g., formulation into therapeutic products, intermediates for use in production of therapeutic compounds, isolatable or storable intermediate compounds, treating a disease or condition responsive to therapeutic agents).

"D" refers to deuterium. "Stereoisomer" refers to both enantiomers and 20 diastereomers. "Tert", " ¹ ", and "t-" each refer to tertiary. "US" refers to the United States of America.

As used herein the term "alkylene" means a straight or branched chain divalent hydrocarbon radical, preferably having from one to six carbon atoms (Ci_ ₆ alkylene). In some embodiments, the alkylene group has from one to four 25 carbon atoms (Ci_ ₄ alkylene). Examples of "alkylene" as used herein include, but are not limited to, methylene (-CH ₂ -), ethylene (-CH ₂ CH ₂ -), propylene (-CH ₂ CH ₂ CH ₂ -), and branched versions thereof such as (-CH(CH ₃ )-), -CH ₂ CH(CH ₃ )- and the like.

"Halo" means chloro, bromo, fluoro, or iodo.

"Alkyl" means an aliphatic hydrocarbon group which may be straight or 30 branched having 1 to 15 carbon atoms in the chain. Preferred alkyl groups have 1 to 12 carbon atoms in the chain, and more preferably 1 to 6 carbon atoms. Branched means that one or more lower alkyl groups such as methyl, ethyl or propyl are attached to a linear alkyl chain. "Lower alkyl" means about 1 to about 4 carbon atoms in the chain which may be straight or branched. Exemplary alkyl groups

BOST 1635628.1 _ 7 _ include methyl, fluoromethyl, difluoromethyl, trifluoromethyl, cyclopropylmethyl, cyclopentylmethyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, n-pentyl, 3-pentyl, heptyl, octyl, nonyl, decyl and dodecyl; preferred are methyl, difluoromethyl and i- propyl. Alkyl groups may be optionally substituted with one or more groups 5 selected from halo, cyano, hydroxyl, carboxy, alkoxy, alkoxycarbonyl, oxo, amino, alkylamino, dialkylamino, cycloheteroalkyl, alkylcycloheteroalkyl, aryl, alkylaryl, heteroaryl, and alkylheteroaryl. Typically any alkyl or alkoxy moiety of the alkyl substituent group has 1 to 6 carbon atoms.

"Aryl" means an aromatic carbocyclic radical containing 6 to 10 carbon 10 atoms. Exemplary aryl groups include phenyl or naphthyl. Aryl groups may be optionally substituted with one or more groups which may be the same or different, and which are selected from alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, halo, and nitro.

Typically any alkyl or alkoxy moiety of the aryl substituent group has 1 to 6 carbon 15 atoms.

"Heteroaryl" means a 5- to a 10-membered aromatic monocyclic or multicyclic hydrocarbon ring system in which one or more of the carbon atoms in the ring system is or are element(s) other than carbon, for example nitrogen, oxygen or sulfur. Heteroaryl groups may be optionally substituted with one or more groups 20 which may be the same or different, and which are selected from alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkyloxy, halo, and nitro. Exemplary heteroaryl groups include pyrazinyl, furanyl, thienyl, pyridyl, pyrimidinyl, isoxazolyl, isothiazolyl, pyridazinyl, 1,2,4-triazinyl, quinolinyl, and isoquinolinyl.

"Aralkyl" means an aryl-alkyl group in which the aryl and alkyl components 25 are as previously described. Preferred aralkyls contain a lower alkyl moiety.

Exemplary aralkyl groups include benzyl and 2-phenethyl.

"Heteroaralkyl" means a heteroaryl-alkyl group in which the heteroaryl and alkyl components are as previously described.

"Cycloalkyl" means a non-aromatic mono-, multicyclic, or bridged ring 30 system of 3 to 10 carbon atoms. The cycloalkyl group is optionally substituted by one or more halo, or alkyl. Exemplary monocyclic cycloalkyl rings include cyclopentyl, fluorocyclopentyl, cyclohexyl and cycloheptyl.

"Heterocycloalkyl" means a non-aromatic mono-, bi- or tricyclic, or bridged hydrocarbon ring system in which one or more of the atoms in the ring system is or

BOST 1635628.1 _ 8 _ are element(s) other than carbon, for example nitrogen, oxygen or sulfur. Preferred heterocycloalkyl groups contain rings with a ring size of 3-6 ring atoms. Exemplary heterocycloalkyl groups pyrrolidine, piperidine, tetrahydropyran, tetrahydrofuran, tetrahydrothiopyran, and tetrahydrothiofuran.

"Cycloalkylalkyl" means a group in which the cycloalkyl and alkyl components are as previously described.

"Heteroycloalkylalkyl" means a group in which the cycloalkyl and alkyl components are as previously described.

The term "optionally substituted with deuterium" means that one or more hydrogen atoms in the referenced moiety or compound may be replaced with a corresponding number of deuterium atoms.

Throughout this specification, a variable may be referred to generally

1 2 3

(e.g., "each R") or may be referred to specifically (e.g., R , R , R , etc.). Unless otherwise indicated, when a variable is referred to generally, it is meant to include all specific embodiments of that particular variable.

THERAPEUTIC COMPOUNDS

The resent invention provides a compound of Formula A:

(A), or a pharmaceutically acceptable salt thereof,

20 wherein:

R 1 and R 2" are each independently selected from hydrogen, -(Ci-C4)alkyl, or - (Ci-C4)alkylene-0-(Ci-C2)alkyl, wherein the alkyl and alkylene groups at each instance are independently and optionally substituted with deuterium;

R ³ is selected from -CH ₃ , -CH ₂ D, -CHD ₂ and -CD ₃ ;

25 R ⁴ is n-butylene optionally substituted with deuterium;

R ⁵ is selected from hydrogen, deuterium, alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl, and heteroaryl, wherein each of the alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl, and heteroaryl is optionally substituted and wherein one or more hydrogen atoms in the 30 alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl, or

BOST 1635628.1 O heteroaryl or optional substituent thereof is optionally replaced with a corresponding number of deuterium atoms; and

either (a)Y 1 and Y 2 are each fluorine, or are taken together with the carbon to which they are bound to form C=0 or (b) Y ¹ is selected from fluorine and OH; and 5 Y is selected from hydrogen, deuterium, -CH , -CH ₂ D, -CHD ₂ and -CD ;

with the provisos that:

when Y 1 and Y 2 are taken together with the carbon to which they are bound to form C=0, then at least one of R ¹ , R ² , R ³ , R ⁴ , and R ⁵ bears at least one deuterium atom; and

10 when Y ¹ is OH and Y ² is hydrogen or CH ₃ , then at least one of R ¹ , R ² , R ³ , R ⁴ and R ⁵ bears at least one deuterium atom.

In another embodiment, the compound of Formula A is other than the following:

In another embodiment of Formula A, when R 1 and R 2 are each methyl optionally substituted with deuterium and R ⁵ is hydrogen or deuterium, then either: 20 (i) Y ¹ is fiuoro _; or (ii) Y ¹ is OH, and Y ² is selected from -CH ₃ , -CH ₂ D, -CHD ₂ and -CD . In one aspect of this embodiment, the compound is not

2 1 2 3

In a more specific aspect of this embodiment, at least one of Y\ R , R\ R\ and R ⁴ bears at least one deuterium atom.

25 In still another embodiment of Formula A, R 1 and R 2 are each methyl

optionally substituted with deuterium; R ⁵ is hydrogen or deuterium; and either: (a)

Y 1 and Y 2" are taken together with the carbon atom to which they are bound to form

BOST 1635628.1 10 - =0, or (b) Y 1 is -OH and Y 2 is selected from hydrogen and deuterium, with the provisos that:

when Y 1 and Y 2 are taken together with the carbon to which they are bound to form C=0, then at least one of R ¹ , R ² , R ³ , R ⁴ , and R ⁵ bears at least one deuterium atom; and

when Y ¹ is OH, then at least one of Y ² , R ¹ , R ² , R ³ , R ⁴ and R ⁵ bears at least one deuterium atom.

In another embodiment of Formula A, R ⁵ is D, the compound having

Formula Al : l), or a salt thereof, wherein R ¹ , R ² , R ³ , R ⁴ ,

1 2

10 Y' and Y" are as defined for Formula A.

In one aspect of Formula Al , R 1 and R 2 are each independently selected from -CH ₃ , -CH ₂ D, -CHD ₂ and -CD ₃ ; R ³ is selected from -CH ₃ , -CH ₂ D, -CHD ₂ and -CD ₃ ; R ⁴ is selected from -(CH ₂ ) ₄ -, -(CD ₂ ) ₄ -,†- (CD ₂ ) ₃ CH ₂ , and†-CD ₂ (CH ₂ ) ₃ -, wherein

4 1 2

"†" represents the portion of the R moiety bound to C(Y )(Y ) in the compound;

1 2 1

15 and either (a) Y is OH and Y is selected from hydrogen and deuterium; or (b) Y and Y are taken together with the carbon to which they are attached to form C=0.

In a more specific aspect of Formula A 1, R 1 and R 2 are each independently selected from -CH ₃ and -CD ₃ ; R ³ is selected from -CH ₃ and -CD ₃ ; R ⁴ is selected from -(CH ₂ ) ₄ - and†-CD ₂ (CH ₂ ) ₃ -; and either (a) Y ¹ is OH and Y ² is selected from

20 hydrogen and deuterium; or (b) Y 1 and Y 2 are taken together with the carbon to which they are attached to form C=0.

1 2

In another aspect of Formula A 1, R and R are each independently selected from -CH ₃ and -CD ₃ ; R ³ is selected from -CH ₃ and -CD ₃ ; R ⁴ is selected from

-(CH ₂ ) ₄ - and†-CD ₂ (CH ₂ ) ₃ -; and Y 1 and Y 2 are taken together with the carbon to 25 which they are attached to form C=0.

In another embodiment, the present invention provides a compound of Formula A, wherein R ⁵ is hydrogen, the compound having Formula I:

(I), or a salt thereof, wherein:

BOST 1635628.1 11 - R 1 and R 2" are each independently selected from hydrogen, -(Ci-C ₄ )alkyl, or - (Ci-C ₄ )alkylene-0-(Ci-C2)alkyl, wherein the alkyl and alkylene groups at each instance are independently and optionally substituted with deuterium;

R ³ is selected from -CH ₃ , -CH ₂ D, -CHD ₂ and -CD ₃ ;

5 R ⁴ is n-butylene optionally substituted with deuterium; and

either (a) Y 1 and Y 2 are each fluorine, or taken together with the carbon to which they are attached, form C=0; or (b) Y ¹ is selected from fluorine and OH; and Y is selected from hydrogen, deuterium, -CH ₃ , -CH ₂ D, -CHD ₂ and -CD ₃ , with the provisos that:

1 2

10 when Y ¹ and Y" are taken together with the carbon to which they are

attached to form C=0, at least one of R ¹ , R ² , R ³ and R ⁴ bears at least one deuterium atom; and

when Y 1 is OH and Y2 is hydrogen or -CH ₃ , then at least one of R 1 , R2 , R 3 and R ⁴ bears at least one deuterium atom.

15 In a more specific embodiment of Formula I, R 1 and R 2 are each

independently selected from -CH ₃ , -CH ₂ D, -CHD ₂ and -CD ₃ ; R is selected from -CH ₃ , -CH ₂ D, -CHD ₂ and -CD ₃ ; R ⁴ is selected from -(CH ₂ ) ₄ -, -(CD ₂ ) ₄ -,†- (CD ₂ ) ₃ CH ₂ , and†-CD ₂ (CH ₂ ) ₃ -, wherein "†" represents the portion of the R ⁴ moiety

1 2 1 2

bound to C(Y )(Y ) in the compound; and either: Y is OH and Y is selected from

20 hydrogen and deuterium; or Y 1 and Y 2 are taken together with the carbon to which they are attached to form C=0.

1 2

In another aspect of Formula I, R and R are each independently selected from -CH ₃ and -CD ₃ ; R ³ is selected from -CH ₃ and -CD ₃ ; R ⁴ is selected from

-(CH ₂ ) ₄ - and†-CD ₂ (CH ₂ ) ₃ -; and either: Y 1 is OH and Y 2 is selected from hydrogen

25 and deuterium; or Y 1 and Y 2 are taken together with the carbon to which they are attached to form C=0.

1 2

In another aspect of Formula I, R and R are each independently selected from -CH ₃ and -CD ₃ ; R ³ is selected from -CH ₃ and -CD ₃ ; R ⁴ is selected from

-(CH ₂ ) ₄ - and†-CD ₂ (CH ₂ ) ₃ -; and Y 1 and Y 2 are taken together with the carbon to 30 which they are attached to form C=0.

In another embodiment, in any of the aspects set forth above, the compound of Formula I is other than the following:

BOST 1635628.1

In yet another embodiment, in any of the aspects set forth above, the compound of Formula I is other than the following:

5

10 In yet another embodiment, in any of the aspects set forth above, the

compound of Formula I is other than the following:

15 Another embodiment of the present invention provides a compound of

Formula II:

O ° R ¹

R (II), or a salt thereof, wherein:

R 1 and R 2" are each independently selected from hydrogen, -(Ci-C ₄ )alkyl, or - (Ci-C ₄ )alkylene-0-(Ci-C2)alkyl, wherein the alkyl and alkylene groups at each 20 instance are independently and optionally substituted with deuterium;

R ³ is selected from -CH ₃ , -CH ₂ D, -CHD ₂ and -CD ₃ ;

R ⁴ is n-butylene optionally substituted with deuterium; and

BOST 1635628.1 n 2 3 4

wherein at least one of R , R and R bears at least one deuterium atom. One embodiment relates to a compound of Formula A, A 1 , 1, or II, wherein

2 3

R" and R ^J are each independently selected from -CH ₃ , -CH ₂ D, -CHD ₂ and -CD ₃ .

Another embodiment relates to a compound of Formula A, A 1 , 1, or II,

2 3

5 wherein R and R are each independently selected from -CH ₃ , and -CD ₃ .

Another embodiment relates to a compound of Formula A, A 1 , 1, or II, wherein R ¹ is selected from hydrogen, (Ci-C ₃ )alkyl, and (Ci-C ₂ )alkylene-0(Ci- C ₂ )alkyl.

Another embodiment relates to a compound of Formula A, A 1 , 1, or II, 10 wherein R ¹ is hydrogen, -CH ₃ , -CD ₃ , -CH ₂ CH ₂ CH ₃ , -CD ₂ CH ₂ CH ₃ , -CD ₂ CD ₂ CH ₃ , - CD ₂ CD ₂ CD ₃ , -CH ₂ OCH ₂ CH ₃ , -CH ₂ OCD ₂ CH ₃ , -CH ₂ OCD ₂ CD ₃ , -CD ₂ OCH ₂ CH ₃ , - CD ₂ OCD ₂ CH ₃ , or -CD ₂ OCD ₂ CD ₃ .

Another embodiment relates to a compound of Formula A, wherein R ⁵ is selected from hydrogen, deuterium, alkyl, cycloalkyl, heterocycloalkyl,

15 cycloalkylalkyl, and heterocycloalkylalkyl, wherein each of alkyl, cycloalkyl,

heterocycloalkyl, cycloalkylalkyl, and heterocycloalkylalkyl may be optionally substituted.

In other embodiments of Formula A, Al or I:

a) each methylene unit in R ⁴ is selected from -CH ₂ - and -CD ₂ -; more 20 specifically R ⁴ is selected from -(CH ₂ ) ₄ -, -(CD ₂ ) ₄ -, ^† -CD ₂ (CH ₂ ) ₃ - and

^† -(CD ₂ ) ₃ CH ₂ -, wherein " ^† " represents the point where R ⁴ is attached to C(Y ^l )(Y ² ) in the compound;

b) when Y ¹ is F, Y ² is selected from hydrogen, -CH ₃ , -CH ₂ D, -CHD ₂ and -CD ₃ ; or

1 2

25 c) when Y is F, Y fluorine; or

1 2 2 3 1 d) when Y and Y are not the same and Y and R are not the same and Y and

Y ¹ _Ν γ2

3 R ³ ^R ⁴ - R are not the same, the stereochemistry at "*" is represented by: * ; or

1 2 2 3 1 e) when Y and Y are not the same and Y and R are not the same and Y and γΐ γ2

3 R ³ ^R ⁴ - R are not the same, the stereochemistry at "*" is represented by: ⁵ .

1 2 3

In other embodiments of Formula A, Al or I, R is -CD ₃ ; R and R are

BOST 1635628.1 - 14 - 1 2 4

-CH ₃ ; Y and Ύ ^Δ are taken together to form C=0; and R is selected from -(CH ₂ )4-, -(CD ₂ ) ₄ -, ^† -CD ₂ (CH ₂ ) ₃ - and ^† -(CD ₂ ) ₃ CH ₂ -.

In other embodiments of Formula A, Al or I, R 1 is -CD ₃ ; R 2 and R 3 are

-CH ₃ ; Y 1 and 2 ; and R 4

Ύ ^Δ are taken together to form C=0 is selected from -(CH ₂ ) ₄ -, 5 and -(CD ₂ ) ₄ -.

In other embodiments of Formula A, Al or I, R 1 is -CD ₃ ; R 2 and R 3 are -

CH ₃ ; R 4 is -(CH ₂ ) ₄ -; Y 1 is fluoro; and Y 2 is selected from deuterium, -CH ₂ D, -CHD ₂ and -CD ₃ .

In other embodiments of Formula A, Al or I, R 1 is -CD ₃ ; R 2 and R 3 are - 10 CH ₃ ; R ⁴ is -(CH ₂ ) ₄ -; Y ¹ is fluoro; and Y ² is fluorine.

In other embodiments of Formula A or A 1, R 1 is -CD ₃ ; R 2 and R 3 are -CH ₃ ; R ⁴ is -(CH ₂ ) ₄ -; R ⁵ is deuterium; Y ¹ is fluoro; and Y ² is selected from deuterium, -CH ₂ D, -CHD ₂ and -CD ₃ .

In other embodiments of Formula A or A 1, R 1 is -CD ₃ ; R 2 and R 3 are -CH ₃ ; 15 R ⁴ is -(CH ₂ ) ₄ -; R ⁵ is deuterium; Y ¹ is fluoro; and Y ² is fluorine.

In other embodiments of Formula A, Al or I, Y 1 is F; Y 2 is selected from hydrogen; R ³ is -CH ₃ ; and R ⁴ is -(CH ₂ ) ₄ -.

In other embodiments of Formula A, Al or I, Y 1 is F; Y2 is fluorine; R 3 is - CH ₃ ; and R ⁴ is -(CH ₂ ) ₄ -.

One embodiment provides a compound of Formula B:

B, or a pharmaceutically

1 2

acceptable salt thereof, wherein each of R and R is independently selected from

25 -CH ₃ and -CD ₃ ; R 5 is hydrogen or deuterium; each Z 3 is hydrogen or deuterium; each Z ⁴ is hydrogen or deuterium; each Z ⁵ is hydrogen or deuterium; and either (a)

Y 1 is OH, and Y2 is hydrogen or deuterium, or (b) Y 1 and Y2 are taken together with the carbon to which they are attached to form C=0.

One embodiment provides a compound of Formula B, wherein each Z ³ , Z ⁴

30 and Z 5 is hydrogen. In one aspect, R 1 and R2 are each -CD ₃ . In another aspect R 5 is

BOST 1635628.1 - 15 - deuterium. In another aspect, Y 1 and Y 2 are taken together with the carbon to which

1 2 they are attached to form C=0. In still another aspect, Y and is OH, and Y is hydrogen or deuterium.

Another embodiment provides a compound of Formula B, wherein each Z , 5 Z ⁴ and Z ⁵ is deuterium. In one aspect, R ¹ and R ² are each -CD3. In another aspect

R 5 is deuterium. In another aspect, Y 1 and Y2 are taken together with the carbon to which they are attached to form C=0. In still another aspect, Y 1 and is OH, and Y 2 is hydrogen or deuterium.

Yet another embodiment provides a compound of Formula B, wherein R ¹ 10 and R ² are each -CD ₃ . In one aspect, R ⁵ is deuterium. In another aspect, each Z , Z and Z ⁵ is hydrogen and R ⁵ is deuterium. In another aspect, each Z ³ , Z ⁴ and Z ⁵ is deuterium and R ⁵ is deuterium.

A further embodiment provides a compound of Formula B, wherein Y ¹ and

Y are taken together with the carbon to which they are attached to form C=0. In 15 one aspect, R ⁵ is deuterium. In another aspect, each Z ³ , Z ⁴ and Z ⁵ is hydrogen and R ⁵ is deuterium. In another aspect, each Z ³ , Z ⁴ and Z ⁵ is deuterium and R ⁵ is deuterium. In another aspect, R 1 and R2 are each ₃ . In another aspect, R 1

-CD and R2 are each 5 In another aspect, R 1

-CD ₃ and R is deuterium. and R2 are each -CD ₃ , and each Z ³ , Z ⁴ and Z ⁵ is deuterium. In another aspect, R ¹ and R ² are each -CD ₃ , each 20 Z ³ , Z ⁴ and Z ⁵ is deuterium and R ⁵ is deuterium. In another aspect, R ¹ and R ² are each -CD ₃ , and each Z ³ , Z ⁴ and Z ⁵ is hydrogen. In another aspect, R ¹ and R ² are each -CD ₃ , each Z ³ , Z ⁴ and Z ⁵ is hydrogen and R ⁵ is deuterium

A still further embodiment provides a compound of Formula B, Y ¹ and is

OH, and Y 2 is hydrogen or deuterium. In one aspect, R 5 is deuterium. In another 25 aspect, each Z ³ , Z ⁴ and Z ⁵ is hydrogen and R ⁵ is deuterium. In another aspect, each Z ³ , Z ⁴ and Z ⁵ is deuterium and R ⁵ is deuterium. In another aspect, R ¹ and R ² are each 1 2 h 5

-CD ₃ . In another aspect, R and R are eac -CD ₃ and R is deuterium. In another aspect, R ¹ and R ² are each -CD ₃ , and each Z ³ , Z ⁴ and Z ⁵ is deuterium. In another aspect, R ¹ and R ² are each -CD ₃ , each Z ³ , Z ⁴ and Z ⁵ is deuterium and R ⁵ is 30 deuterium. In another aspect, R ¹ and R ² are each -CD ₃ , and each Z ³ , Z ⁴ and Z ⁵ is hydrogen. In another aspect, R ¹ and R ² are each -CD ₃ , each Z ³ , Z ⁴ and Z ⁵ is hydrogen and R ⁵ is deuterium

Another embodiment provides a compound of Formula B, wherein R ⁵ is deuterium.

BOST 1635628.1 Another embodiment provides a compound of Formula B, wherein R ⁵ is deuterium, Z ³ , Z ⁴ and Z ⁵ is hydrogen and R ¹ is -CD ₃ .

Specific examples of compounds of Formula A, Al , I, or II include those shown in Tables 1-6 (below) or pharmaceutically acceptable salts thereof, wherein

† 4 1 2

5 "'" represents the portion of the R moiety bound to C(Y )(Y ) in the compound. In the tables, compounds designated as "(R)" or "(5)" refer to the stereochemistry at the carbon bearing the Y ¹ substituent. Compounds lacking either designation and

1 2

containing a chiral carbon atom bound to Y and Y are intended to represent a racemic mixture of enantiomers.

Table 1 : Examples of Specific Compounds of Formula I. Deuterated and/or

Fluorinated Analogs of Pentoxifylline and its Metabolites.

BOST 1635628.1 - 17 -

Table 1 above shows examples of specific compounds of Formula I. These examples are deuterated and/or fluorinated analogs of pentoxifylline and its metabolites.

5

1 2

Table 2: Examples of Specific Compounds of Formula I Where R is H and Y is

CHs or CDs.

BOST 1635628.1 - 18 -

Table 2 above shows examples of specific compounds of Formula I where R is H and Y is CH ₃ or CD ₃ . These compounds include deuterated and fluorinated analogs of Albifylline (HWA-138). Albifylline has been studied for uses that are 5 associated with pentoxifylline.

Table 3: Specific Examples of Formula I Where R ¹ is -CH ₂ -0-CH ₂ CH ₃ Optionally

Substituted with Deuterium.

BOST 1635628.1 - 19 - Compound R R R R Y Y

279 CD ₂ OCD ₂ CD ₃ CD ₃ CD ₃ (CD ₂ ) ₄ F CD ₃

280 CD ₂ OCH ₂ CH ₃ CD ₃ CD ₃ (CD ₂ ) ₄ F CD ₃

281 CH ₂ OCH ₂ CH ₃ CD ₃ CD ₃ (CD ₂ ) ₄ F CD ₃

282 CD ₂ OCD ₂ CD ₃ CH ₃ CD ₃ (CD ₂ ) ₄ F CD ₃

283 CD ₂ OCH ₂ CH ₃ CH ₃ CD ₃ (CD ₂ ) ₄ F CD ₃

284 CH ₂ OCH ₂ CH ₃ CH ₃ CD ₃ (CD ₂ ) ₄ F CD ₃

Table 3 above shows examples of specific compounds of Formula I where R is -CH ₂ -0-CH ₂ CH ₃ , optionally substituted with deuterium. In these examples, Y ¹ is OH or F and Y is CH ₃ or CD ₃ . These compounds include deuterated and

5 fluorinated analogs of torbafylline (HWA-448). Torbafylline has been studied for the treatment of depression, urinary incontinence, irritable bowel syndrome and multiple sclerosis.

Table 4: Specific Examples of Formula I Where R ¹ is -CH ₂ CH ₂ CH ₃ Optionally

Substituted With Deuterium and Y ¹ is OH or F.

BOST 1635628.1 - 20 - Compound R R R R Y Y

326 CD2CD2CD3 CH ₃ CH ₃ (CH ₂ ) ₄ F CH ₃

327 CD ₂ CH ₂ CH ₃ CH ₃ CH ₃ (CH ₂ ) ₄ F CH ₃

328 CH ₂ CH ₂ CH ₃ CH ₃ CH ₃ (CH ₂ ) ₄ F CH ₃

329 CD ₂ CD ₂ CD ₃ CD ₃ CD ₃ (CD ₂ ) ₄ F CD ₃

330 CD2CH2CH3 CD ₃ CD ₃ (CD ₂ ) ₄ F CD ₃

331 CH ₂ CH ₂ CH ₃ CD ₃ CD ₃ (CD ₂ ) ₄ F CD ₃

332 CD2CD2CD3 CH ₃ CD ₃ (CD ₂ ) ₄ F CD ₃

333 CD2CH2CH3 CH ₃ CD ₃ (CD ₂ ) ₄ F CD ₃

334 CH ₂ CH ₂ CH ₃ CH ₃ CD ₃ (CD ₂ ) ₄ F CD ₃

Table 4 above shows examples of specific compounds of Formula I where R is -CH ₂ CH ₂ CH ₃ optionally substituted with deuterium. In these examples, Y ¹ is OH or F and Y is CH ₃ or CD ₃ . These compounds include deuterated and fluorinated 5 analogs of A-802715. A-802715 has been studied for the treatment of septic shock and inhibition of effects of allograft reaction.

Table 5 : Specific Examples of Formula I where R ¹ is -CH ₂ CH ₂ CH ₃ Optionally

1 2

Substituted With Deuterium and Y and Y Are Taken Together As = O

BOST 1635628.1 - 21 - Table 5 above shows examples of specific compounds of Formula I where R is -CH ₂ CH ₂ CH ₃ optionally substituted with deuterium. In these examples, Y ¹ and Y are taken together with their intervening carbon to form a carbonyl. These compounds include deuterated analogs of propentofylline. Propentofylline has been 5 studied for the treatment of Alzheimer's disease, neuropathic pain, traumatic brain injury, dysuria, retinal or optic nerve head damage, and peptic ulcers. It has also been studied for controlling intraocular pressure, stabilization of auto-regulation of cerebral blood flow and inhibition of effects of allograft reaction.

Table 6: Examples of Specific Compounds of Formula A. Deuterated and/or

Fluorinated Analogs of Pentoxifylline and its Metabolites where R ⁵ is D

BOST 1635628.1 - 22 - Compound R R R R R ⁵ Y Y

429 CH ₃ CH ₃ CD ₃ ^T (CD ₂ ) ₃ CH ₂ D OH H

430 CD ₃ CD ₃ CH ₃ (CH ₂ ) ₄ D OH D

431 CD ₃ CH ₃ CH ₃ (CH ₂ ) ₄ D OH D

432 CH ₃ CD ₃ CH ₃ (CH ₂ ) ₄ D OH D

433 CH ₃ CH ₃ CH ₃ (CH ₂ ) ₄ D OH D

434 CD ₃ CD ₃ CD ₃ ^T CD ₂ (CH ₂ ) ₃ D OH D

435 CD ₃ CH ₃ CD ₃ ^T CD ₂ (CH ₂ ) ₃ D OH D

435(R) CD ₃ CH ₃ CD ₃ ^T CD ₂ (CH ₂ ) ₃ D (R)OH D

435(5) CD ₃ CH ₃ CD ₃ ^T CD ₂ (CH ₂ ) ₃ D (5)OH D

436 CH ₃ CD ₃ CD ₃ ^T CD ₂ (CH ₂ ) ₃ D OH D

437(R) CH ₃ CH ₃ CD ₃ ^T CD ₂ (CH ₂ ) ₃ D (R)OH D

437(5) CH ₃ CH ₃ CD ₃ ^T CD ₂ (CH ₂ ) ₃ D (5)OH D

437 CH ₃ CH ₃ CD ₃ ^T CD ₂ (CH ₂ ) ₃ D OH D

438 CD ₃ CD ₃ CD ₃ (CD ₂ ) ₄ D OH D

439 CD ₃ CH ₃ CD ₃ (CD ₂ ) ₄ D OH D

440 CH ₃ CD ₃ CD ₃ (CD ₂ ) ₄ D OH D

441 CH ₃ CH ₃ CD ₃ (CD ₂ ) ₄ D OH D

442 CD ₃ CD ₃ CD ₃ ^T (CD ₂ ) ₃ CH ₂ D OH D

443 CD ₃ CH ₃ CD ₃ ^T (CD ₂ ) ₃ CH ₂ D OH D

444 CH ₃ CD ₃ CD ₃ ^T (CD ₂ ) ₃ CH ₂ D OH D

445 CH ₃ CH ₃ CD ₃ ^T (CD ₂ ) ₃ CH ₂ D OH D

446 CD ₃ CD ₃ CH ₃ (CH ₂ ) ₄ D F H

447 CH ₃ CH ₃ CH ₃ (CH ₂ ) ₄ D F H

448 CD ₃ CH ₃ CH ₃ (CH ₂ ) ₄ D F H

449 CH ₃ CD ₃ CH ₃ (CH ₂ ) ₄ D F H

450 CD ₃ CD ₃ CH ₃ (CH ₂ ) ₄ D F F

451 CD ₃ CH ₃ CH ₃ (CH ₂ ) ₄ D F F

452 CH ₃ CD ₃ CH ₃ (CH ₂ ) ₄ D F F

453 CH ₃ CH ₃ CH ₃ (CH ₂ ) ₄ D F F

Table 6 above shows examples of specific compounds of Formula A. These examples are deuterated and/or fluorinated analogs of pentoxifylline and its metabolites where R ⁵ is deuterium.

5 In one aspect of the above embodiment, the compound is not any one of

Compounds 100, 1 16, or 149.

Examples of specific compounds of this invention include the following:

BOST 1635628.1 - 23 -

The present invention also provides a compound of Formula C:

C

or a pharmaceutically acceptable salt thereof, wherein R ¹ is selected from -CH ₃ and -CD ₃ ; R ⁵ is hydrogen or deuterium; and Y is fluorine , hydrogen or deuterium.

One embodiment provides a compound of Formula C, wherein R ¹ is -CH ₃ One embodiment provides a compound of Formula C, wherein R ¹ is -CD ₃

BOST 1635628.1 - 24 - One embodiment provides a compound of Formula C, wherein R is hydrogen.

One embodiment provides a compound of Formula C, wherein R ⁵ is deuterium.

One embodiment provides a compound of Formula C, wherein Y is fluorine.

One embodiment provides a compound of Formula C, wherein Y is hydrogen. In one aspect of this embodiment, the compound of Formula C or a pharmaceutically acceptable salt thereof has the structure

10 In another aspect of this embodiment, the compound of Formula C has the structur

One embodiment provides a compound of Formula C, wherein Y is deuterium. In one aspect of this embodiment, the compound of Formula C or a

15 pharmaceutically acceptable salt thereof has the structure

In another aspect of this embodiment, the compound of Formula C has the structur

20 Examples of the compounds of the formula C include the following

BOST 1635628.1 25 - compounds and pharmaceutically acceptable salts thereof:

BOST 1635628.1 -26-

The present invention also provides a compound of Formula D(i):

D(i)

or a pharmaceutically acceptable salt thereof, wherein R is selected from -CH and -CD and R ⁵ is hydrogen or deuterium.

The present invention also provides a compound of Formula D(ii):

10

D(ii)

or a pharmaceutically acceptable salt thereof, wherein R ¹ is selected from -CH and -CD and R ⁵ is hydrogen or deuterium.

Examples of the compounds of the formula D(i) include the following compounds or pharmaceutically acceptable salts thereof:

BOST 1635628.1 - 27 - and

Examples of the compounds of the formula D(ii) include the following compounds or pharmaceutically acceptable salts thereof:

In another set of embodiments, any atom not designated as deuterium in any

10 of the embodiments set forth above is present at its natural isotopic abundance.

The synthesis of compounds of this invention can be achieved by synthetic chemists of ordinary skill. Relevant procedures and intermediates are disclosed, for instance in Sidzhakova, D et al., Farmatsiya, (Sofia, Bulgaria) 1988, 38(4): 1-5; Davis, PJ et al, Xenobiotica, 1985, 15(12): 1001-10; Akgun, H et al, J Pharm Sci, 2001, 26(2): 67-71; German Patent publication DD 274334; Czech Patent Nos. CS 237719, CS201558; PCT patent publication WO9531450; and in Japanese Patent publication Nos. JP58150594, JP58134092, JP58038284, JP57200391, JP57098284, JP57085387, JP57062278, JP57080385, JP57056481, JP57024385, JP57011981,

BOST 1635628.1 28 - JP57024386, JP57024382, JP56077279, JP56032477, JP56007785, JP56010188, JP56010187, JP55122779, and JP55076876.

Such methods can be carried out utilizing corresponding deuterated and optionally, other isotope-containing reagents and/or intermediates to synthesize the compounds delineated herein, or invoking standard synthetic protocols known in the art for introducing isotopic atoms to a chemical structure.

EXEMPLARY SYNTHESIS

Methods for synthesizing compounds of Formula I are depicted in the

10 following schemes.

Scheme 1A. Synthesis of Compounds of Formula I

15 As depicted in Scheme 1 A, deuterated compound 10 is alkylated with

deuterated intermediate 11 (wherein X is chloride, bromide or iodide) in the presence of potassium carbonate to afford compounds of Formula I. Alternatively, sodium hydroxide in aqueous methanol may be employed to afford compounds of Formula I according to the methods of US Patent 4289776.

20

Scheme IB. Preparation of Compounds Where Y ¹ = OH From Compounds of

Formula II

Formula II

BOST 1635628.1 - 29 - As depicted in Scheme IB, compounds of Formula II can be used to make compounds where Y ¹ is OH. Thus, compounds of Formula II are reduced with either sodium borohydride or sodium borodeuteride (commercially available at 99 atom %D) according to the general method of European Patent publication 0330031

5 to form compounds wherein Y 1 is OH and Y 2 is hydrogen or deuterium. The

enantiomeric alcohol products may be separated, for example through the method of Nicklasson, M et al, Chirality, 2002, 14(8): 643-652. In an alternate method, enzymatic reduction affords an enantiomerically-enriched alcohol product using the methods disclosed in Pekala, E et al., Acta Poloniae Pharmaceutica, 2007, 64(2): 10 109-113, or in Pekala, E et al, Biotech J, 2007, 2(4): 492-496.

Synthesis of Compound 10

Referring to Scheme 1 A, compounds that can be used as compound 10 to make compounds of Formula I are known and include, but are not limited to, the

15 following: theobromine (wherein R 1 and R 2 are CH ₃ ) which is commercially

1 2 1 available. Isotopologues of 10 wherein: (a) R is -CD ₃ and R is -CH ₃ ; (b) R is -

CH ₃ and R 2" is -CD ₃ ; and (c) R 1 and R2" are -CD ₃ are all known. See Benchekroun, Y et al, J Chromatogr B, 1977, 688: 245; Ribon, B et al, Coll INSERM, 1988, 164: 268; and Horning, MG et al, Proc Int Conf Stable Isot 2 ^nd , 1976, 41-54. 3-Methyl-

1 2

20 7-propylxanthine, wherein R is n-propyl and R is -CH ₃ , is commercially available.

Compound 10 wherein R 1 is CH ₂ OCH ₃ and R 2 is CH ₃ is also known. See German patent application DE 3942872A1.

Scheme 2. Synthesis of Compound 10

25

1 . NaOH, H ₂ Q

2. HCI, H ₂ 0

BOST 1635628.1 - 30 - HCOOH

A synthesis of compound 10 is depicted in Scheme 2 starting with

5 commercially-available N-nitroso-N-methylurea. Treatment with appropriately deuterated amine 12 in water affords N-alkylurea 13 following the methods of Boivin, JL et al., Canadian Journal of Chemistry, 1951, 29: 478-81. Urea 13 may be treated with 2-cyanoacetic acid and acetic anhydride to provide cyanoacetamide derivative 14, which is treated first with aqueous NaOH and then with aqueous HC1

10 to provide cyclized pyrimidinedione 15 according to the methods of Dubey, PK et al., Indian Journal of Heterocyclic Chemistry, 2005, 14(4): 301-306. Alternatively, cyanoacetamide 14 may be treated with trimethylsilylchloride and

hexamethyldisilazane to afford the cyclized product 15 via the methods of Fulle, F et al, Heterocycles, 2000, 53(2): 347-352.

15 Following the methods of Merlos, M et al., European Journal of Medicinal

Chemistry, 1990, 25(8): 653-8, treatment of pyrimidinedione 15 with sodium nitrite in acetic acid, and then by ammonium hydroxide and sodium dithionite, yields compound 16, which is treated with formic acid to provide purine derivative 17. Following the methods disclosed by Rybar, A et al, in Czech patent application CS

20 263595B1, alkylation of 17 with appropriately deuterated electrophile 18 (X is

chloro, bromo, or iodo) in the presence of potassium carbonate and optionally in the presence of additives such as NaBr, KBr, Nal, KI, or iodine, affords compound 10.

Referring to Scheme 2, useful deuterated amine reagents 12 include, but are not limited to, commercially-available compounds such as n-propyl-d7-amine, or

25 known compounds such as l-propan-l,l-d2-amine (Moritz, F et al, Organic Mass Spectrometry, 1993, 28(3): 207-15). Useful deuterated urea reagents 13 may include, but are not limited to, commercially-available compounds such as N-

O O methyl-d3-urea H , or methylurea-d ₆ ^D

Useful deuterated electrophiles 18 may include, but are not limited to,

BOST 1635628.1 31 - commercially-available compounds such as iodomethane-d3, or bromomethane-d3, or l-bromopropane-d ₇ , or l-bromopropane-l,l-d2, or known compounds such as (chloromethoxy-d ₂ )-ethane (Williams, AG, WO 2002059070A1), or

bromomethoxymethane-d ₂ (Van der Veken, BJ et al., Journal of Raman

5 Spectroscopy, 1992, 23(4): 205-23, or (bromomethoxy-d ₂ )-methane-d3 (Van der Veken, BJ et al, Journal of Raman Spectroscopy, 1992, 23(4): 205-23. The commercially available deuterated intermediates 12, 13 and 18 mentioned above are available having an isotopic purity of at least 98 atom % D.

10 Synthesis of Intermediate lla-i/ ₅ (cf. Scheme 1A)

Scheme 3. Synthesis of Intermediate lla-i/g

21 11a-d,

An approach to the preparation of compound lla-i ₅ (cf. Scheme 1A)

3 4 † 1 2

(wherein R is CD ₃ ; R is '-CD ₂ (CH ₂ )3-, and Y and Y are taken together to form =0), is depicted in Scheme 3. Thus, methyllithium is added to commercially-

20 available delta-valerolactone 19 according to the procedure of Zhang, Q et al.,

Tetrahedron, 2006, 62(50): 11627-11634 to afford ketone 20. Treatment of 20 with TFA-i _/ (99 atom %D) in D ₂ 0 (99 atom %D) under microwave conditions provides deuterated ketone 21 according to the general method of Fodor-Csorba K, Tet Lett, 2002, 43: 3789-3792. The alcohol moiety in 21 is converted to the chloride upon

25 treatment with triphenylphosphine and carbon tetrachloride to yield chloride lla-i ₅ following the general procedures of Clement, J-L, Org Biomol Chem, 2003, 1 : 1591-1597.

BOST 1635628.1 - 32 - Scheme 4. Synthesis of Intermediates 11a

22 23

24 25

1. L1AIH4

Scheme 4 depicts a synthesis of compound lla-dg and compound lla-djj. Thus, commercially-available 4-phenylbutyric acid 22 may be heated in D ₂ 0 (99

10 atom %D) in the presence of Pd/C and hydrogen gas to afford deuterated acid 23 according to the general methods of Esaki, et al, Chem Eur J, 2007, 13: 4052-4063. Addition of deuterated methyllithium in the presence of trimethylsilyl chloride provides ketone 24, according to the general method of Porta, A et al., J Org Chem, 2005, 70(12): 4876-4878. Ketone 24 is converted to acetal 25 by treatment with

15 D ₂ S0 ₄ (99 atom %D) and commercially-available ethyleneglycol-^ (99 atom %D).

Treatment of 25 with NaI0 ₄ and RuCl ₃ according to the general method of Gamier, J-M et al., Tetrahedron: Asymmetry, 2007, 18(12): 1434-1442 provides carboxylic acid 26. Reduction with either L1AIH4 or L1AID4 (98 atom %D) provides the alcohols (not shown), which are then chlorinated using either phosphorus

20 oxychloride or triphenylphosphine and N-chlorosuccinimide (Naidu, SV et al, Tet Lett, 2007, 48(13): 2279-2282), followed by acetal cleavage with D ₂ S0 ₄

(Heathcock, CH et al, J Org Chem, 1995, 60(5): 1120-30) to provides chlorides

BOST 1635628.1 - 33 - lla-i 9 and lla-i n, respectively.

Scheme 4a. Synthesis of Intermediates llb-(R)

27 28 11 b-(R)

Scheme 4b. Synthesis of Chloride llb-(S)

SOCI,

R ^3' ^R ⁴ — OH R ³ ^R ⁴ — CI

29

11 b-(S)

10

Schemes 4a and 4b depict the synthesis of specific enantiomers of chlorides

1 2

llb-(R) (wherein Y is fluorine; Y is selected from hydrogen and deuterium; and

1 2 the compound is in the (R) configuration) and llb-(S) (wherein Y is fluorine; Y is selected from hydrogen and deuterium; and the compound is in the (S)

15 configuration). In Scheme 4a, a deuterated (or nondeuterated) benzyl-protected alcohol 27, such as known [[[(5R)-5-fluorohexyl]oxy]methyl]-benzene (PCT publication WO2000031003) is deprotected by hydrogenation in the presence of Pd/C to provide alcohol 28. The alcohol is chlorinated with thionyl chloride according to the general procedure of Lacan, G et al., J Label Compd Radiopharm,

20 2005, 48(9): 635-643 to afford chloride llb-(R).

In Scheme 4b, a deuterated (or nondeuterated) alcohol 29, such as known (S)-(+)-5-fluorohexanol (Riswoko, A et al, Enantiomer, 2002, 7(1): 33-39) is chlorinated to afford chloride llb-(S).

BOST 1635628.1 - 34 - Scheme 5. Synthesis of Intermediates 11c and lie

Scheme 5 depicts a synthesis of other intermediates 11c and lie. Thus, following the methods of either Kutner, Andrzej et al., Journal of Organic

Chemistry, 1988, 53(15): 3450-7, or of Larsen, SD et al, Journal of Medicinal Chemistry, 1994, 37(15): 2343-51, compounds 30 or 31 (wherein X is a halide) may be treated with deuterated Grignard reagent 32 to afford intermediate 11c wherein

3 2 1

R ^J and are the same, Y is OH, and X is a halide. Treatment with

diethylaminosulfur trifluoride (DAST) in dichloromethane or toluene provides

3 2 1

intermediate lie wherein R and Y are the same, Y is F, and X is a halide according to the general procedures of either Karst, NA et al, Organic Letters, 2003, 5(25): 4839-4842, or of Kiso, M et al, Carbohydrate Research, 1988, 177: 51-67.

Commercially available halides can be used to make compounds 11 as disclosed in Scheme 5. For example, commercially-available 5-chlorovaleryl chloride, or commercially-available 5-bromovaleryl chloride, or commercially- available ethyl 5-bromovalerate, may be useful as reagents 30 or 31. Referring again to Scheme 5, use of commercially-available methyl-<¾-magnesium iodide as

3 2

Grignard reagent 32 affords electrophile 11 wherein R and Y are simultaneously CD ₃ .

Scheme 6. Synthesis of Intermediate lie (X=Br)

33 or acetone-d 4

35 36 11e (X=Br)

25 Scheme 6 depicts an alternate synthesis of intermediate lie, wherein R and

Y are the same and X=Br. Thus, according to the procedures of Hester, JB et al,

BOST 1635628.1 - 35 - Journal of Medicinal Chemistry, 2001 , 44(7): 1099-1 1 15, commercially-available 4-chloro-l-butanol is protected via treatment with 3,4-dihydro-2H-pyran (DHP) and camphorsulfonic acid (CSA) to provide chloride 33. Generation of the

corresponding Grignard reagent with magnesium, followed by addition of acetone 5 (R ³ = Y ² = CH ₃ ) or acetone-d ₆ (Y ² = R ³ = CD ₃ ), affords alcohol 34. Fluorination with diethylaminosulfur trifluoride (DAST) in DCM provides fluoride 35.

Deprotection with CSA in MeOH provides alcohol 36, and treatment with N- bromosuccinimide and triphenyl phosphine affords intermediate lie.

Scheme 7. Alternative Synthesis of Intermediate lie (X=Br)

EtC\ DHP, CSA, Et ₂ 0 LiAID HO

OH ΌΤΗΡ OTHP

O or O Et,0 D D

37 DHP, TsOH,

38

r, CH ₂ CI ₂ 39

3 2

15 Scheme 7 depicts the synthesis of intermediate lie wherein R and Y are the same and X=Br. Thus, commercially-available 4-hydroxy-butanoic acid ethyl ester 37 is treated with DHP and CSA, or with DHP, TsOH, and pyridine to provide ester 38. Reduction with L1AID ₄ affords deuterated alcohol 39, which is treated with either triphenyl phosphine in CC1 ₄ (Sabitha, G et al., Tetrahedron Letters, 2006,

20 (volume date 2007), 48(2): 313-315) or with methanesulfonyl chloride, lithium

chloride, and 2,6-lutidine in DMF (Blaszykowski, C et al, Organic Letters, 2004, 6(21): 3771-3774) to afford chloride 40. Following the same methods as in Scheme 6, chloride 40 may be converted to lie.

Scheme 8. Synthesis of Intermediate lle-dx (X=Br)

D ₂ D ₂

D ₂ C" ^X CD ₂ DCI, ZnCI ₂ D ₂ D D ₂ C-CD ₂ ^{C^} C ^C OH

D ₂ D ₂ D ₂

41 42 11e- 8

BOST 1635628.1 - 36 - Scheme 8 depicts the synthesis of intermediate lle 3 2

-i 8 wherein R and Y are the same and X=Br. Thus, commercially-available THF-<¾ 41 may be treated with DCl and ZnCl ₂ according to the general methods of Yang, A et al., Huagong Shikan, 2002, 16(3): 37-39 to afford known chloride 42 (Aiken, Rudolf-Giesbert, WO 5 2003080598A1). Following the same methods as in Scheme 6, chloride 42 may be converted to lle-dg.

Scheme 9. Synthesis of Intermediate llc-fifs (X=Br)

43 44 Hc-d,

Scheme 9 depicts the synthesis of intermediate llc 3 2

10 -i 8 wherein R and Y are the same and X=Br. Thus, known carboxylic acid 43 (Lompa-Krzymien, L et al., Proc. Int. Conf. Stable Isot. 2 ^nd , 1976, Meeting Date 1975, 574-8) is treated with either diazomethane (according to the general method of Garrido, NM et al., Molecules, 2006, 11(6): 435-443.) or with trimethylsilyl chloride and methanol-<i/ 15 (according to the general method of Doussineau, T et al, Synlett, 2004, (10): 1735- 1738) to provide methyl ester 44. As in Scheme 5, treatment of the ester with deuterated Grignard reagent 45 affords intermediate llc-i s- For example, use of commercially-available methyl-d3-magnesium iodide as Grignard reagent 45 affords

3 2

llc-i 8 wherein R and Y are simultaneously CD3.

20

Scheme 10. Synthesis of Intermediate llc-di.

25 Scheme 10 depicts a preparation of llc 3 2

-i 2, wherein R and Y are the same.

Thus, known deuterated ester 46 (Feldman, KS et al, Journal of Organic Chemistry, 2000, 65(25): 8659-8668) is treated with carbon tetrabromide and

triphenylphosphine (Brueckner, AM et al., European Journal of Organic Chemistry, 2003, (18): 3555-3561) to afford ester 47 wherein X is bromide, or is treated with

BOST 1635628.1 - 37 - methanesulfonyl chloride and triethylamine, followed by lithium chloride and DMF (Sagi, K et al, Bioorganic & Medicinal Chemistry, 2005, 13(5): 1487-1496) to afford ester 47 wherein X is chloride. As in Scheme 5, treatment of ester 47 with deuterated Grignard reagent 48 affords llc-i/2. For example, use of commercially- 5 available methyl-d3 -magnesium iodide as Grignard reagent 48 affords 11c- wherein R 3 and Y 2

d are simultaneously CD3.

Additional known chlorides that may be utilized as reagent 11 in Scheme 1 A include:

l-chloro-5,5-difluoro-hexane (Rybczynski, PJ et al, J Med Chemistry, 2004, 47(1): 10 196-209); l-chloro-5-fluorohexane (Chambers, RD et al., Tetrahedron, 2006,

62(30): 7162-7167); 6-chloro-2-hexanol (European Patent publication 0412596); (S)-6-chloro-2-hexanol (Keinan, E et al, J Am Chem Soc, 1986, 108(12): 3474- 3480); commercially-available (R)-6-chloro-2-hexanol; commercially available 6- chloro-2-hexanone; known 6-chloro-2-methylhexan-2-ol (Kutner, A et al., Journal 15 of Organic Chemistry, 1988, 53(15): 3450-7); known 6-bromo-2-methylhexan-2-ol (Kutner, A et al, Journal of Organic Chemistry, 1988, 53(15): 3450-7); known 1- bromo-5-fluoro-5-methylhexane (Hester, JB et al., Journal of Medicinal Chemistry, 2001, 44(7): 1099-1115).

20 Scheme 11. Synthesis of Compounds of Formula Al

Formula I Formula A1

Scheme 11 depicts the synthesis of a compound of Formula Al . Thus, a 25 compound of Formula I is treated with potassium carbonate in D ₂ 0 to effect a

hydrogen-to-deuterium exchange reaction, providing a compound of Formula Al . One skilled in the art will appreciate that additional hydrogen-to-deuterium exchange reactions may also occur elsewhere in the molecule.

BOST 1635628.1 38 - Scheme 12. Alternative Synthesis of Com ounds of Formula Al

10 50 Formula A1

An alternative synthesis of a compound of Formula Al is depicted in Scheme 12. Thus, intermediate 10 (cf. Scheme 1 A) is treated with potassium carbonate in D ₂ 0 to effect a hydrogen-to-deuterium exchange reaction, providing compound 50 as either the N-D or N-H species. Alkylation with intermediate 11 in the presence of potassium carbonate affords compounds of Formula Al .

Scheme 12b. Alternative Synthesis of Compounds of Formula A, wherein Y ¹ and

2

Y are each Fluorine.

Formula A, Formula A,

wherein -C(Y ¹ )(Y ² )- are carbonyl wherein Y ¹ and Y ² are each F

1 2

An alternative synthesis of compounds of Formula A, wherein Y and Y are 15 each fluorine, is depicted in Scheme 12b. Thus, a compound of Formula A, wherein

1 2

-C(Y )(Y )- is carbonyl, is treated with bis(2-methoxyethyl)aminosulfur trifluoride "Deoxo-fluor" and boron trifluoride etherate in dichloromethane to afford a

1 2

compound of Formula A, wherein Y and Y are each fluorine.

BOST 1635628.1 - 39 - Scheme 12c. Alternative Synthesis of Compounds of Formula A, wherein Y ¹ is

2

Fluorine and Y is not Fluorine.

Formula A, Formula A,

wherein Y ¹ is OH wherein Y ¹ is F and Y ² is not F

An alternative synthesis of compounds of Formula A, wherein Y is fluorine and Y is not fluorine, is depicted in Scheme 12c. Thus, a compound of Formula A, wherein Y ¹ is OH, is treated with bis(2-methoxyethyl)aminosulfur trifluoride "Deoxo-fluor" and boron trifluoride etherate in dichloromethane to afford a

1 2

compound of Formula A, wherein Y is fluorine and Y is not fluorine.

Scheme 12d. Alternative Synthesis of Compounds of Formula I.

Formula A1 Formula I

An alternative synthesis of compounds of Formula I is depicted in Scheme 12d. Thus, a compound of Formula Al is treated with potassium carbonate in water to effect a deuterium-to-hydrogen exchange, which affords a compound of Formula I.

A number of novel intermediates can be used to prepare compounds of Formula A. Thus, the invention also provides such a compound which is selected from the following:

BOST 1635628.1 - 40 -

BOST 1635628.1 -41 -

m

Compounds a-d above may be prepared as generally described in Org. Lett., 10 2005, 7: 1427-1429 using appropriately-deuterated starting materials. Compounds e- o may be prepared from the appropriate bromides listed above by reference to Scheme 15 shown below.

Certain xanthine intermediates useful for this invention are also novel. Thus, the invention provides a deuterated xanthine intermediate III:

15 III, where W is hydrogen or deuterium, and each of R ¹ and

R is independently selected from hydrogen, deuterium, Ci_ ₃ alkyl optionally substituted with deuterium, and Ci_ ₃ alkoxyalkyl optionally substituted with deuterium. Examples of R 1 and R 2 Ci_ ₃ alkyl include -CH ₃ , -CD ₃ , -CH ₂ CH ₂ CH ₃ , and -CD ₂ CD ₂ CD ₃ . Examples of Ci_ ₃ alkoxyalkyl include -CH ₂ OCH ₂ CH ₃ , - 20 CD ₂ OCH ₂ CH ₃ , -CD ₂ OCD ₂ CH ₃ , and -CD ₂ OCD ₂ CD ₃ .

Specific examples of formula III include the following:

BOST 1635628.1 - 42 - CH,

O ^' " ΊΜ ^{' ' N} O ^' " ΊΜ ^{' ' N} O ^' " ΊΜ ^{' ' N}

CH ₃ CD ₃ CD ₃

Ill-a, Ill-b, III-c,

Ill-d, Ill-e, Ill-f,

ni-g, Ill-h, Ill-i,

ni-j, Ill-k, and III-l

In each of the above examples of formula III, W is hydrogen. In a set of

corresponding examples, W is deuterium. Salts of compounds of Formula III are also useful, including salts that are known to be useful with respect to known 5 xanthines. Examples of useful salts include, but are not limited to, the lithium salt, sodium salt, potassium salt, and cesium salt. An example of a particularly useful salt is the potassium salt.

The specific approaches and compounds shown above are not intended to be 10 limiting. The chemical structures in the schemes herein depict variables that are hereby defined commensurately with chemical group definitions (moieties, atoms, etc.) of the corresponding position in the compound formulae herein, whether

1 2

identified by the same variable name (i.e., R , R , R 3 , etc.) or not. The suitability of

BOST 1635628.1 - 43 - a chemical group in a compound structure for use in the synthesis of another compound is within the knowledge of one of ordinary skill in the art.

Additional methods of synthesizing compounds of this invention and their synthetic precursors, including those within routes not explicitly shown in schemes herein, are 5 within the means of chemists of ordinary skill in the art. Synthetic chemistry

transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the applicable compounds are known in the art and include, for example, those described in Larock R, Comprehensive Organic Transformations, VCH Publishers (1989); Greene TW et al, Protective Groups in Organic Synthesis,

10 3 ^rd Ed., John Wiley and Sons (1999); Fieser L et al, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and Paquette L, ed.,

Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and subsequent editions thereof.

Combinations of substituents and variables envisioned by this invention are

15 only those that result in the formation of stable compounds.

COMPOSITIONS

The invention also provides pyrogen-free compositions comprising an effective amount of a compound of this invention or pharmaceutically acceptable

20 salts thereof; and an acceptable carrier. Preferably, a composition of this invention is formulated for pharmaceutical use ("a pharmaceutical composition"), wherein the carrier is a pharmaceutically acceptable carrier. The carrier(s) are "acceptable" in the sense of being compatible with the other ingredients of the formulation and, in the case of a pharmaceutically acceptable carrier, not deleterious to the recipient

25 thereof in an amount used in the medicament.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic

30 acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances,

BOST 1635628.1 . AA . polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

If required, the solubility and bioavailability of the compounds of the present invention in pharmaceutical compositions may be enhanced by methods well-known 5 in the art. One method includes the use of lipid excipients in the formulation. See "Oral Lipid-Based Formulations: Enhancing the Bioavailability of Poorly Water- Soluble Drugs (Drugs and the Pharmaceutical Sciences)," David J. Hauss, ed.

Informa Healthcare, 2007; and "Role of Lipid Excipients in Modifying Oral and Parenteral Drug Delivery: Basic Principles and Biological Examples," Kishor M.

10 Wasan, ed. Wiley-Interscience, 2006.

Another known method of enhancing bioavailability is the use of an amorphous form of a compound of this invention optionally formulated with a poloxamer, such as LUTROL™ and PLURONIC™ (BASF Corporation), or block copolymers of ethylene oxide and propylene oxide. See United States patent

15 7,014,866; and United States patent publications 20060094744 and 20060079502.

The pharmaceutical compositions of the invention include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous and intradermal)

administration. In certain embodiments, the compound of the formulae herein is

20 administered transdermally (e.g., using a transdermal patch or iontophoretic

techniques). Other formulations may conveniently be presented in unit dosage form, e.g., tablets, sustained release capsules, and in liposomes, and may be prepared by any methods well known in the art of pharmacy. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, PA (17th ed.

25 1985).

Such preparative methods include the step of bringing into association with the molecule to be administered ingredients such as the carrier that constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, 30 liposomes or finely divided solid carriers, or both, and then, if necessary, shaping the product.

In certain embodiments, the compound is administered orally. Compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, sachets, or tablets each containing a predetermined amount of

BOST 1635628.1 - 45 - the active ingredient; a powder or granules; a solution or a suspension in an aqueous liquid or a non-aqueous liquid; an oil-in-water liquid emulsion; a water-in-oil liquid emulsion; packed in liposomes; or as a bolus, etc. Soft gelatin capsules can be useful for containing such suspensions, which may beneficially increase the rate of 5 compound absorption.

In the case of tablets for oral use, carriers that are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are administered orally,

10 the active ingredient is combined with emulsifying and suspending agents. If

desired, certain sweetening and/or flavoring and/or coloring agents may be added.

Compositions suitable for oral administration include lozenges comprising the ingredients in a flavored basis, usually sucrose and acacia or tragacanth; and pastilles comprising the active ingredient in an inert basis such as gelatin and

15 glycerin, or sucrose and acacia.

Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may

20 include suspending agents and thickening agents. The formulations may be

presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared

25 from sterile powders, granules and tablets.

Such injection solutions may be in the form, for example, of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable

30 preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3- butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending

BOST 1635628.1 - 46 - medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically- acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated 5 versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant.

The pharmaceutical compositions of this invention may be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a compound of this invention with a suitable non-irritating

10 excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.

The pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques

15 well-known in the art of pharmaceutical formulation and may be prepared as

solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art. See, e.g.: Rabinowitz, JD and Zaffaroni, AC, US Patent 6,803,031, assigned to Alexza Molecular Delivery

20 Corporation.

Topical administration of the pharmaceutical compositions of this invention is especially useful when the desired treatment involves areas or organs readily accessible by topical application. For topical application topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment

25 containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax, and water.

Alternatively, the pharmaceutical composition can be formulated with a suitable

30 lotion or cream containing the active compound suspended or dissolved in a carrier.

Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. The pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable

BOST 1635628.1 _ 47 _ enema formulation. Topically-transdermal patches and iontophoretic administration are also included in this invention.

Application of the subject therapeutics may be local, so as to be administered at the site of interest. Various techniques can be used for providing the subject 5 compositions at the site of interest, such as injection, use of catheters, trocars,

projectiles, pluronic gel, stents, sustained drug release polymers or other device which provides for internal access.

Thus, according to yet another embodiment, the compounds of this invention may be incorporated into compositions for coating an implantable medical device,

10 such as prostheses, artificial valves, vascular grafts, stents, or catheters. Suitable coatings and the general preparation of coated implantable devices are known in the art and are exemplified in US Patents 6,099,562; 5,886,026; and 5,304,121. The coatings are typically biocompatible polymeric materials such as a hydrogel polymer, polymethyldisiloxane, polycaprolactone, polyethylene glycol, polylactic

15 acid, ethylene vinyl acetate, and mixtures thereof. The coatings may optionally be further covered by a suitable topcoat of fluorosilicone, polysaccharides,

polyethylene glycol, phospholipids or combinations thereof to impart controlled release characteristics in the composition. Coatings for invasive devices are to be included within the definition of pharmaceutically acceptable carrier, adjuvant or

20 vehicle, as those terms are used herein.

According to another embodiment, the invention provides a method of coating an implantable medical device comprising the step of contacting said device with the coating composition described above. It will be obvious to those skilled in the art that the coating of the device will occur prior to implantation into a mammal.

25 According to another embodiment, the invention provides a method of

impregnating an implantable drug release device comprising the step of contacting said drug release device with a compound or composition of this invention.

Implantable drug release devices include, but are not limited to, biodegradable polymer capsules or bullets, non-degradable, diffusible polymer capsules and

30 biodegradable polymer wafers.

According to another embodiment, the invention provides an implantable medical device coated with a compound or a composition comprising a compound of this invention, such that said compound is therapeutically active.

BOST 1635628.1 - 48 - According to another embodiment, the invention provides an implantable drug release device impregnated with or containing a compound or a composition comprising a compound of this invention, such that said compound is released from said device and is therapeutically active.

5 Where an organ or tissue is accessible because of removal from the patient, such organ or tissue may be bathed in a medium containing a composition of this invention, a composition of this invention may be painted onto the organ, or a composition of this invention may be applied in any other convenient way.

In another embodiment, a composition of this invention further comprises a

10 second therapeutic agent. The second therapeutic agent may be selected from any compound or therapeutic agent known to have or that demonstrates advantageous properties when administered with a compound having the same mechanism of action as pentoxifylline. Such agents include those indicated as being useful in combination with pentoxifylline, including but not limited to, those described in WO

15 1997019686, EP 0640342, WO 2003013568, WO 2001032156, WO 2006035418, and WO 1996005838.

Preferably, the second therapeutic agent is an agent useful in the treatment or prevention of a disease or condition selected from peripheral obstructive vascular disease; glomerulonephritis; nephrotic syndrome; nonalcoholic steatohepatitis;

20 Leishmaniasis; cirrhosis; liver failure; Duchenne's muscular dystrophy; late

radiation induced injuries; radiation induced lymphedema; radiation-associated necrosis; alcoholic hepatitis; radiation-associated fibrosis; necrotizing enterocolitis in premature neonates; diabetic nephropathy, hypertension-induced renal failure, and other chronic kidney disease; Focal Segmental Glomerulosclerosis; pulmonary

25 sarcoidosis; recurrent aphthous stomatitis; chronic breast pain in breast cancer

patients; brain and central nervous system tumors; malnutrition-inflammation- cachexia syndrome; interleukin-1 mediated disease; graft versus host reaction and other allograft reactions; diet-induced fatty liver conditions, atheromatous lesions, fatty liver degeneration and other diet-induced high fat or alcohol-induced tissue-

30 degenerative conditions; human immunodeficiency virus type 1 (HIV-1) and other human retroviral infections; multiple sclerosis; cancer; fibroproliferative diseases; fungal infection; drug-induced nephrotoxicity; collagenous colitis and other diseases and/or conditions characterized by elevated levels of platelet derived growth factor (PDGF) or other inflammatory cytokines; endometriosis; optic neuropathy and CNS

BOST 1635628.1 . AQ . impairments associated with acquired immunodeficiency syndrome (AIDS), immune disorder diseases, or multiple sclerosis; autoimmune disease; upper respiratory viral infection; depression; urinary incontinence; irritable bowel syndrome; septic shock; Alzheimers Dementia; neuropathic pain; dysuria; retinal or 5 optic nerve damage; peptic ulcer; insulin-dependent diabetes; non-insulin-dependent diabetes; diabetic nephropathy; metabolic syndrome; obesity; insulin resistance; dyslipidemia; pathological glucose tolerance; hypertension; hyperlipidemia;

hyperuricemia; gout; hypercoagulability; and inflammation or injury associated with neutrophil chemotaxis and/or degranulation. The compounds of this invention can 10 also be used to control intraocular pressure or to stabilize auto-regulation of cerebral blood flow in subjects who require such control as determined by medical examination.

In one embodiment, the second therapeutic agent is selected from a- tocopherol and hydroxyurea.

15 In another embodiment, the second therapeutic agent is useful in the

treatment of diabetes or an associated disorder, and is selected from insulin or insulin analogues, glucagon- like-peptide-1 (GLP-1) receptor agonists, sulfonylurea agents, biguanide agents, alpha-glucosidase inhibitors, PPAR agonists, meglitinide agents, dipeptidyl-peptidase (DPP) IV inhibitors, other phosphodiesterase (PDE1,

20 PDE5, PDE9, PDE10 or PDE1) inhibitors, amylin agonists, CoEnzyme A inhibitors, and antiobesity agents.

Specific examples of insulin include, but are not limited to Humulin® (human insulin, rDNA origin), Novolin® (human insulin, rDNA origin), Velosulin® BR (human buffered regular insulin, rDNA origin), Exubera® (human insulin,

25 inhaled), and other forms of inhaled insulin, for instance, as delivered by

Mannkind's "Technosphere Insulin System".

Specific examples of insulin analogues include, but are not limited to, novarapid, insulin detemir, insulin lispro, insulin glargine, insulin zinc suspension and Lys-Pro insulin.

30 Specific examples of Glucagon-Like -Peptide- 1 receptor agonists include, but are not limited to BIM-51077 (CAS-No. 275371-94-3), EXENATIDE (CAS-No. 141758-74-9), CJC-1131 (CAS-No. 532951 -64-7), LIRAGLUTIDE (CAS-No. 20656-20-2) and ZP-10 (CAS-No. 320367-13-3).

BOST 1635628.1 Specific examples of sulfonylurea agents include, but are not limited to, TOLBUTAMIDE (CAS- No. 000064-77-7), TOLAZAMIDE (CAS-No. 001156-19- 0), GLIPIZIDE (CAS-No. 029094-61-9), CARBUT AMIDE (CAS-No. 000339-43- 5), GLISOXEPIDE (CAS-No. 025046-79-1), GLISENTIDE (CAS-No. 032797-92- 5 5), GLIBORNURIDE (CAS-No. 026944-48-9), GLIBENCLAMIDE (CAS-NO.

010238-21 -8), GLIQUIDONE (CAS-No. 033342-05-1), GLIMEPIRIDE (CAS-No. 093479-97-1) and GLICLAZIDE (CAS-No. 021187-98-4).

A specific example of a biguanide agent includes, but is not limited to METFORMIN (CAS-No. 000657-24-9).

10 Specific examples of alpha-glucosidase-inhibitors include, but are not limited to ACARBOSE (Cas-No. 056180-94-0), MIGLITOL (CAS-No. 072432-03-2) and VOGLIBOSE (CAS-No. 083480-29-9).

Specific examples of PPAR-agonists include, but are not limited to

MURAGLITAZAR (CAS-No. 331741 -94-7), ROSIGLITAZONE (CAS-NO.

15 122320-73-4), PIOGLITAZONE (CAS-No.111025-46-8), RAGAGLITAZAR

(CAS-NO. 222834-30-2), FARGLITAZAR (CAS-No. 196808-45-4),

TESAGLITAZAR (CAS- No. 251565-85-2), NAVEGLITAZAR (CAS-No.

476436-68-7), NETOGLITAZONE (CAS-NO. 161600-01 -7), RIVOGLITAZONE (CAS-NO. 185428-18-6), K-l 11 (CAS-No. 221564-97-2), GW-677954 (CAS-No.

20 622402-24-8), FK-614 (CAS-No 193012-35-0) and (-)-Halofenate (CAS-No.

024136-23-0). Preferred PPAR- agonists are ROSGLITAZONE and

PIOGLITAZONE.

Specific examples of meglitinide agents include, but are not limited to REPAGLINIDE (CAS-No. 135062-02-1 ), NATEGLINIDE (CAS-No. 105816-04-

25 4) and MITIGLINIDE (CAS-No. 145375-43-5).

Specific examples of DPP IV inhibitors include, but are not limited to SITAGLIPTIN (CAS-No. 486460-32-6), SAXAGLIPTIN (CAS-No. 361442-04-8), VILDAGLIPTIN (CAS-No. 274901 -16-5), DENAGLIPTIN (CAS-No. 483369-58- 0), P32/98 (CAS-No. 251572-70-0) and NVP-DPP-728 (CAS-No. 247016-69-9).

30 Specific examples of PDE5 inhibitors include, but are not limited to

SILDENAFIL (CAS-No. 139755-83-2), VARDENAFIL (CAS-No. 224785-90-4) and TADALAFIL (CAS-No. 171596-29-5). Examples of PDE1, PDE9, PDE10 or PDE11 inhibitors which may be usefully employed according to the present invention can be found, for example, in US20020160939, WO2003037432,

BOST 1635628.1 - 51 - US2004220186, WO2005/003129, WO2005012485, WO2005120514 and

WO03077949.

A specific example of an amylin agonist includes, but is not limited to PRAMLINITIDE (CAS-No. 151126-32-8).

5 A specific example of a Coenzyme A inhibitor includes, but is not limited to

ETOMOXIR (CAS- No. 082258-36-4).

Specific examples of anti-obesity drugs include, but are not limited to HMR- 1426 (CAS-No. 262376-75-0), CETILISTAT (CAS-No. 282526-98-1) and

SIBUTRAMINE (CAS-No. 106650-56-0).

10 In another embodiment, the invention provides separate dosage forms of a compound of this invention and one or more of any of the above-described second therapeutic agents, wherein the compound and second therapeutic agent are associated with one another. The term "associated with one another" as used herein means that the separate dosage forms are packaged together or otherwise attached to

15 one another such that it is readily apparent that the separate dosage forms are

intended to be sold and administered together (within less than 24 hours of one another, consecutively or simultaneously).

In the pharmaceutical compositions of the invention, the compound of the present invention is present in an effective amount. As used herein, the term

20 "effective amount" refers to an amount which, when administered in a proper dosing regimen, is sufficient to treat (therapeutically or prophylactically) the target disorder. For example, and effective amount is sufficient to reduce or ameliorate the severity, duration or progression of the disorder being treated, prevent the advancement of the disorder being treated, cause the regression of the disorder being treated, or enhance

25 or improve the prophylactic or therapeutic effect(s) of another therapy.

The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described in Freireich et al., Cancer Chemother. Rep, 1966, 50: 219. Body surface area may be determined

approximately from height and weight of the patient. See, e.g., Scientific Tables,

30 Geigy Pharmaceuticals, Ardsley, N.Y., 1970, 537.

In one embodiment, an effective amount of a compound of this invention is in the range of 20 mg to 2000 mg per treatment. In more specific embodiments the amount is in the range of 40 mg to 1000 mg, or in the range of 100 mg to 800 mg, or

BOST 1635628.1 _ _ more specifically in the range of 200 mg to 400 mg per treatment. Treatment typically is administered from one to three times daily.

Effective doses will also vary, as recognized by those skilled in the art, depending on the diseases treated, the severity of the disease, the route of

5 administration, the sex, age and general health condition of the patient, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents and the judgment of the treating physician. For example, guidance for selecting an effective dose can be determined by reference to the prescribing information for pentoxifylline.

10 For pharmaceutical compositions that comprise a second therapeutic agent, an effective amount of the second therapeutic agent is between about 20% and 100% of the dosage normally utilized in a monotherapy regime using just that agent.

Preferably, an effective amount is between about 70% and 100% of the normal monotherapeutic dose. The normal monotherapeutic dosages of these second

15 therapeutic agents are well known in the art. See, e.g., Wells et al, eds.,

Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000), each of which references are incorporated herein by reference in their entirety.

20 It is expected that some of the second therapeutic agents referenced above will act synergistically with the compounds of this invention. When this occurs, it will allow the effective dosage of the second therapeutic agent and/or the compound of this invention to be reduced from that required in a monotherapy. This has the advantage of minimizing toxic side effects of either the second therapeutic agent of a

25 compound of this invention, synergistic improvements in efficacy, improved ease of administration or use and/or reduced overall expense of compound preparation or formulation.

METHODS OF TREATMENT

30 In one embodiment, the invention provides a method of inhibiting the

activity of phosphodiesterase (PDE) in a cell, comprising contacting a cell with one or more compounds of Formula A, Al, I, II, B, C, D(i) or D(ii).

In addition to its PDE inhibitory activity, pentoxifylline is known to suppress the production of a number of other biological agents such as interleukin-1 (IL-1),

BOST 1635628.1 _ ^ _ IL-6, IL-12, TNF-alpha, fibrinogen, and various growth factors. Accordingly, in another embodiment, the invention provides a method of suppressing the production of interleukin-1 (IL-1), IL-6, IL-12, TNF-alpha, fibrinogen, and various growth factors in a cell, comprising contacting a cell with one or more compounds of 5 Formula A, Al , I, II or B.

According to another embodiment, the invention provides a method of treating a disease in a patient in need thereof that is beneficially treated by pentoxifylline comprising the step of administering to said patient an effective amount of a compound of Formula A, Al, I, II, B, C, D(i) or D(ii) or a

10 pharmaceutical composition comprising a compound of Formula A, Al, I, II, B, C, D(i) or D(ii) and a pharmaceutically acceptable carrier.

Such diseases are well known in the art and are disclosed in, but not limited to the following patents and published applications: WO 1988004928, EP 0493682, US 5112827, EP 0484785, WO 1997019686, WO 2003013568, WO 2001032156,

15 WO 1992007566, WO 1998055110, WO 2005023193, US 4975432, WO

1993018770, EP 0490181, and WO 1996005836. Such diseases include, but are not limited to, peripheral obstructive vascular disease; glomerulonephritis; nephrotic syndrome; nonalcoholic steatohepatitis; Leishmaniasis; cirrhosis; liver failure; Duchenne's muscular dystrophy; late radiation induced injuries; radiation induced

20 lymphedema; radiation-associated necrosis; alcoholic hepatitis; radiation-associated fibrosis; necrotizing enterocolitis in premature neonates; diabetic nephropathy, hypertension-induced renal failure, and other chronic kidney disease; Focal

Segmental Glomerulosclerosis; pulmonary sarcoidosis; recurrent aphthous stomatitis; chronic breast pain in breast cancer patients; brain and central nervous

25 system tumors; malnutrition-inflammation-cachexia syndrome; interleukin-1

mediated disease; graft versus host reaction and other allograft reactions; diet- induced fatty liver conditions, atheromatous lesions, fatty liver degeneration and other diet-induced high fat or alcohol-induced tissue-degenerative conditions;

human immunodeficiency virus type 1 (HIV-1) and other human retroviral

30 infections; multiple sclerosis; cancer; fibroproliferative diseases; fungal infection; drug-induced nephrotoxicity; collagenous colitis and other diseases and/or conditions characterized by elevated levels of platelet derived growth factor (PDGF) or other inflammatory cytokines; endometriosis; optic neuropathy and CNS impairments associated with acquired immunodeficiency syndrome (AIDS),

BOST 1635628.1 immune disorder diseases, or multiple sclerosis; autoimmune disease; upper respiratory viral infection; depression; urinary incontinence; irritable bowel syndrome; septic shock; Alzheimers Dementia; neuropathic pain; dysuria; retinal or optic nerve damage; peptic ulcer; insulin-dependent diabetes; non-insulin-dependent 5 diabetes; diabetic nephropathy; metabolic syndrome; obesity; insulin resistance; dyslipidemia; pathological glucose tolerance; hypertension; hyperlipidemia;

hyperuricemia; gout; hypercoagulability; acute alcoholic hepatitis; olfaction disorders; patent ductus arteriosus; and inflammation or injury associated with neutrophil chemotaxis and/or degranulation.

10 The compounds of Formula A, Al, I, II, B, C, D(i) or D(ii) can also be used to control intraocular pressure or to stabilize auto-regulation of cerebral blood flow in subjects who require such control as determined by medical examination.

In one particular embodiment, the method of this invention is used to treat a disease or condition in a patient in need thereof selected from intermittent

15 claudication on the basis of chronic occlusive arterial disease of the limbs and other peripheral obstructive vascular diseases; glomerulonephritis; Focal Segmental Glomerulosclerosis; nephrotic syndrome; nonalcoholic steatohepatitis;

Leishmaniasis; cirrhosis; liver failure; Duchenne's muscular dystrophy; late radiation induced injuries; radiation induced lymphedema; alcoholic hepatitis;

20 radiation-induced fibrosis; necrotizing enterocolitis in premature neonates; diabetic nephropathy, hypertension-induced renal failure and other chronic kidney diseases; pulmonary sarcoidosis; recurrent aphthous stomatitis; chronic breast pain in breast cancer patients; brain and central nervous system tumors; obesity; acute alcoholic hepatitis; olfaction disorders; endometriosis-associated infertility; malnutrition-

25 inflammation-cachexia syndrome; and patent ductus arteriosus.

In one embodiment, the method of this invention is used to treat diabetic nephropathy, hypertensive nephropathy or intermittent claudication on the basis of chronic occlusive arterial disease of the limbs. In another particular embodiment, the method of this invention is used to treat a disease or condition in a patient in

30 need thereof selected from intermittent claudication on the basis of chronic occlusive arterial disease of the limbs.

In one embodiment, the method of this invention is used to treat chronic kidney disease. The chronic kidney disease may be selected from

glomerulonephritis, focal segmental glomerulosclerosis, nephrotic syndrome, reflux

BOST 1635628.1 _ _ uropathy, or polycystic kidney disease.

In one embodiment, the method of this invention is used to treat chronic disease of the liver. The chronic disease of the liver may be selected from nonalcoholic steatohepatitis, fatty liver degeneration or other diet-induced high fat or 5 alcohol-induced tissue-degenerative conditions, cirrhosis, liver failure, or alcoholic hepatitis.

In one embodiment, the method of this invention is used to a diabetes-related disease or condition. This disease may be selected from insulin resistance, retinopathy, diabetic ulcers, radiation-associated necrosis, acute kidney failure or 10 drug-induced nephrotoxicity.

In one embodiment, the method of this invention is used to treat a patient suffering from cystic fibrosis, including those patients suffering from chronic Pseudomonas bronchitis.

In one embodiment, the method of this invention is used to aid in wound 15 healing. Examples of types of wounds that may be treated include venous ulcers, diabetic ulcers and pressure ulcers.

In another particular embodiment, the method of this invention is used to treat a disease or condition in a patient in need thereof selected from insulin dependent diabetes; non-insulin dependent diabetes; metabolic syndrome; obesity; 20 insulin resistance; dyslipidemia; pathological glucose tolerance; hypertension;

hyperlipidemia; hyperuricemia; gout; and hypercoagulability.

In one embodiment, the method of this invention is used to treat a disease or condition in a patient in need thereof wherein the disease or condition is selected from anemia, Graves disease, retinal vein occlusion, lupus nephritis, macular 25 degeneration, myelodysplasia, pruritis of HIV origin, pulmonary hypertension,

retinal artery occlusion, intestinal inflammation, ischemic optic neuropathy, acute pancreatitis, sickle cell anemia and beta thalassemia.

Methods delineated herein also include those wherein the patient is identified as in need of a particular stated treatment. Identifying a patient in need of such 30 treatment can be in the judgment of a patient or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).

In another embodiment, any of the above methods of treatment comprises the further step of co-administering to the patient one or more second therapeutic agents.

BOST 1635628.1 The choice of second therapeutic agent may be made from any second therapeutic agent known to be useful for co-administration with pentoxifylline. The choice of second therapeutic agent is also dependent upon the particular disease or condition to be treated. Examples of second therapeutic agents that may be employed in the 5 methods of this invention are those set forth above for use in combination

compositions comprising a compound of this invention and a second therapeutic agent.

In particular, the combination therapies of this invention include coadministering a compound of Formula A, Al, I, II, B, C, D(i) or D(ii) and a second

10 therapeutic agent for treatment of the following conditions (with the particular

second therapeutic agent indicated in parentheses following the indication): late radiation induced injuries (a-tocopherol), radiation-induced fibrosis (a-tocopherol), radiation induced lymphedema (a-tocopherol), chronic breast pain in breast cancer patients (a-tocopherol), type 2 diabetic nephropathy (captopril), malnutrition-

15 inflammation-cachexia syndrome (oral nutritional supplement, such as Nepro; and oral anti-inflammatory module, such as Oxepa); and brain and central nervous system tumors (radiation therapy and hydroxyurea).

The combination therapies of this invention also include co-administering a compound of Formula A, Al, I, II, B, C, D(i) or D(ii) and a second therapeutic

20 agent for treatment of insulin dependent diabetes; non-insulin dependent diabetes; metabolic syndrome; obesity; insulin resistance; dyslipidemia; pathological glucose tolerance; hypertension; hyperlipidemia; hyperuricemia; gout; and

hyp erco agul ability .

The term "co-administered" as used herein means that the second therapeutic 25 agent may be administered together with a compound of this invention as part of a single dosage form (such as a composition of this invention comprising a compound of the invention and an second therapeutic agent as described above) or as separate, multiple dosage forms. Alternatively, the additional agent may be administered prior to, consecutively with, or following the administration of a compound of this 30 invention. In such combination therapy treatment, both the compounds of this

invention and the second therapeutic agent(s) are administered by conventional methods. The administration of a composition of this invention, comprising both a compound of the invention and a second therapeutic agent, to a patient does not preclude the separate administration of that same therapeutic agent, any other second

BOST 1635628.1 therapeutic agent or any compound of this invention to said patient at another time during a course of treatment.

Effective amounts of these second therapeutic agents are well known to those skilled in the art and guidance for dosing may be found in patents and published 5 patent applications referenced herein, as well as in Wells et al, eds.,

Pharmacotherapy Handbook, 2nd Edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000), and other medical texts. However, it is well within the skilled artisan's purview to determine the second

10 therapeutic agent's optimal effective-amount range.

In one embodiment of the invention, where a second therapeutic agent is administered to a subject, the effective amount of the compound of this invention is less than its effective amount would be where the second therapeutic agent is not administered. In another embodiment, the effective amount of the second

15 therapeutic agent is less than its effective amount would be where the compound of this invention is not administered. In this way, undesired side effects associated with high doses of either agent may be minimized. Other potential advantages (including without limitation improved dosing regimens and/or reduced drug cost) will be apparent to those of skill in the art.

20 In yet another aspect, the invention provides the use of a compound of

Formula A, Al, I, II, B, C, D(i) or D(ii) alone or together with one or more of the above-described second therapeutic agents in the manufacture of a medicament, either as a single composition or as separate dosage forms, for treatment or prevention in a patient of a disease, disorder or symptom set forth above. Another

25 aspect of the invention is a compound of Formula A, Al, I, II, B, C, D(i) or D(ii) for use in the treatment or prevention in a patient of a disease, disorder or symptom thereof delineated herein.

DIAGNOSTIC METHODS AND KITS

30 The present invention also provides kits for use to treat peripheral obstructive vascular disease, in particular intermittent claudication on the basis of chronic occlusive arterial disease of the limbs; glomerulonephritis; nephrotic syndrome; nonalcoholic steatohepatitis; Leishmaniasis; cirrhosis; liver failure; Duchenne's muscular dystrophy; late radiation induced injuries; radiation induced lymphedema;

BOST 1635628.1 alcoholic hepatitis; radiation fibrosis; necrotizing enterocolitis in premature neonates; chronic kidney disease; pulmonary sarcoidosis; recurrent aphthous stomatitis; chronic breast pain in breast cancer patients; brain and central nervous system tumors; malnutrition-inflammation-cachexia syndrome; insulin dependent 5 diabetes; non-insulin dependent diabetes; metabolic syndrome; obesity; insulin

resistance; dyslipidemia; pathological glucose tolerance; hypertension;

hyperlipidemia; hyperuricemia; gout; and hypercoagulability. These kits comprise (a) a pharmaceutical composition comprising a compound of Formula A, Al, I, II, B, C, D(i) or D(ii) or a salt thereof, wherein said pharmaceutical composition is in a

10 container; and (b) instructions describing a method of using the pharmaceutical composition to treat peripheral obstructive vascular disease, in particular intermittent claudication on the basis of chronic occlusive arterial disease of the limbs;

glomerulonephritis; nephrotic syndrome; nonalcoholic steatohepatitis;

Leishmaniasis; cirrhosis; liver failure; Duchenne's muscular dystrophy; late

15 radiation induced injuries; radiation induced lymphedema; alcoholic hepatitis;

radiation fibrosis; necrotizing enterocolitis in premature neonates; chronic kidney disease; pulmonary sarcoidosis; recurrent aphthous stomatitis; chronic breast pain in breast cancer patients; brain and central nervous system tumors; malnutrition- inflammation-cachexia syndrome; insulin dependent diabetes; non-insulin dependent

20 diabetes; metabolic syndrome; obesity; insulin resistance; dyslipidemia; pathological glucose tolerance; hypertension; hyperlipidemia; hyperuricemia; gout; and hyp erco agul ability .

The container may be any vessel or other sealed or sealable apparatus that can hold said pharmaceutical composition. Examples include bottles, ampules,

25 divided or multi-chambered holders bottles, wherein each division or chamber

comprises a single dose of said composition, a divided foil packet wherein each division comprises a single dose of said composition, or a dispenser that dispenses single doses of said composition. The container can be in any conventional shape or form as known in the art which is made of a pharmaceutically acceptable material,

30 for example a paper or cardboard box, a glass or plastic bottle or jar, a re-sealable bag (for example, to hold a "refill" of tablets for placement into a different container), or a blister pack with individual doses for pressing out of the pack according to a therapeutic schedule. The container employed can depend on the exact dosage form involved, for example a conventional cardboard box would not

BOST 1635628.1 generally be used to hold a liquid suspension. It is feasible that more than one container can be used together in a single package to market a single dosage form. For example, tablets may be contained in a bottle, which is in turn contained within a box. In one embodiment, the container is a blister pack.

5 The kits of this invention may also comprise a device to administer or to measure out a unit dose of the pharmaceutical composition. Such device may include an inhaler if said composition is an inhalable composition; a syringe and needle if said composition is an injectable composition; a syringe, spoon, pump, or a vessel with or without volume markings if said composition is an oral liquid

10 composition; or any other measuring or delivery device appropriate to the dosage formulation of the composition present in the kit.

In certain embodiment, the kits of this invention may comprise in a separate vessel of container a pharmaceutical composition comprising a second therapeutic agent, such as one of those listed above for use for co-administration with a

15 compound of this invention.

SYNTHETIC EXAMPLES

The synthetic examples below provide detailed procedures for making certain compounds of this invention. It will be apparent to one skilled in the art that

20 further compounds of this invention may be prepared through the use of other

reagents or intermediates by reference to these procedures and the schemes described above. The prepared compounds were analyzed by NMR, mass spectrometry, and/or elemental analysis as indicated. ¹ HNMR were taken on a 300 MHz instrument, which was useful for determining deuterium incorporation. Unless

25 otherwise stated, the absence of an NMR signal as noted in the examples below

indicates a level of deuterium incorporation that is at least 90%.

BOST 1635628.1 Example 1. Synthesis of 3-Methyl-7-(methyl-<ij)- 1 -(5-oxohexyD- 1H- purine-2,6(3H,7H)-dione (Compound 100).

Scheme 13. Preparation of Compounds 100 and 409.

Step 1. 3-Methyl-7-(methyl- j -lH-purine-2,6(3HJH)-dione (51). A suspension of 3-methylxanthine 50 (5.0 g, 30.1 mmol, 1 equiv) and powdered

10 K ₂ C0 ₃ (5.0 g, 36.0 mmol, 1.2 equiv) in DMF (95 mL) was heated to 60 °C and iodomethane-d3 (Cambridge Isotopes, 99.5 atom% D, 2.2 mL, 36.0 mmol, 1.2 equiv) was added via syringe. The resulting mixture was heated at 80 °C for 5 hours (h). The reaction mixture was cooled to room temperature (rt) and the DMF was evaporated under reduced pressure. The crude residue was dissolved in 5% aqueous

15 NaOH (50 mL), resulting in a dull yellow solution. The aqueous solution was

washed with DCM three times (500 mL total). The aqueous layer was acidified to pH 5 with acetic acid (6 mL), resulting in formation of a tan precipitate. The mixture was cooled in an ice-water bath, and the solids were filtered and washed with cold water. The solid was dried in a vacuum oven to give 2.9 g of 51 as a tan solid. The

20 filtrate was concentrated to approximately 25 mL and a second crop (0.70 g) of 51 was collected by filtration. The total yield of 51 was 3.6 g. The crude material was used without further purification.

Step 2. 3-Methyl-7-(methyl- j -l-(5-oxohexyn-lH-purine-2,6(3HJH)- dione (Compound 100). Crude 51 (1.50 g, 8.2 mmol, 1 equiv) and powdered K ₂ CO ₃

25 (2.28 g, 16.4 mmol, 2 equiv) were suspended in DMF (30 mL) and heated to 50 °C.

To the resulting tan suspension was added 6-chloro-2-hexanone (52, 1.2 mL, 9.0

BOST 1635628.1 - 61 - mmol, 1.1 equiv) and the reaction temperature was raised to 130 °C. Heating was continued at 130 °C for 2 h, during which time the suspension became finer and darker in color. The reaction mixture was cooled to rt and DMF was evaporated under reduced pressure. The residual tan paste was suspended in EtOAc (250 mL) 5 and filtered to remove insoluble material. The filtrate was concentrated under

reduced pressure resulting in a yellow oil. The crude product was purified using an Analogix chromatography system eluting with 100% EtOAc (10 minutes) followed by a gradient of 0 to 25% MeOH/EtOAc over 50 minutes (min). Product fractions were concentrated under reduced pressure to give a slightly yellow oil that solidified

10 after standing for several minutes. The solid was triturated with heptanes (100 mL) and filtered to give 2.00 g of 100 as an off-white solid, mp 101.8-103.0 °C. 1H- NMPv (300 MHz, CDC1 ₃ ): δ 1.64-1.68 (m, 4H), 2.15 (s, 3H), 2.51 (t, J = 7.0, 2H), 3.57 (s, 3H), 4.01 (t, J = 7.0, 2H), 7.52 (s, 1H). ¹³ C-NMR (75 MHz, CDC1 ₃ ): δ 20.95, 27.41, 29.69, 29.98, 40.80, 43.18, 107.63, 141.41, 148.75, 151.45, 155.26,

15 208.80. HPLC (method: 20 mm C18-RP column - gradient method 2 to 95% ACN + 0.1% formic acid in 3.3 min with 1.7 min hold at 95% ACN; Wavelength: 254 nm): retention time: 2.54 min; 98.5% purity. MS (M+H): 282.0. Elemental Analysis Calculated: C=55.50, H=6.45, N=19.92. Found:

C=55.58, H=6.48, N=19.76.

20 Due to the presence of a triplet at 4.01 ppm in the above ¹ H-NMR spectrum, determination of the presence or absence of a singlet peak at around 3.99 ppm corresponding to the presence or absence of hydrogens on the N-methyl group at the 7 position (R ¹ ) of the purine ring was not possible.

25

Example 2. Synthesis of 8- 3-methyl-7-(methyl- j)-l-(6- j-4- 7-5- oxohexyl)-lH-purine-2,6(3H,7H)-dione (Compound 409).

8- y_-3-methyl-7-(methyl- j)-l-(6-(ij-4-(i?-5-oxohexyn-lH-purine- 2.6(3H.7H -dione (Compound 409). A suspension of 100 (1.80 g, 6.4 mmol, 1 30 equiv) and powdered K ₂ CO ₃ (0.23 g, 1.7 mmol, 0.25 equiv) in D ₂ 0 (Cambridge Isotope Labs, 99 atom%> D) (45 mL) was stirred under reflux conditions for 24 h during which time the suspension became a slightly yellow solution. The reaction mixture was cooled to rt, saturated with sodium chloride, and extracted four times

BOST 1635628.1 - 62 - with dichloromethane (400 mL total). The combined organic solution was dried over Na ₂ S0 ₄ , filtered, and evaporated under reduced pressure to provide 1.7 g of a slightly yellow oil that solidified upon standing. The crude material was re-subjected to the hydrogen/deuterium exchange conditions described above with fresh K ₂ CO ₃ 5 and D ₂ 0. After an identical workup, the off-white solid was triturated with hexanes (100 mL) and filtered to give 1.61 g of 409 as an off white solid, mp 99.6-99.8 °C. 1H-NMR (300 MHz, CDC1 ₃ ): δ 1.64-1.69 (m, 4H), 3.57 (s, 3H), 4.01 (t, J = 7.0, 2H). 1 ³ C-NMR (75 MHz, CDCI ₃ ): δ 21.05, 27.61, 29.90, 41.02, 107.83, 148.99, 151.69, 155.50, 209.28. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη CI 8-RP 10 column - gradient method 5-95% ACN + 0.1% formic acid in 14 min (1.0 mL/min) with 4 min hold at 95% ACN; Wavelength: 254 nm): retention time: 3.26 min; 98% purity. MS (M+H): 288.3. Elemental Analysis (CisHpDpN^s): Calculated:

C=54.35, H=6.31, N=19.50. Found: C=54.36, H=6.32, N=19.10.

Notable in the H-NMR spectrum above was the absence of the following

15 peaks: a singlet at around 2.15 ppm indicating an absence of methyl ketone

hydrogens; a triplet at around 2.51 ppm indicating an absence of methylene ketone hydrogens; and a singlet at around 7.52 ppm indicating an absence of hydrogen at the number 8 position on the purine ring. Due to the presence of a triplet at 4.01 ppm in the above ¹ H-NMR spectrum, determination of the presence or absence of a

20 singlet peak at around 3.99 ppm corresponding to the presence or absence of

hydrogens on the N-methyl group at the 7 position (R ¹ ) of the purine ring was not possible.

25 Example 3. Synthesis of 3,7-Di(methyl-6 -l-(5-oxohexyl)-lH-purine-

2,6(3H,7H)-dione (Compound 101).

Scheme 14. Preparation of Compounds 101 and 413.

BOST 1635628.1 - 63 -

Step 1. 3,7-Di(methyl-(ij)-lH-purine-2,6(3H,7H)-dione (55). A suspension of xanthine 53 (2.00 g, 13.2 mmol, 1.0 equiv) and hexamethyldisilazane (32 mL) in toluene (60 mL) was heated to reflux and stirred for 4 days. The reaction mixture 5 was cooled to room temperature, diluted with additional toluene (50 mL) and filtered through Celite to remove any unreacted starting material. The filtrate was evaporated to dryness under reduced pressure to produce 54 as a white solid (4.1 g). A portion of this material (3.00 g) was placed in a 100 mL sealed tube reaction vessel, followed by the addition of toluene (60 mL) and CD ₃ I (4 mL, Cambridge

10 Isotopes, 99.5 atom% D). The reaction mixture was heated in a 120 °C oil bath and stirred for 24 hours, during which time the reaction mixture turned yellow and a solid formed. The reaction mixture was cooled to room temperature, resulting in the entire reaction mixture solidifying to a yellow solid. The mixture was diluted with acetone (30 mL) and MeOH (5 mL) and filtered under a stream of N ₂ . The solids

15 were washed with acetone (100 mL) which removed the yellow color to afford an off- white solid. The solid was dried on the filter under a stream of N ₂ to give a mixture of 55 and monoalkylated side product, 7-(methyl-<¾)-xanthine in a roughly 1 : 1 ratio. Total mass recovery was 2.6 g (42% crude yield). Due to the poor solubility of this mixture, it was carried forward without further purification.

20 Step 2. 3,7-Di(methyl- ^-l-(5-oxohexyn-lH-purine-2,6(3H,7H)-dione

(Compound 101). A suspension of crude 55 (2.50 g, 13.4 mmol, 1.0 equiv) and powdered K ₂ C0 ₃ (2.20 g, 16 mmol, 1.2 equiv) in DMF (50 mL) was heated to 60 °C. To the resulting tan suspension was added 6-chloro-2-hexanone 52 (2.0 mL, 14.8 mmol, 1.1 equiv) and the mixture was heated to 140 °C. Heating was continued

25 at 140 °C for 4 hours during which time the suspension became finer and darker in color. The reaction mixture was cooled to room temperature and the DMF was evaporated under reduced pressure. The resulting tan paste was suspended in 1 : 1 dichloromethane/ethyl acetate (200 mL) and filtered to remove insoluble material. The filtrate was concentrated under reduced pressure giving a yellowish-brown oil

30 (3.0 g). This crude reaction product was adsorbed onto silica gel and dry-loaded onto

BOST 1635628.1 - 64 - a silica gel column packed with 100% dichloromethane. The column was eluted with a gradient of 0-5% MeOH/dichloromethane. Fractions containing product were concentrated under reduced pressure to give 0.75 g of a yellow oil. LCMS showed the material to be about 90% pure. The yellow oil was further purified using an 5 Analogix chromatography system eluting initially with 60% EtO Ac/heptanes

followed by a gradient of 60-100% EtO Ac/heptanes over 20 min. The desired product eluted at about 20 minutes. Fractions containing product were concentrated under reduced pressure to give 0.55 g (16%>) of Compound 101 as a slightly yellow oil which solidified upon standing. 1H-NMR (300 MHz, CDC1 ₃ ): δ 1.64-1.69 (m,

10 4H), 2.15 (s, 3H), 2.51 (t, J = 7.0, 2H), 4.02 (t, J = 7.0, 2H), 7.51 (s, 1H). ¹³ C-NMR (75 MHz, CDC1 ₃ ): δ 20.97, 27.43, 29.97, 40.80, 43.19, 107.64, 141.40, 148.78, 151.48, 155.29, 208.77. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 min (1.0 mL/min) with 4 min hold at 95% ACN + 0.1%> formic acid; Wavelength: 305 nm):

15 retention time: 3.24 min; 98.6% purity. MS (M+H): 285.3, (M+Na): 307.2.

Elemental Analysis (Ci ₃ Hi ₂ D ₆ N ₄ 0 ₃ ): Calculated: C=54.92, H=6.38, N=19.71. Found: C=54.90, H=6.40, N=19.50.

Notable in the ¹ H-NMR spectrum above was the absence of a singlet at around 3.57 ppm indicating an absence of N-methyl hydrogens at the 3 position of

20 the purine ring. Due to the presence of a triplet at 4.01 ppm in the above ¹ H-NMR spectrum, determination of the presence or absence of a singlet peak at around 3.99 ppm corresponding to the presence or absence of hydrogens on the N-methyl group at the 7 position (R ¹ ) of the purine ring was not possible.

25 Example 4. Synthesis of 8- -3J-Di(methyl-^Vl-(4,4,6.6.6-^-5- oxohexyl)-lH-purine-2,6(3HJH)-dione (Compound 413).

8- -3J-Di(methyl- j)-l-(4- ₂ -6-^-5-oxohexyl)-lH-purine-2.6(3HJH)- dione (Compound 413). A suspension of Compound 101 (0.60 g, 2.1 mmol, 1.0 equiv) and powdered K ₂ C0 ₃ (0.10 g, 0.72 mmol, 0.30 equiv) in D ₂ 0 (15 mL,

30 Cambridge Isotopes, 99 atom%> D) was heated and stirred at reflux for 16 hours during which time the suspension became a slightly yellow solution. The reaction mixture was cooled to room temperature, saturated with sodium chloride, and extracted four times with dichloromethane (200 mL). The combined organic extracts were dried over Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure to

BOST 1635628.1 - 65 - provide 0.53 g of a slightly yellow oil that solidified upon standing. The crude reaction product was re-subjected to the above reaction conditions with fresh powdered K ₂ CO ₃ and D ₂ 0. After an identical workup, the off-white solid was triturated with hexanes (50 mL) and filtered to give 0.45 g (74%) of Compound 413 5 as an off-white solid, mp 99.2-99.3 °C. 1H-NMR (300 MHz, CDCI ₃ ): δ 1.64-1.71 (m, 4H), 4.01 (t, J = 7.0, 2H). ¹³ C-NMR (75 MHz, CDC1 ₃ ): δ 20.85, 27.41, 40.81, 107.63, 148.80, 151.50, 155.31, 209.09. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 minutes (1.0 mL/min) with a 4 minute hold at 95% ACN + 0.1% formic acid;

10 Wavelength: 254 nm): retention time: 3.25 min; 98.7% purity. MS (M+H): 291.3, (M+Na): 313.2. Elemental Analysis (Ci3H ₆ Di ₂ N ₄ 0 ₃ ): Calculated: C=53.78, H=6.25, N=19.30. Found: C=53.76, H=6.39, N=19.11.

Notable in the ¹ H-NMR spectrum above was the absence of the following peaks: a singlet at around 2.15 ppm indicating an absence of methyl ketone

15 hydrogens; a triplet at around 2.51 ppm indicating an absence of methylene ketone hydrogens; a singlet around 3.57 ppm indicating an absence of N-methyl hydrogens at the 3 position on the purine ring; and a singlet at around 7.51 ppm indicating an absence of hydrogen at the number 8 position on the purine ring. Due to the presence of a triplet at 4.01 ppm in the above ¹ H-NMR spectrum, determination of

20 the presence or absence of a singlet peak at around 3.99 ppm corresponding to the presence or absence of hydrogens on the N-methyl group at the 7 position (R ¹ ) of the purine ring was not possible.

Example 5. Synthesis of 3-Methyl-7-(methyl- j)-l-(6,6,6- j-5-oxohexyl)- 25 lH-purine-2,6(3H,7H)-dione (Compound 99).

Scheme 15. Preparation of Compound 99.

30

BOST 1635628.1 - 66 -

Step 1. 5-(3-Methyl-7-(methyl- ^-2,3.6J-tetrahvdro-lH-purin-l-vn-N- methoxy-N-methylpentanamide (58). A suspension of 51 (1.50 g, 8.2 mmol, 1.0 5 equiv, see Example 1 for preparation) and powdered K ₂ CO ₃ (1.80 g, 12.9 mmol, 1.6 equiv) in DMF (40 mL) was heated to 60 °C. 5-Bromo-N-methoxy-N- methylpentanamide 57 (2.21 g, 9.8 mmol, 1.2 equiv, prepared as outlined in Org. Lett., 2005, 7: 1427-1429) was added and the mixture was heated at 110 °C for 4 hours during which time the suspended solid became finer and tan in color. The

10 reaction mixture was cooled to room temperature and DMF was evaporated under reduced pressure. The resulting tan paste was suspended in 1 : 1 CH ₂ Cl ₂ :ethyl acetate (250 mL) and the suspension was filtered to remove insoluble material. The filtrate was concentrated under reduced pressure to a yellow oil. This crude reaction product was purified using an Analogix automated chromatography system eluting with

15 100% CH ₂ C1 ₂ for 8 minutes followed by a gradient of 0-5% MeOH/ CH ₂ C1 ₂ over 40 minutes. The desired product eluted at approximately 24 minutes. Fractions containing product were concentrated under reduced pressure to a slightly yellow oil. 1H NMR of the oil indicated it contained approximately 10% unreacted 51. A second purification on an Analogix automated chromatography system eluting with

20 100% CH ₂ C1 ₂ for 10 minutes followed by a gradient of 0-5% MeOH/ CH ₂ C1 ₂ over 50 minutes allowed for removal of the impurity. Fractions containing product were concentrated under reduced pressure to a slightly yellow oil that crystallized as an off- white solid on standing. The solid was triturated with heptanes (100 mL) and filtered to give 1.29 g (49%>) of 58 as an off-white solid.

25 Step 2. 3-Methyl-7-(methyl-(ij)-l-(6,6,6-(ij-5-oxohexyl)-lH-purine-

2,6(3H,7H)-dione (Compound 99). A suspension of 58 (0.72 g, 2.2 mmol, 1.0 equiv) in THF (20 mL) was cooled to 2 °C and 1M CD ₃ MgI in ether (2.4 mL, 2.4 mmol, 1.1 equiv, Aldrich >99 atom%> D) was added drop-wise via syringe at a rate to maintain the temperature below 5 °C. During the addition, the mixture became a

30 fine, slightly yellow suspension. When addition was complete, the reaction mixture was warmed to room temperature and was stirred for 3 hours. The mixture was

BOST 1635628.1 cooled to 2 °C and an additional portion of CD ₃ MgI solution (0.4 mL, 0.4 mmol) was added. The mixture was allowed to warm to room temperature and was stirred an additional 3 hours. The reaction was quenched with IN HC1 (4 mL) and diluted with H ₂ 0 (10 mL) resulting in a slightly yellow solution that was extracted with 5 CH ₂ C1 ₂ (3X, 200 mL). The combined organic extracts were dried over Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure to a yellow oil. The crude product was purified using an Analogix automated chromatography system eluting with 100% CH ₂ C1 ₂ for 8 minutes and then a gradient of 0-5% MeOH/ CH ₂ C1 ₂ over 40 minutes. The desired product elutes first at about 22 minutes, followed by unreacted

10 starting material. Fractions containing the desired product were concentrated under reduced pressure to a yellow oil that solidified upon standing. The solid was triturated with hexane (25 mL) and collected via vacuum filtration to give 0.33 g (53%>) of Compound 99 as a white solid, mp 93.7 - 94.4 °C. Fractions containing unreacted starting material were also collected and concentrated to give 0.21 g of 58

15 as a clear, colorless oil. The recovered material was re-subjected to the above

alkylation reaction to give, after workup and purification, an additional 0.06 g (33%, 62% overall based on total starting material) of Compound 99, mp 93.3 - 94.0 °C. 1H-NMR (300 MHz, CDC1 ₃ ): δ 1.64-1.68 (m, 4H), 2.50 (t, J = 7.0, 2H), 3.58 (s, 3H), 4.02 (t, J = 7.0, 2H), 7.51 (s, 1H). ¹³ C-NMR (75 MHz, CDC1 ₃ ): δ 21.16, 27.65,

20 29.91, 41.03, 43.41, 107.87, 141.62, 149.00, 151.69, 155.50, 209.12. HPLC

(method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 min (1.0 mL/min) with 4 min hold at 95% ACN + 0.1%) formic acid; Wavelength: 305 nm): retention time: 3.24 min; 99.0%> purity. MS (M+H): 285.3, (M+Na): 307.2. Elemental Analysis (Ci ₃ Hi ₂ D ₆ N ₄ 0 ₃ ):

25 Calculated: C=54.92, H=6.38, N=19.71. Found: C=54.85, H=6.36, N=19.49.

Notable in the ¹ H-NMR spectrum above was the absence of a singlet at around 2.15 ppm indicating an absence of methyl ketone hydrogens. Due to the presence of a triplet at 4.01 ppm in the above ¹ H-NMR spectrum, determination of the presence or absence of a singlet peak at around 3.99 ppm corresponding to the

30 presence or absence of hydrogens on the N-methyl group at the 7 position (R ¹ ) of the purine ring was not possible.

BOST 1635628.1 Example 6. Synthesis of (±)8-^-l-(4,4,6,6,6- -5-Hvdroxyhexyl)-3- methyl-7-(methyl-(i _j )-lH-purine-2,6(3H,7H)-dione (Compound 419).

Scheme 16. Preparation of Compounds 419, 419(R), and 419(5).

(±)8-(i^-l-(4,4,6,6,6-(i -5-Hydroxyhexyl)-3-methyl-7-(methyl-(ij)-lH-purine- 2,6(3H,7H)-dione (Compound 419). Compound 409 (0.50 g, 1.7 mmol, 1.0 equiv,

10 see Example 2) was dissolved in EtOD (13 mL, Aldrich 99.5 atom% D) and NaBH ₄ (0.07 g, 1.9 mmol, 1.1 equiv) was added. An increase in temperature from 24 to 28 °C was observed. The reaction was stirred 2 hours at room temperature, then was quenched by the addition of D ₂ 0 (30 mL, Cambridge Isotope Labs, 99 atom% D). A white suspension formed that was extracted with MTBE (4X, 200 mL total). The

15 combined organic extracts were dried over Na ₂ S0 ₄ , filtered, and concentrated under reduced pressure to a clear, colorless oil (0.45 g). The crude product was purified by silica gel chromatography eluting first with 1% MeOH/ CH ₂ C1 ₂ followed by a gradient of 1-5% MeOH/ CH ₂ C1 ₂ . Fractions containing product were concentrated under reduced pressure to give (0.41 g, 83 %) of Compound 419 as a clear colorless

20 oil that solidified on standing.

Example 7. Chiral Separation of (i?)-8- -l-(4,4,6,6,6- -5-Hydroxyhexyl)- 3-methyl-7-(methyl- ^-lH-purine-2,6(3HJH)-dione (Compound 419(R) and (6^- 8-^-l-(4,4,6,6,6-(i -5-Hvdroxyhexyl)-3-methyl-7-(methyl-(i _j )-lH-purine- 2,6(3H,7H)-dione (Compound 419(S)V

Separation of Enantiomers of Compound 419. Compound 419 obtained from Example 6 above (0.38 g) was dissolved in a minimal amount of iPrOH (6 mL,

BOST 1635628.1 69 - HPLC grade, heating required) and diluted with hexane (4 mL, HPLC grade).

Enantiomeric separation was achieved using a Waters HPLC system equipped with a preparative Daicel Chiralpak AD column (20 X 250 mm). For the first minute of the run, the mobile phase was 80% hexane and 20% iPrOH along with 0.1%

5 diethylamine. After the first minute a gradient to 75% hexane and 25% iPrOH along with O. P/o diethylamine over 15 minutes was used, followed by holding at this solvent ratio for 17 minutes at a flow rate of 18 mL/min. This method resulted in baseline separation with 419(R) eluting first (21.0 min), followed by 419(5) (24.1 min). Fractions containing each enantiomer were concentrated under reduced

10 pressure to give 0.16 g each of 419(R) (mp 107.8-108.8 °C) and 419(5) (mp 108.3- 108.4 °C) as off-white solids.

A). (R)-8-d,-l -(4,4,6,6,6- ^-5-HydroxyhexylV3-methyl-7-(methyl-^yiH- purine-2,6(3H,7H)-dione (Compound 419(R)V 1H-NMR (300 MHz, CDC1 ₃ ): δ 1.36-1.50 (m, 2H), 1.60-1.74 (m, 3H), 3.58 (s, 3H), 3.80 (s, 1H), 4.02 (t, J = 7.3,

15 2H). ¹³ C-NMR (75 MHz, CDC1 ₃ ): δ 22.70, 27.86, 29.71 , 41.14, 67.66, 107.66, 148.78, 151.54, 155.40. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 min (1.0 mL/min) with 4 min hold at 95%> ACN + 0.1%> formic acid; Wavelength: 254 nm): retention time: 3.26 min; 99.9% purity. Chiral HPLC (method: Chiralpak AD 25

20 cm column - isocratic method 78% hexane/ 22% isopropanol/0.01% diethylamine for 40 min at 1.00 mL/min; Wavelength: 254 nm): retention time: 27.51 min (major enantiomer); 31.19 min (expected for minor enantiomer): >99.9% ee purity. MS (M+H): 290.1 , (M+Na): 312.3. Elemental Analysis (Ci ₃ HnD ₉ N ₄ 0 ₃ ): Calculated: C=53.97, H=6.97, N=19.36. Found: C=54.39, H=7.1 1 , N=18.98.

25 Notable in the ¹ H-NMR spectrum above was the absence of the following peaks: a peak at around 1.19 ppm indicating an absence of methyl hydrogens alpha to the hydroxyl group; and a singlet at around 7.51 ppm indicating an absence of hydrogen at the number 8 position on the purine ring. Due to the presence of a multiplet at 1.36-1.50 ppm and a triplet at 4.01 ppm in the above ¹ H-NMR spectrum,

30 determination of the presence or absence a peak at 1.51 ppm corresponding to the presence or absence of methylene hydrogens alpha to the hydroxyl group and of a singlet peak at around 3.99 ppm corresponding to the presence or absence of

BOST 1635628.1 hydrogens on the N-methyl group at the 7 position (R ) of the purine ring was not possible.

B). (^-8- -l-(4,4,6,6,6- -5-Hvdroxyhexyn-3-methyl-7-(methyl- -lH- purine-2,6(3HJH)-dione (Compound 419(5 1H-NMR (300 MHz, CDC1 ₃ ): δ 1.41- 5 1.48 (m, 2H), 1.64-1.72 (m, 3H), 3.58 (s, 3H), 3.79 (s, 1H), 4.02 (t, J = 7.4, 2H).

1 ³ C-NMR (75 MHz, CDC1 ₃ ): δ 22.70, 27.86, 29.71, 41.15, 67.66, 107.67, 148.78, 151.54, 155.41. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη CI 8-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 min (1.0 mL/min) with 4 min hold at 95% ACN + 0.1% formic acid; Wavelength: 254 nm): retention

10 time: 3.26 min; 99.9% purity. Chiral HPLC (method: Chiralpak AD 25 cm column - isocratic method 78% hexane/ 22% isopropanol/0.01% diethylamine for 40 min at 1.00 mL/min; Wavelength: 254 nm): retention time: 31.19 min (major enantiomer); 27.51 min (expected for minor enantiomer): >99.9% ee purity. MS (M+H): 290.1, (M+Na): 312.3. Elemental Analysis (C ₁₃ H ₁₁ D ₉ N ₄ O ₃ ): Calculated: C=53.97,

15 H=6.97, N=19.36. Found: C=54.35, H=7.28, N=18.75.

Notable in the ¹ H-NMR spectrum above was the absence of the following peaks: a peak at around 1.19 ppm indicating an absence of methyl hydrogens alpha to the hydroxyl group; and a singlet at around 7.51 ppm indicating an absence of hydrogen at the number 8 position on the purine ring. Due to the presence of a

20 multiplet at 1.36-1.50 ppm and a triplet at 4.01 ppm in the above ¹ H-NMR spectrum, determination of the presence or absence a peak at 1.51 ppm corresponding to the presence or absence of methylene hydrogens alpha to the hydroxyl group and of a singlet peak at around 3.99 ppm corresponding to the presence or absence of hydrogens on the N-methyl group at the 7 position (R ¹ ) of the purine ring was not

25 possible.

Example 8. Synthesis of (±)8- y_-l-(4,4,5,6,6,6-^-5-Hydroxyhexyl)-3- methyl-7-(methyl-(ij)-lH-purine-2,6(3H,7H)-dione (Compound 435).

30 Scheme 17. Preparation of Compounds 435, 435(i?), and 435(61.

BOST 1635628.1 - 71 - Chiral HPLC

Separation

(± 8- -l-(4,4,5.6.6.6-A-5-Hvdroxyhexyn-3-methyl-7-(methyl- _j -lH- purine-2,6(3H,7H)-dione (Compound 435). To a solution of Compound 409 (0.50 g, 1.7 mmol, 1.0 equiv) in EtOD (13 mL, Aldrich 99.5 atom% D) was added NaBD ₄ 5 (0.08 g, 1.9 mmol, 1.1 equiv, Cambridge Isotope Labs, 99 atom% D). An increase in temperature from 24 to 27 °C was observed. The reaction was stirred 2 hours at room temperature then was quenched by the addition of of D ₂ 0 (30 mL) (Cambridge Isotope, 99 atom% D). A white suspension formed that was extracted with MTBE (4X, 200 mL total). The combined organic extracts were dried over Na ₂ S0 ₄ , 10 filtered, and concentrated under reduced pressure to a clear, colorless oil (0.45 g).

The crude product was purified by silica gel chromatography eluting first with 1% MeOH/ CH ₂ C1 ₂ followed by a gradient of 1 -5% MeOH/ CH ₂ C1 ₂ . Fractions containing product were concentrated under reduced pressure to give 0.40 g (81 %) of Compound 435 as a clear colorless oil that solidified on standing.

15

Example 9. Chiral Separation of (i?)-8-^-l-(4,4,5,6,6,6-^-5- Hvdroxyhexyl)-3 -methyl-7-(methyl-<i _j )- lH-purine-2,6(3H,7H)-dione (Compound 435(R) and (^-8- -l-(4.4.5.6.6.6-A-5-Hvdroxyhexyn-3-methyl-7-(methyl- ^- lH-purine-2.6(3H.7H)-dione (Compound 435(S)).

20

Separation of Enantiomers of Compound 435. Compound 435 obtained from Example 8 above (0.32 g) was dissolved in a minimal amount of iPrOH (5 mL, HPLC grade, heating was required) and diluted with hexane (4 mL, HPLC grade). Enantiomer separation was achieved using a Waters HPLC system equipped with a

25 preparative Daicel Chiralpak AD column (20 X 250 mm). For the first minute of the run, the mobile phase was 80% hexane and 20% iPrOH along with 0.1%

diethylamine. After the first minute a gradient to 75% hexane and 25% iPrOH along with O. P/o diethylamine over 15 minutes was used, followed by holding at this solvent ratio for 17 minutes at a flow rate of 18 mL/min. This method resulted in

30 baseline separation with Compound 435(R) eluting first (21.9 min), followed by Compound 435(5) (25.2 min). Fractions containing each enantiomer were

BOST 1635628.1 . 79 . concentrated under reduced pressure to give 0.12 g each of 435(R) (mp 108.0-108.1 °C) and 435(5) (mp 107.6- 107.7 °C) as off-white solids.

A) . (i? -8- -l-(4,4,5,6,6,6-A-5-Hvdroxyhexyn-3-methyl-7-(methyl- ^-lH- purine-2,6(3HJH)-dione (Compound 435(R)V 1H-NMR (300 MHz, CDC1 ₃ ): δ 1.40-

5 1.48 (m, 3H), 1.66-1.70 (m, 2H), 3.58 (s, 3H), 4.02 (t, J = 7.5, 2H). ¹³ C-NMR (75 MHz, CDCI ₃ ): δ 22.66, 27.86, 29.71 , 41.15, 107.67, 148.80, 151.54, 155.41. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C 18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 min (1.0 mL/min) with 4 min hold at 95% ACN + 0.1% formic acid; Wavelength: 254 nm): retention time: 3.25 min; 99.8%

10 purity. Chiral HPLC (method: Chiralpak AD 25 cm column - isocratic method 78% hexane/ 22% isopropanol/0.01% diethylamine for 40 min at 1.00 mL/min;

Wavelength: 254 nm): retention time: 27.24 min (major enantiomer); 31.1 1 min (expected for minor enantiomer): >99.9%> ee purity. MS (M+H): 291.3, (M+Na): 313.2. Elemental Analysis (C ₁₃ H ₁₀ D ₁₀ N ₄ O ₃ ): Calculated: C=53.78, H=6.94,

15 N=19.30. Found: C=54.01 , H=7.07, N=18.90.

Notable in the ¹ H-NMR spectrum above was the absence of the following peaks: a peak at around 1.19 ppm indicating an absence of methyl hydrogens alpha to the hydroxyl group; a peak at around 3.80 ppm indicating an absence of hydrogen at the methinyl hydroxyl position; and a singlet at around 7.51 ppm indicating an

20 absence of hydrogen at the number 8 position on the purine ring. Due to the

presence of a multiplet at 1.36-1.50 ppm and a triplet at 4.01 ppm in the above 1H- NMR spectrum, determination of the presence or absence a peak at 1.51 ppm corresponding to the presence or absence of methylene hydrogens alpha to the hydroxyl group and of a singlet peak at around 3.99 ppm corresponding to the

25 presence or absence of hydrogens on the N-methyl group at the 7 position (R ¹ ) of the purine ring was not possible.

B . (^-8- -l-(4,4,5,6,6,6-A-5-Hvdroxyhexyn-3-methyl-7-(methyl- ^-lH- purine-2,6(3HJH)-dione (Compound 435(5 1H-NMR (300 MHz, CDCI ₃ ): δ 1.41- 1.48 (m, 3H), 1.62-1.72 (m, 2H), 3.58 (s, 3H), 4.03 (t, J = 7.4, 2H). ¹³ C-NMR (75

30 MHz, CDCI ₃ ): δ 22.69, 27.90, 29.70, 41.17, 107.69, 148.82, 151.58, 155.43. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C 18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 min (1.0 mL/min) with 4 min hold at 95% ACN + 0.1% formic acid; Wavelength: 254 nm): retention time: 3.25 min; 99.5%

BOST 1635628.1 purity. Chiral HPLC (method: Chiralpak AD 25 cm column - isocratic method 78% hexane/ 22% isopropanol/0.01% diethylamine for 40 min at 1.00 mL/min;

Wavelength: 254 nm): retention time: 31.11 min (major enantiomer); 27.24 min (expected for minor enantiomer): >99.9%> ee purity. MS (M+H): 291.3, (M+Na): 5 313.2. Elemental Analysis (C ₁₃ H ₁₀ D ₁₀ N ₄ O ₃ ): Calculated: C=53.78, H=6.94,

N=19.30. Found: C=54.01, H=7.11, N=18.78.

Notable in the ¹ H-NMR spectrum above was the absence of the following peaks: a peak at around 1.19 ppm indicating an absence of methyl hydrogens alpha to the hydroxyl group; a peak at around 3.80 ppm indicating an absence of hydrogen

10 at the methinyl hydroxyl position; and a singlet at around 7.51 ppm indicating an absence of hydrogen at the number 8 position on the purine ring. Due to the presence of a multiplet at 1.36-1.50 ppm and a triplet at 4.01 ppm in the above 1H- NMR spectrum, determination of the presence or absence a peak at 1.51 ppm corresponding to the presence or absence of methylene hydrogens alpha to the

15 hydroxyl group and of a singlet peak at around 3.99 ppm corresponding to the

presence or absence of hydrogens on the N-methyl group at the 7 position (R ¹ ) of the purine ring was not possible.

Example 10. Synthesis of 8-(^-3,7-Dimethyl-l-(4,4,6,6,6-( 5-oxohexyl)- 20 lH-purine-2.6(3H.7H)-dione (Compound 407).

Scheme 18. Preparation of Compounds 407. 437. 437(R). and 437(51

BOST 1635628.1 - 74 - 8- -3J-Dimethyl-l-(4,4,6,6,6-^-5-oxohexyn-lH-purine-2,6(3HJH)-d ione (Compound 407). A mixture of commercially-available 59 (7.95 g, 28.6 mmol) and potassium carbonate (990 mg, 7.2 mmol) in D ₂ 0 (195 mL, Cambridge Isotopes, 5 99.9 atom% D) was heated to reflux for 24 hours. The suspended solid dissolved gradually giving a yellow solution. The solution was cooled to approximately 40 °C and was concentrated under reduced pressure to a tan solid. The solid was dissolved in D ₂ 0 (195 mL) and the solution was heated to reflux for another 24 hours. The solution was cooled to room temperature and concentrated under reduced pressure to

10 a tan solid. Ethyl acetate (200 mL) was added and the mixture was stirred 0.5 hours at approximately 40 °C. The insoluble materials were filtered off and the filtrate was concentrated under reduced pressure to a pale yellow solid, which was triturated with MTBE (40 mL) to give 7.5 g (93%) of Compound 407 as an off-white solid. 1H-NMR (300 MHz, CDC1 ₃ ): δ 1.64-1.68 (m, 4H), 3.57 (s, 3H), 3.99 (s, 3H), 3.99-

15 4.04 (m, 2H). ¹³ C-NMR (75 MHz, CDCI3): δ 20.84, 27.40, 29.69, 33.57, 40.81,

107.62, 148.77, 151.48, 155.28, 209.07. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 min (1.0 mL/min) with 4 min hold at 95% ACN + 0.1% formic acid;

Wavelength: 305 nm): retention time: 3.24 min; 99.9% purity. MS (M+H): 285.3,

20 (M+Na): 307.2. Elemental Analysis (Ci ₃ Hi ₂ D ₆ N ₄ 0 ₃ ): Calculated: C=54.92,

H=6.38, N=19.71. Found: C=54.89, H=6.38, N=19.70.

Notable in the ¹ H-NMR spectrum above was the absence of the following peaks: a singlet at around 2.15 ppm indicating an absence of methyl ketone hydrogens; a triplet at around 2.51 ppm indicating an absence of methylene ketone

25 hydrogens; and a singlet at around 7.52 ppm indicating an absence of hydrogen at the number 8 position on the purine ring.

Example 11. Synthesis of (± 8-< ,-l-(4A5A6.6-<^-5-Hvdroxyhexyr)-3.7- dimethyl-lH-purine-2,6(3H,7H)-dione (Compound 437).

30 (± 8- -l-(4,4,5.6.6.6-A-5-Hvdroxyhexyn-3J-dimethyl-lH-purine-

2,6(3H,7H)-dione (Compound 437). Sodium borodeuteride (1.06 g, 25.3 mmol, Cambridge Isotopes, 99 atom%> D) was added to a suspension of 407 (6.5 g, 22.9 mmol) in ethanol-di (65 mL, Aldrich, 99.5 atom% D) at 0 °C. The mixture was

BOST 1635628.1 - 75 - warmed to room temperature and stirred until a clear solution had developed (approximately 1 hour). The reaction was quenched with a saturated solution of ammonium chloride-d4 (Cambridge Isotopes, 98 atom% D) in D ₂ 0 (8 mL,

Cambridge Isotope, 99.9 atom% D), ethanol-di was evaporated under reduced 5 pressure and the residue was extracted with EtOAc (160 mL). The organic phase was washed with D ₂ 0 (20 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure to give 4.8 g (73%) of Compound 437 as a pale yellow solid.

10 Hvdroxyhexyl)-3,7-dimethyl-lH-purine-2,6(3H,7H)-dione (Compound 437(RV) and (^-8- -l-(4,4,5,6,6,6-A-5-Hvdroxyhexyn-3J-dimethyl-lH-purine-2,6(3 H,7H - dione (Compound 437(5)).

Separation of Enantiomers of Compound 437. Compound 437 obtained from Example 1 1 above (1.60 g) was dissolved in iPrOH (20 mL, HPLC grade,

15 heating required). Enantiomeric separation was achieved using a Waters HPLC

system equipped with a preparative Chiralpak AD column (20 x 250 mm Daicel, 10 μΜ) with a preparative Chiralpak AD guard column (20 x 50 mm Daicel, 10 μΜ) preceding it. For the first minute of the run, the sample was eluted with 20% iPrOH/hexanes (henceforth, with 0.1% diethylamine as co-eluent) while ramping up

20 from a flow rate of 15 mL/min to 18 mL/min. Over the next 15 minutes, the sample was eluted at a flow rate of 18 mL/min with a gradient of 20% to 25%

iPrOH/hexanes. For the next 19 minutes the sample was eluted at a flow rate of 18 mL/min with 25% iPrOH/hexanes. Over the next 0.5 minutes, the sample was eluted at a flow rate of 18 mL/min with a gradient of 25% to 20% iPrOH/hexanes. For the

25 next 4.5 minutes, the sample was eluted at a flow rate of 18 mL/min with 20%

iPrOH/hexanes. This elution method resulted in baseline separation of Compound 437(R) eluting first (retention time approximately 29 min) and Compound 437(5) eluting second (retention time approximately 33 min). Fractions containing each enantiomer were collected and concentrated under reduced pressure to give 340 mg

30 of 437(R) (mp 1 12.0-1 14.5 °C) and 375 mg of 437(5) (mp 1 1 1.9-1 12.3 °C) as off- white solids. [Note: only 1.0 g of 437 was injected from the solution prepared above.]

BOST 1635628.1 A. (RV8- /-l-(4.4.5.6.6.6-^-5-HvdroxyhexylV3J-dimethyl-lH-purine- 2.6(3H.7HVdione (Compound 437(R)V 1H-NMR (300 MHz, CDC1 ₃ ): δ 1.36-1.50 (m, 2H), 1.54 (s, 1H), 1.64-1.74 (m, 2H), 3.58 (s, 3H), 3.99 (s, 3H), 4.00-4.05 (m, 2H). ¹³ C-NMR (75 MHz, CDCI ₃ ): δ 22.66, 27.86, 29.70, 33.59, 41.14, 107.65,

5 148.76, 151.52, 155.40. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 min (1.0 mL/min) with 4 min hold at 95%> ACN + 0.1%> formic acid; Wavelength: 305 nm): retention time: 3.28 min; 99.9% purity. Chiral HPLC (method: Chiralpak AD 25 cm column - isocratic method 78% hexane/ 22% isopropanol/0.01% diethylamine

10 for 40 min at 1.00 mL/min; Wavelength: 254 nm): retention time: 25.20 min (major enantiomer); 28.39 min (expected for minor enantiomer): >99.9% ee purity. MS (M+H): 288.3, (M+Na): 310.2. Elemental Analysis (C ₁₃ H ₁₃ D ₇ N ₄ O ₃ ): Calculated: C=54.34, H=7.02, N=19.50. Found: C=54.32, H=7.23, N=19.35.

Notable in the ¹ H-NMR spectrum above was the absence of the following

15 peaks: a peak at around 1.19 ppm indicating an absence of methyl hydrogens alpha to the hydroxyl group; a peak at around 3.80 ppm indicating an absence of hydrogen at the methinyl hydroxyl position; and a singlet peak at around 7.51 ppm indicating an absence of hydrogen at the number 8 position on the purine ring. Due to the presence of a multiplet at 1.36-1.50 ppm in the above ¹ H-NMR spectrum,

20 determination of the presence or absence a peak at 1.51 ppm corresponding to the presence or absence of methylene hydrogens alpha to the hydroxyl group was not possible.

B. ( _t ^-8- -l-(4,4,5.6.6.6-A-5-Hvdroxyhexyn-3J-dimethyl-lH-purine- 2,6(3HJH -dione (Compound 437(S)V 1H-NMR (300 MHz, CDCI ₃ ): δ 1.38-1.48

25 (m, 2H), 1.55 (s, 1H), 1.64-1.72 (m, 2H), 3.58 (s, 3H), 3.99 (s, 3H), 4.00-4.05 (m, 2H). ¹³ C-NMR (75 MHz, CDCI ₃ ): δ 22.65, 27.84, 29.71 , 33.59, 41.13, 107.64, 148.75, 151.52, 155.39. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 min (1.0 mL/min) with 4 min hold at 95%> ACN + O. P/o formic acid; Wavelength: 305 nm):

30 retention time: 3.27 min; 99.9% purity. Chiral HPLC (method: Chiralpak AD 25 cm column - isocratic method 78% hexane/ 22% isopropanol/0.01% diethylamine for 40 min at 1.00 mL/min; Wavelength: 254 nm): retention time: 28.39 min (major enantiomer); 25.20 min (expected for minor enantiomer): >99.9% ee purity. MS

BOST 1635628.1 - 77 - (M+H): 288.3, (M+Na): 310.2. Elemental Analysis (Ci ₃ Hi3D ₇ N ₄ 03): Calculated: C=54.34, H=7.02, N=19.50. Found: C=54.33, H=7.30, N=19.36.

Notable in the 1H-NMR spectrum above was the absence of the following peaks: a peak at around 1.19 ppm indicating an absence of methyl hydrogens alpha 5 to the hydroxyl group; a peak at around 3.80 ppm indicating an absence of hydrogen at the methinyl hydroxyl position; and a singlet peak at around 7.51 ppm indicating an absence of hydrogen at the number 8 position on the purine ring. Due to the presence of a multiplet at 1.36-1.50 ppm in the above 1H-NMR spectrum, determination of the presence or absence a peak at 1.51 ppm corresponding to the 10 presence or absence of methylene hydrogens alpha to the hydroxyl group was not possible.

Example 13. Synthesis of (±)l-(5-(i 5-Hvdroxyhexyl)-3-methyl-7-(methyl- (ij)-lH-purine-2,6(3H,7H)-dione (Compound 131).

15

Scheme 19. Preparation of Compounds 131, 13KR), and 131(5).

(± l-(5- -5-Hvdroxyhexyn-3-methyl-7-(methyl- -lH-purine-2,6(3H,7H -

20 dione (Compound 131). Following the same general method as for the synthesis of Compound 437 above, Compound 100 (see Example 1) was treated with NaBD ₄ in EtOH to afford Compound 131.

Example 14. Chiral Separation of (iQ-l-(5-^-5-Hydroxyhexyl)-3-methyl-7- 25 (methyl-^yiH-purine-2,6(3H,7Fn-dione (Compound 13KR) and (S)- 5-d,-5- Hvdroxyhexyl)-3 -methyl-7-(methyl-<ij)- 1 H-purine-2,6(3H,7H)-dione (Compound 13US

BOST 1635628.1 - 78 - Separation of Enantiomers of Compound 131. A portion of racemic Compound 131 obtained from Example 13 above was separated in the same manner as racemic Compound 437 above, to afford separated enantiomers Compound

131(R) (mp 1 12.2-1 12.7 °C) (210 mg) and Compound 131(5) (mp 1 12.0-1 12.1 °C) 5 (220 mg).

A. (R)- 1 -(5 -dy-5 -Hvdroxyhexyl)-3 -methyl-7-(methyl-<i _j )- 1 H-purine- 2.6(3H.7HVdione (Compound 13UR) . 1H-NMR (300 MHz, CDC1 ₃ ): δ 1.19 (s, 3H), 1.39-1.56 (m, 5H), 1.64-1.74 (m, 2H), 3.58 (s, 3H), 4.03 (t, J=7.3, 2H), 7.51 (s, 1H). ¹³ C-NMR (75 MHz, CDC1 ₃ ): δ 22.87, 23.40, 27.89, 29.71 , 38.64, 41.13,

10 107.68, 141.40, 148.76, 151.52, 155.39. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 min (1.0 mL/min) with 4 min hold at 95% ACN + 0.1% formic acid;

Wavelength: 305 nm): retention time: 3.29 min; 99.9% purity. Chiral HPLC (method: Chiralpak AD 25 cm column - isocratic method 78% hexane/ 22%

15 isopropanol/0.01% diethylamine for 40 min at 1.00 mL/min; Wavelength: 254 nm): retention time: 25.14 min (major enantiomer); 28.51 min (expected for minor enantiomer): >99.9% ee purity. MS (M+H): 285.3, (M+Na): 307.2. Elemental Analysis (Ci ₃ Hi ₆ D ₄ N ₄ 0 ₃ ): Calculated: C=54.92, H=7.09, N=19.71. Found:

C=54.67, H=7.04, N=19.35.

20 Notable in the ¹ H-NMR spectrum above was the absence of a peak at around

3.80 ppm indicating an absence of hydrogen at the methinyl hydroxyl position. Due to the presence of a triplet at 4.01 ppm in the above ¹ H-NMR spectrum,

determination of the presence or absence of a singlet peak at around 3.99 ppm corresponding to the presence or absence of hydrogens on the N-methyl group at the

25 7 position (R ¹ ) of the purine ring was not possible.

B . (S)- 1 -(5 - -5 -Hydroxyhexyl)-3 -methyl-7-(methyl-<ij)- 1 H-purine- 2,6(3H,7H -dione (Compound 13 S)). 1H-NMR (300 MHz, CDC1 ₃ ): δ 1.18 (s, 3H), 1.39-1.55 (m, 5H), 1.67-1.72 (m, 2H), 3.58 (s, 3H), 4.03 (t, J=7.3, 2H), 7.51 (s, 1H). ¹³ C-NMR (75 MHz, CDC1 ₃ ): δ 23.10, 23.63, 28.12, 29.94, 38.87, 41.36,

30 107.91 , 141.63, 148.99, 151.75, 155.62. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 min (1.0 mL/min) with 4 min hold at 95% ACN + 0.1% formic acid;

Wavelength: 305 nm): retention time: 3.29 min; 99.9% purity. Chiral HPLC

BOST 1635628.1 . 7Q . (method: Chiralpak AD 25 cm column - isocratic method 78% hexane/ 22% isopropanol/0.01% diethylamine for 40 min at 1.00 mL/min; Wavelength: 254 nm): retention time: 28.51 min (major enantiomer); 25.14 min (expected for minor enantiomer): >99.9% ee purity. MS (M+H): 285.3, (M+Na): 307.2. Elemental 5 Analysis (Ci ₃ Hi ₆ D ₄ N ₄ 0 ₃ ): Calculated: C=54.92, H=7.09, N= 19.71. Found:

C=54.65, H=7.04, N=19.32.

Notable in the 1H-NMR spectrum above was the absence of a peak at around 3.80 ppm indicating an absence of hydrogen at the methinyl hydroxyl position. Due to the presence of a triplet at 4.01 ppm in the above 1H-NMR spectrum,

10 determination of the presence or absence of a singlet peak at around 3.99 ppm

corresponding to the presence or absence of hydrogens on the N-methyl group at the 7 position (R ¹ ) of the purine ring was not possible.

15 lH-purine-2,6(3H,7H)-dione (Compound 421).

Scheme 20. Pre aration of Compounds 421, 421(R) and 421(5).

20 Synthesis of (± l-(4A6A6-d 5-hvdroxyhexylV3 J-dimethyl-8-d-lH- purine-2,6(3H,7H)-dione (Compound 421). Following the same general method as for the synthesis of Compound 437 in Example 11 above, Compound 407 (see Example 10) was treated with NaBH ₄ in EtOD and extracted with CH ₂ CI ₂ to afford Compound 421.

25

BOST 1635628.1 - 80 - Example 16. Chiral Separation of (i?)-l-(4,4,6,6,6-ds-5-hvdroxyhexyl)-3,7- dimethyl-8-d-lH-purine-2,6(3HJH)-dione (Compound 42UR)) and (6^-1- (4,4,6.6.6-ds-5-hvdroxyhexyn-3J-dimethyl-8-d-lH-purine-2,6(3 H.7H)-dione (Compound 42US)).

5 Separation of Enantiomers of Compound 421. A portion of racemic

Compound 421 obtained as described above was separated in the same manner as racemic Compound 437 (see Example 12) to afford separated enantiomers

Compound 421(R) (560 mg) and Compound 421(5) (520 mg).

10 A. (i? -l-(4,4,6,6,6-d^-5-hvdroxyhexyl -3,7-dimethyl-8-d-lH-purine-

2,6(3H,7H)-dione (Compound 42UR) . 1H-NMR (300 MHz, CDC1 ₃ ): δ 1.41-1.48 (m, 2H), 1.64-1.72 (m, 3H), 3.58 (s, 3H), 3.79 (s, 1H), 3.99 (s, 3H), 4.03 (t, J=7.3, 2H). ¹³ C-NMR (75 MHz, CDC1 ₃ ): δ 22.69, 27.84, 29.72, 33.60, 41.14, 67.62, 107.64, 148.74, 151.51 , 155.38. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3

15 μιη C 18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14

minutes (1.0 mL/min) with 4 minute hold at 95% ACN; Wavelength: 254 nm): retention time: 3.33 min; >99.9% purity. Chiral HPLC (method: Chiralpak AD 25 cm column - isocratic method 78% hexane/ 22% isopropanol/0.1% diethylamine for 40 minutes at 1.00 mL/min; Wavelength: 254 nm): retention time: 24.77 min (R

20 enantiomer); 28.16 min (expected for S enantiomer); >99.9% ee purity. MS (M+H- H ₂ 0): 269.1; (M+H): 287.1 ; (M+Na): 309.3. Elemental Analysis

Calculated: C=54.53, H=7.04, N=19.57. Found: C=54.44, H=7.18, N=19.32.

B. ( _t ^-l-(4,4,6,6,6-ds-5-hvdroxyhexyl -3,7-dimethyl-8-d-lH-purine- 2,6(3H,7H)-dione (Compound 42 S)). 1H-NMR (300 MHz, CDCI3): δ 1.37-1.48

25 (m, 2H), 1.64-1.74 (m, 3H), 3.58 (s, 3H), 3.79 (s, 1H), 3.99 (s, 3H), 4.03 (t, J=7.4, 2H). ¹³ C-NMR (75 MHz, CDCI ₃ ): δ 22.70, 27.84, 29.71 , 33.60, 41.14, 67.61 , 107.64, 148.74, 151.51 , 155.38. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C 18-RP column - gradient method 5-95%> ACN + 0.1%> formic acid in 14 minutes (1.0 mL/min) with 4 minute hold at 95% ACN; Wavelength: 254 nm):

30 retention time: 3.34 min; >99.9% purity. Chiral HPLC (method: Chiralpak AD 25 cm column - isocratic method 78% hexane/ 22% isopropanol/0.1% diethylamine for 40 minutes at 1.00 mL/min; Wavelength: 254 nm): retention time: 28.16 min (S enantiomer); 24.77 min (expected for R enantiomer); >99.9% ee purity. MS (M+H-

BOST 1635628.1 - 81 - H ₂ 0): 269.1; (M+H): 287.1 ; (M+Na): 309.3. Elemental Analysis (Ci ₃ Hi ₄ D ₆ N ₄ 0 ₃ ): Calculated: C=54.53, H=7.04, N=19.57. Found: C=54.54, H=7.18, N=19.31.

Example 17. Synthesis of (±)-! -(4,4,5.6.6.6-dg-5-Hvdroxyhexyl)-3 J-dimethyl-lH- 5 purine-2,6(3H,7H)-dione (Compound 137). heme 21. Preparation of Compound 137.

Synthesis of (±)-l-(4,4,5,6,6,6-dfi-5-Hydroxyhexyl)-3,7-dimethyl-lH-puri ne- 10 2,6(3H,7H)-dione (Compound 137). Compound 437 (560 mg, approximately 2 mmol, see Example 1 1) was stirred with K ₂ CO ₃ (270 mg, 2 mmol) in water (10 mL). The mixture was heated at 120-130 °C to give a clear solution and was heated overnight. The solution was extracted with CH ₂ C1 ₂ (1 x 50 mL, 2 x 20 mL) and the CH ₂ C1 ₂ solution was dried (Na ₂ S0 ₄ ) and filtered. After removal of solvent, the solid 15 was stirred with K ₂ CO ₃ (140 mg, 1 mmol) in water (10 mL) and was heated

overnight as above to ensure complete deuterium-to-hydrogen exchange. After extraction with CH ₂ C1 ₂ (1 x 50 mL, 2 x 20 mL), the CH ₂ C1 ₂ solution was dried (Na ₂ S0 ₄ ), filtered and concentrated. The crude product was purified by

chromatography on silica gel eluting with 2-3% MeOH/CH ₂ Cl ₂ to give 480 mg 20 (86%) of 137.

HPLC (method: Zorbax 4.6x50 mm SB-Aq 3.5 μιη column - gradient method 2- 98% ACN + 0.1% formic acid in 6.0 min with MSD in ESI positive mode; 0.63 mL/min; Wavelength: 254 nm): retention time: 2.51 min; 98.7% purity. MS (M+H): 287.1 ; (M+Na): 309.0.

25

Example 18. Synthesis of (R)- l-(4,4,5.6.6.6-dfi-5-HvdroxyhexylV3.7-dimethyl- lH-purine-2,6(3HJH)-dione (Compound 137(R)V

Scheme 22. Preparation of Compound 137(R).

BOST 1635628.1 - 82 -

437(R) 137(R)

Synthesis of (i?)-l-(4,4,5,6,6,6-dfi-5-Hvdroxyhexyl)-3,7-dimethyl-lH-puri ne- 2,6(3H,7H)-dione (Compound 137(R)V A solution of 437(R) (650 mg, 2.26 mmol, see Example 12) and K ₂ CO ₃ (320 mg, 2.3 mmol) in water (40 mL) was heated at 5 110 °C (bath temperature) for 26 hours. The solution was concentrated to dryness, redissolved in water (30 mL) and heated to 100 °C for a further 6 hours. After cooling to ambient temperature the solution was extracted with CH ₂ CI ₂ (4 x 50 mL). The organic solution was dried (Na ₂ S0 ₄ ), filtered, concentrated, then dried under vacuum to afford 565 mg of 137(R) as an off-white solid.

10 1H-NMR (300 MHz, CDC1 ₃ ): δ 1.38-1.48 (m, 2H), 1.64-1.72 (m, 3H), 3.58 (s, 3H), 3.99 (d, J=0.5, 3H), 4.02 (t, J=7.4, 2H), 7.51 (d, J=0.6, 1H). ¹³ C-NMR (75 MHz, CDCI ₃ ): δ 22.65, 27.84, 29.71, 33.61, 41.13, 107.67, 141.43, 148.73, 151.50, 155.37. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 minutes (1.0 mL/min) with 4 minute

15 hold at 95% ACN; Wavelength: 305 nm): retention time: 3.30 min; >99.9% purity.

MS (M+H-H ₂ 0): 269.4; (M+H): 287.1; (M+Na): 309.3. Elemental Analysis (Ci ₃ Hi ₄ D ₆ N ₄ 0 ₃ ): Calculated: C=54.53, H=7.04, N=19.57. Found: C=54.43, H=6.93, N=19.44.

20 Example 19. Synthesis of (S)- l-(4A5A6,6-d _fi -5-HvdroxyhexylV3 J-dimethyl-lH- .

Following the same general method as for the synthesis of Compound

137(R) in Example 18 above, a portion of Compound 437(5) (see Example 12) was converted to 310 mg of Compound 137(5).

1H-NMR (300 MHz, CDCI3): δ 1.36-1.45 (m, 2H), 1.62 (s, 1H), 1.64-1.74 (m, 2H), 3.58 (s, 3H), 3.99 (s, 3H), 4.02 (t, J=7.3, 2H), 7.50 (s, 1H). ¹³ C-NMR (75 MHz,

BOST 1635628.1 - 83 - CDCI ₃ ): δ 23.05, 28.24, 30.07, 33.95, 41.49, 107.92, 141.57, 148.93, 151.68, 155.53. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 minutes (1.0 mL/min) with 4 minute hold at 95% ACN; Wavelength: 305 nm): retention time: 3.34 min; 99.6%> purity. 5 MS (M+H-H ₂ 0): 269.1 ; (M+H): 287.1 ; (M+Na): 309.3. Elemental Analysis

(Ci ₃ Hi ₄ D ₆ N ₄ 0 ₃ ): Calculated: C=54.53, H=7.04, N=19.57. Found: C=54.71 , H=7.28, N=19.53.

Example 20. Synthesis of (±Vl-(4.4.6.6.6-ds-5-HvdroxyhexylV3 J-dimethyl-lH- 10 .

Following the same general method as for the synthesis of Compound 137 in Example 17 above, a portion of Compound 421 (see Example 15) was converted to 2.1 g of Compound 121.

15 1H-NMR (300 MHz, CDCI ₃ ): δ 1.41-1.48 (m, 2H), 1.64-1.72 (m, 2H), 1.85 (bs, 1H), 3.58 (s, 3H), 3.79 (s, 1H), 3.99 (d, J=0.5, 3H), 4.02 (t, J=7.3, 2H), 7.52 (d, J=0.6, 1H). ¹³ C-NMR (75 MHz, CDCI ₃ ): δ 22.69, 27.82, 29.70, 33.61 , 41.14, 67.55, 107.66, 141.44, 148.72, 151.49, 155.35. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C 18-RP column - gradient method 5-95% ACN + 0.1% formic acid in

20 14 minutes (1.0 mL/min) with 4 minute hold at 95% ACN; Wavelength: 305 nm): retention time: 3.31 min; 99.3% purity. MS (M+H-H ₂ 0): 268.2; (M+H): 286.2; (M+Na): 308.1. Elemental Analysis (Ci ₃ Hi ₅ D ₅ N ₄ 0 ₃ ): Calculated: C=54.72, H=7.07, N=19.64. Found: C=54.75, H=6.85, N=19.54.

25 Example 21. R- 1 -(4 A6,6,6-d 5-Hvdroxyhexyl)-3 ,7-dimethyl- lH-purine-

2 6(3H,7H)-dione (Compound 12UR)).

BOST 1635628.1 - 84 - Following the same general method as for the synthesis of Compound

137(R) in Example 18 above, a portion of Compound 419(R) (see Example 7) was converted to 1.3 g of Compound 121(R).

1H-NMR (300 MHz, CDC1 ₃ ): δ 1.37-1.48 (m, 2H), 1.64-1.73 (m, 2H), 1.72 (bs, 5 0.5H), 3.58 (s, 3H), 3.79 (s, 1H), 3.99 (s, 3H), 4.00 (t, J=7.5, 2H), 7.51 (d, J=0.6, 1H). ¹³ C-NMR (75 MHz, CDC1 ₃ ): δ 22.67, 27.83, 29.67, 33.57, 41.12, 67.60, 107.66, 141.40, 148.75, 151.51 , 155.37. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C 18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 4.5 minutes (1.0 mL/min) with 1.5 minute hold at 95% CAN (1.5 mL/min);

10 Wavelength: 305 nm): retention time: 3.29 min; 99.7% purity. Chiral HPLC

(method: Chiralpak AD 25 cm column - isocratic method 78% hexane/ 22% isopropanol/0.1% diethylamine for 40 minutes at 1.00 mL/min; Wavelength: 254 nm): retention time: 25.20 min (R enantiomer); 28.78 min (expected for S enantiomer); >99% ee purity. MS (M+H-H ₂ 0): 268.2; (M+H): 286.2; (M+Na):

15 308.1.

Example 22. 6 ^, -l-(4,4,6,6,6-d 5-Hvdroxyhexyl)-3,7-dimethyl-lH-purine- 2 6(3H,7H)-dione (Compound 121f5)V

20 Following the same general method as for the synthesis of Compound

137(R) in Example 18 above, a portion of Compound 419(5) (see Example 7) was converted to 590 mg of Compound 121(5).

1H-NMR (300 MHz, CDCI ₃ ): δ 1.37-1.48 (m, 2H), 1.64-1.73 (m, 2H), 1.86 (bs, 0.5H), 3.58 (s, 3H), 3.79 (s, 1H), 3.99 (d, J=0.6, 3H), 4.02 (t, J=7.4, 2H), 7.52 (d,

25 J=0.7, 1H). ¹³ C-NMR (75 MHz, CDCI ₃ ): δ 22.70, 27.84, 29.71 , 33.62, 41.14, 67.59, 107.67, 141.43, 148.73, 151.50, 155.37. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C 18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 minutes (1.0 mL/min) with 4 minute hold at 95%> ACN; Wavelength: 305 nm): retention time: 3.37 min; 99.5% purity. Chiral HPLC (method: Chiralpak AD 25

30 cm column - isocratic method 78% hexane/ 22% isopropanol/0.1% diethylamine for

BOST 1635628.1 - 85 - 40 minutes at 1.00 mL/min; Wavelength: 254 nm): retention time: 25.20 min (expected for R enantiomer); 28.78 min (S enantiomer); >99% ee purity. MS (M+H- H ₂ 0): 268.2; (M+H): 286.2; (M+Na): 308.1. Elemental Analysis (Ci ₃ Hi ₅ D ₅ N ₄ 03): Calculated: C=54.72, H=7.07, N=19.64. Found: C=54.77, H=7.13, N=19.59.

5

Example 23. Synthesis of 3,7-Dimethyl-l-(4,4,6,6,6-ds-5-oxohexyl)-lH-purine- 2,6(3H,7H)-dione (Compound 107). heme 23. Preparation of Compound 107.

Synthesis of 3,7-Dimethyl-l-(4,4,6,6,6-d_s-5-oxohexyl)-lH-purine-

2,6(3H,7H)-dione (Compound 107). Compound 121 (0.49 g, 1.72 mmol, see Example 20) and N-methylmorpholine N-oxide "NMO" (301 mg, 2.58 mmol) were dissolved in CH ₂ C1 ₂ (20 mL). Tetrapropylammonium perruthenate "TPAP" (27 mg,

15 0.086 mmol) was added and the solution was stirred for 2.5 hours at ambient

temperature. TLC (EtOAc) showed the reaction was complete. The reaction was concentrated and purified by silica gel chromatography eluting with EtOAc. The material was dried in a vacuum oven (50 °C) for 4 hours to afford 400 mg (82%) of Compound 107. The material was further purified by crystallization

20 (EtO Ac/heptane) to give 320 mg of 107. NMR and LCMS analysis indicated no loss of deuterium.

1H-NMR (300 MHz, CDC1 ₃ ): δ 1.64-1.70 (m, 4H), 3.57 (s, 3H), 3.99 (d, J=0.6, 3H), 4.01-4.04 (m, 2H), 7.51 (d, J=0.6, 1H). ¹³ C-NMR (75 MHz, CDC1 ₃ ): δ 20.82, 27.38, 29.69, 33.61, 40.80, 107.75, 141.42, 148.76, 151.46, 155.26. HPLC (method:

25 Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 minutes (1.0 mL/min) with 4 minute hold at 95% ACN; Wavelength: 305 nm): retention time: 3.28 min; >99.9% purity. MS (M+H): 284.1; (M+Na): 306.0.

30 Example 24. Synthesis of (±) l-(4.4.5.6.6.6-d^-5-Hvdroxyhexyn-3.7-di(methyl-d^- lH-purine-2,6(3H.7H)-dione (Compound 434).

BOST 1635628.1 - 86 - Scheme 24. Pre aration of Compounds 434, 434(R) and 434(S).

Synthesis of (± l-(4.4.5.6.6.6-d^-5-Hvdroxyhexyn-3.7-di(methyl-d^-lH- 5 purine-2,6(3H,7H)-dione (Compound 434). Following the same general method as for the synthesis of Compound 437 in Example 1 1 above, a portion of Compound 413 (see Example 4) was treated with NaBD ₄ in EtOD to and extracted with CH ₂ CI ₂ afford 190 mg of Compound 434.

10 Example 25. Chiral Separation of (RVl-(4.4.5.6.6.6-dg-5-Hvdroxyhexyl)-3.7- di(methyl-d^-lH-purine-2,6(3HJH)-dione (Compound 434(R) and (S)-l- (4,4,5,6,6,6-dfi-5-Hvdroxyhexyl)-3,7-di(methyl-d2)-lH-purine -2,6(3H,7H)-dione (Compound 434(5

Separation of Enantiomers of Compound 434. A portion of racemic

15 Compound 434 obtained as described above was separated in the same manner as racemic Compound 437 (see Example 12) to afford separated enantiomers

Compound 434(R) (72 mg) and Compound 434(5) (74 mg).

A. (i? -l-(4,4,5.6.6.6-dfi-5-Hvdroxyhexyn-3.7-di(methyl-d2 -lH-purine- 2,6(3H,7H)-dione (Compound 434(R)V 1H-NMR (300 MHz, CDC1 ₃ ): δ 1.34-1.52

20 (m, 2H), 1.59-1.76 (m, 3H), 4.02 (t, J=7.3, 2H). ¹³ C-NMR (75 MHz, CDC1 ₃ ):

5 22.65, 27.84, 41.12, 107.64, 151.52, 155.40. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 minutes (1.0 mL/min) with 4 minute hold at 95% ACN; Wavelength: 254 nm): retention time: 3.29 min; 99.5% purity. Chiral HPLC (method: Chiralpak AD

25 25 cm column - isocratic method 78% hexane/ 22% isopropanol/0.1% diethylamine for 40 minutes at 1.00 mL/min; Wavelength: 254 nm): retention time: 24.34 min (R

BOST 1635628.1 - 87 - enantiomer); 28.82 min (expected for S enantiomer); >99% ee purity. MS (M+H- H ₂ 0): 276.3; (M+H): 294.3; (M+Na): 316.2.

B. ( _t ^-l-(4,4,5,6,6,6-dfi-5-Hvdroxyhexyn-3J-di(methyl-d ₁ -lH-purine- 2,6(3H,7H)-dione (Compound 434(S)). 1H-NMR (300 MHz, CDC1 ₃ ): δ 1.36-1.50 5 (m, 2H), 1.64-1.76 (m, 3H), 4.02 (t, J=7.5, 2H). ¹³ C-NMR (75 MHz, CDC1 ₃ ):

δ 22.65, 27.84, 41.12, 151.52, 155.40. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 minutes (1.0 mL/min) with 4 minute hold at 95% ACN; Wavelength: 254 nm): retention time: 3.29 min; 99.4% purity. Chiral HPLC (method: Chiralpak AD 25 10 cm column - isocratic method 78% hexane/ 22% isopropanol/0.1% diethylamine for 40 minutes at 1.00 mL/min; Wavelength: 254 nm): retention time: 24.34 min (expected for R enantiomer); 28.82 min (S enantiomer); >99% ee purity. MS (M+H- H ₂ 0): 276.3; (M+H): 294.3; (M+Na): 316.2.

15 Example 26. Synthesis of (±Vl-(4.4.5.6.6.6-dfi-5-hvdroxyhexylV3-methyl-7- methyl-d3-lH-purine-2,6(3H,7H)-dione (Compound 135).

Scheme 25. Pre aration of Compounds 135, 135(R) and 135(5).

20

Synthesis of (±)-! -(4,4,5, 6,6,6-dfi-5-hvdroxyhexyl)-3 -methyl-7-methyl-d3- lH-purine-2,6(3H,7H)-dione (Compound 135). Following the same general method as for the synthesis of Compound 137 in Example 17 above, a portion of Compound 435 (see Example 8) was converted to 0.99 g of Compound 135.

25

BOST 1635628.1 - 88 - Example 27. Chiral Separation of (i -l-(4,4,5,6,6,6-dfi-5-hvdroxyhexyr)-3 -methyl- 7-methyl-d?-lH-purine-2,6(3H,7H)-dione (Compound 135(R) and (S)-l- (4,4, 5,6,6, 6-dfi-5-hydroxyhexyl)-3 -methyl-7-methyl-d^-lH-purine-2,6(3H,7H - dione (Compound 135(5)).

5 Separation of Enantiomers of Compound 135. A portion of racemic

Compound 135 obtained as described above was separated in the same manner as racemic Compound 437 (see Example 12) to afford separated enantiomers

Compound 135(R) (352 mg) and Compound 135(5) (343 mg).

A. (R)- 1 -(4,4,5 ,6,6,6-dfi-5-hvdroxyhexyl)-3 -methyl-7-methyl-d ₃ - lH-purine- 10 2,6(3H,7H)-dione (Compound 135(R)V 1H-NMR (300 MHz, CDC1 ₃ ): δ 1.41-1.48

(m, 2H), 1.64-1.74 (m, 3H), 3.58 (s, 3H), 4.02 (t, J=7.4, 2H), 7.50 (s, 1H). ¹³ C- NMR (75 MHz, CDC1 ₃ ): δ 22.65, 27.84, 29.68, 41.12, 107.67, 141.38, 148.76, 151.52, 155.37. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη CI 8-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 minutes (1.0

15 mL/min) with 4 minute hold at 95% ACN; Wavelength: 305 nm): retention time:

3.27 min; 99.6% purity. Chiral HPLC (method: Chiralpak AD 25 cm column - isocratic method 78% hexane/ 22% isopropanol/0.1% diethylamine for 40 minutes at 1.00 mL/min; Wavelength: 254 nm): retention time: 25.21 min (R enantiomer); 28.42 min (expected for S enantiomer); >99.5% ee purity. MS (M+H-H ₂ 0): 272.1 ;

20 (M+H): 290.1 ; (M+Na): 312.3. Elemental Analysis (Ci ₃ HnD ₉ N ₄ 0 ₃ ): Calculated:

C=53.97, H=6.97, N=19.36. Found: C=53.83, H=6.98, N=19.30.

B. (5)-l-(4,4,5,6,6,6-dfi-5-hvdroxyhexyl)-3 -methyl-7-methyl-d ₃ -lH-purine- 2,6(3H,7H)-dione (Compound 135(5)). 1H-NMR (300 MHz, CDC1 ₃ ): δ 1.38-1.48 (m, 2H), 1.64-1.74 (m, 3H), 3.58 (s, 3H), 4.02 (t, J=7.4, 2H), 7.50 (s, 1H). ¹³ C-

25 NMR (75 MHz, CDC1 ₃ ): δ 22.64, 27.84, 29.68, 41.12, 107.67, 141.38, 148.76,

151.52, 155.37. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη CI 8-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 minutes (1.0 mL/min) with 4 minute hold at 95% ACN; Wavelength: 305 nm): retention time: 3.27 min; 99.8% purity. Chiral HPLC (method: Chiralpak AD 25 cm column -

30 isocratic method 78% hexane/ 22% isopropanol/0.1% diethylamine for 40 minutes at 1.00 mL/min; Wavelength: 254 nm): retention time: 25.39 min (R enantiomer;

minor species); 28.42 min (S enantiomer; major species); 99.1% ee purity. MS (M+H-H ₂ 0): 272.1 ; (M+H): 290.1 ; (M+Na): 312.3. Elemental Analysis

BOST 1635628.1 - 89 - (C ₁₃ H ₁₁ D ₉ N ₄ O ₃ ): Calculated: C=53.97, H=6.97, N=19.36. Found: C=53.93, H=7.03, N=19.29.

Example 28. Synthesis of (±) l-(5-Hvdroxyhexyl)-3-methyl-7-methyl-(iJ-lH- 5 .

Following the same general method as for the synthesis of Compound 437 in Example 11 above, Compound 100 (see Example 1) was treated with NaBH ₄ in EtOH and extracted with CH ₂ CI ₂ to afford Compound 116.

10 MS (M+H-H2O): 266.1; (M+H): 284.1; (M+Na): 306.0.

General Methods

General methods A-D were used for preparing the compounds shown in Exampli 29-39 below. The General Methods are described below and reference to the specific General Method is given in each of the following Examples.

General Method A: General procedure for the preparation of geminal difluoro compounds. A solution of boron trifluoride etherate (several drops) and bis(2- methoxyethyl)aminosulfur trifluoride ("Deoxo-fluor") (4.0 mmol, 1.5 equiv) in

20 dichloromethane (10 mL) was stirred for 30 minutes at room temperature. To the solution was added the appropriate ketone (2.7 mmol, 1 equiv) and the reaction was stirred at room temperature overnight. When TLC analysis revealed no further conversion, the reaction was quenched with saturated aqueous sodium bicarbonate solution (10 mL) and extracted with dichloromethane (2 x 100 mL). The combined

25 organic solution was dried over sodium sulfate, filtered, and evaporated under

reduced pressure to give a yellow-brown oil. The crude product was purified using an Analogix automated chromatography system eluting with a gradient of 0-8% methanol/dichloromethane over 30 minutes. The desired geminal difluoride eluted first, followed closely by unreacted starting material. Fractions containing the

30 desired product were evaporated to give a yellow oil. The oil was dissolved in ethyl

BOST 1635628.1 - 90 - acetate and slowly evaporated to give a white solid that was triturated with heptane, filtered and dried to provide the desired geminal difluoride product.

General Method B: General procedure for the preparation of mono-fluoro 5 compounds. A solution of boron trifluoride etherate (several drops) and bis(2- methoxyethyl)aminosulfur trifluoride ("Deoxo-fluor") (1.2 mmol, 1.4 equiv) in chloroform (5 mL) was stirred for 30 minutes at room temperature. To the solution was added the appropriate alcohol (0.9 mmol, 1 equiv) and the reaction was stirred at room temperature overnight. When TLC analysis revealed no further conversion,

10 the reaction was quenched with saturated aqueous sodium bicarbonate solution (10 mL) and extracted with dichloromethane (2 x 75 mL). The combined organic solution was dried over sodium sulfate, filtered, and evaporated under reduced pressure to give a yellow oil. The crude product was purified using an Analogix automated chromatography system eluting with a gradient of 0-8%

15 methanol/dichloromethane over 30 minutes. The desired fluoride product eluted first, followed by unreacted starting material. Fractions containing the desired product were evaporated to give a yellow solid. LC/MS usually indicated the material was a mixture of the desired mono-fluoride and an olefinic side-product. The olefinic side-product can be removed when the mixture is subjected to chiral

20 HPLC to isolate the two enantiomers (see General Method C).

General Method C: General procedure for the chiral HPLC separation of racemic mono-fluoro compounds to afford single enantiomers. The mixture of racemic mono-fluoride and olefinic side-product (obtained via General Method B)

25 was dissolved in HPLC-grade isopropanol (IP A). Enantiomer separation was

achieved using a Waters HPLC with preparative Daicel Chiralpak AD column (20 x 250 mm). The mobile phase consisted of 6% IPA in hexanes containing 0.5% diethylamine. The flow rate was 18 mL/minute and a typical run time was 80 min. As needed, each isolated single enantiomer was subjected to column

30 chromatography using an Analogix automated chromatography system eluting with a gradient of 0-5% methanol/dichloromethane to afford chemically pure material.

General Method D: General procedure for the deuterium-to-hydrogen exchange reaction on fluorinated 8-deuteroxanthine compounds. A mixture of

BOST 1635628.1 - 91 - the appropriate 8-deuteroxanthine compound (0.32 mmol, 1 equiv), K ₂ CO ₃ (40 mg, 0.3 mmol) and water (5 mL) was heated overnight at 120-125 °C (bath temperature). The mixture was concentrated to near dryness and additional water (4 mL) was added. The mixture was heated at 120-125 °C for 6 hours, then cooled. The mixture 5 was extracted with CH ₂ CI ₂ (5 x 20 mL) and the combined organic solution was dried over Na ₂ S0 ₄ and filtered. The filtrate was concentrated under reduced pressure and the resulting 8-H xanthine product was purified on an Analogix automated chromatography system eluting with 0-4% MeOH/CE^Cb, as needed, to afford the desired product.

10

Example 29. Synthesis of l-(5,5-Difluorohexyl)-3-methyl-7-methyl-(ij-lH-purine-

2 6(3H.7HVdione (Compound 151).

15 Following General Method A, Compound 151 was prepared from Compound 100 (see Example 1).

1H-NMR (300 MHz, CDCI ₃ ): δ 1.53-1.74 (m, 7H), 1.82-2.05 (m, 2H), 3.58 (s, 3H), 4.03 (t, J=7.5, 2H), 7.51 (s, 1H). ¹³ C-NMR (75 MHz, CDCI ₃ ): δ 20.15 (t, J= 4.7), 23.25 (t, J= 27.6), 27.67, 29.71, 37.58 (t, J= 25.4), 40.94, 141.41, 151.60, 155.25. 20 HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 minutes (1.0 mL/min) with 4 minute hold at 95% ACN; Wavelength: 305 nm): retention time: 5.04 min; 99.2% purity. MS (M+H): 304.2.

25 Example 30. Synthesis of (±) l-(5-Fluorohexyl)-3-methyl-7-methyl-(ij-lH-purine-

2 6(3H,7H -dione (Compound 147).

BOST 1635628.1 - 92 - Following General Method B, Compound 147 was prepared from Compound 116 (see Example 28).

MS (M+H): 286.2.

5 Example 31. Synthesis of (R)-l-(5-Fluorohexyl)-3-methyl-7-methyl-(i _j -lH-purine- 2.6(3H.7HVdione (Compound 147(R) and (SH-(5-FluorohexylV3-methyl-7- m thyl- j-lH-purine-2,6(3HJH -dione (Compound 147(S)V

General Method C was followed for the separation of Compounds 147(R) and

10 147(S) from racemic Compound 147.

A: Analytical data for the first eluting enantiomer is as follows:

1H-NMR (300 MHz, CDC1 ₃ ): δ 1.32 (dd, J,= 24.0, J ₂ = 6.1 , 3H), 1.40-1.56 (m, 2H), 1.64-1.76 (m, 4H), 3.58 (s, 3H), 4.02 (t, J= 7.6, 2H), 4.56-4.77 (doublet of multiplets, 1H), 7.51 (s, 1H). ¹³ C-NMR (75 MHz, CDC1 ₃ ): δ 21.01 (d, J= 22.7),

15 22.51 (d, J= 5.0), 27.81 , 29.70, 36.54 (d, J= 21.0), 41.16, 90.83 (d, J = 164.5),

141.36, 151.47, 155.30. HPLC (method: 20 mm CI 8-RP column - gradient method 2-95% ACN + 0.1% formic acid in 3.3 min with 1.7 min hold at 95% ACN;

Wavelength: 254 nm): retention time: 2.92 min; 98.2% purity. Chiral HPLC (method: Chiralpak AD 25 cm column - isocratic method 93% hexane/ 7%

20 isopropanol/0.1% diethylamine for 120 minutes at 0.700 mL/min; Wavelength: 254 nm): retention time: 50.17 min >99% ee purity. MS (M+H): 286.2.

B: Analytical data for the second eluting enantiomer is as follows:

1H-NMR (300 MHz, CDC1 ₃ ): δ 1.32 (dd, J,= 24.0, J ₂ = 6.1 , 3H), 1.50-1.57 (m, 2H), 1.59-1.73 (m, 4H), 3.58 (s, 3H), 4.02 (t, J= 7.6, 2H), 4.55-4.78 (doublet of

25 multiplets, 1H), 7.51 (s, 1H). ¹³ C-NMR (75 MHz, CDCI ₃ ): δ 21.01 (d, J= 22.7), 22.51 (d, J= 5.0), 27.81 , 29.70, 36.54 (d, J= 21.0), 41.16, 90.83 (d, J = 165.0), 141.35, 151.47, 155.29. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 minutes (1.0 mL/min) with 4 minute hold at 95% ACN; Wavelength: 305 nm): retention

30 time: 4.76 min; 99.4% purity. Chiral HPLC (method: Chiralpak AD 25 cm column - isocratic method 93% hexane/ 7% isopropanol/0.1% diethylamine for 120 minutes

BOST 1635628.1 - 93 - at 0.700 mL/min; Wavelength: 254 nm): retention time: 53.84 min >99% ee purity. MS (M+H): 286.2.

Example 32. Synthesis of l-(4,4-^?-6,6,6-^-5,5-Difluorohexyl)-8- -3,7-dimethyl- 5 lH- urine-2,6(3H,7H)-dione (Compound 500)

Following General Method A, Compound 500 was prepared from Compound 407 (see Example 10).

1H-NMR (300 MHz, CDC1 ₃ ): δ 1.51-1.56 (m, 2H), 1.66-1.74 (m, 2H), 3.58 (s, 3H), 10 3.99 (s, 3H), 4.02 (t, J=7.4, 2H). ¹³ C-NMR (75 MHz, CDCI3): δ 19.94, 27.62,

29.70, 33.59, 40.94, 155.27. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 minutes (1.0 mL/min) with 4 minute hold at 95% ACN; Wavelength: 305 nm): retention time: 5.02 min; >99.9% purity. MS (M+H): 307.2. Elemental Analysis

15 (Ci ₃ Hi ₂ D ₆ F ₂ N ₄ 0 ₂ ): Calculated: C=50.98, H=5.92, N=18.29, F=12.40. Found:

C=50.96, H=5.96, N=18.18, F=12.78.

Example 33. Synthesis of (±) l-(4,4-^2-6-6,6- ₃ -5-Fluorohexyl)-8- -3,7-dimethyl- lH- urine-2,6(3H,7H)-dione (Compound 501)

Following General Method B, Compound 501 was prepared from Compound 421 (see Example 15).

MS (M+H): 289.2.

25 Example 34. Synthesis of (R)-l-(4,4-A-6.6.6-^-5-Fluorohexyl)-8- -3J-dimethyl- lH-purine-2,6(3H,7H)-dione (Compound 50UR)) and (S)- 1 -(4,4-^-6,6,6-^-5- Fluorohexyl)-8-(i-3,7-dimethyl-lH-purine-2,6(3H,7H)-dione (Compound 501(SV)

BOST 1635628.1 QA and (R -l-(4,4-A-6,6,6-^-5-Fluorohexyn-3,7-dimethyl-lH-purine-2,6(3 HJH - dione (Compound 503(R)) and (S)-l-(4,4-(i2'6-6,6-(i2-5-Fluorohexyl)-3,7-dimethyl- lH-purine-2,6(3H,7H -dione (Compound 503(S)V

General Method C was followed for the separation of Compounds 501(R) and

501(S) from racemic Compound 501.

A: Analytical data for the first eluting enantiomer is as follows:

1H-NMR (300 MHz, CDC1 ₃ ): δ 1.42-1.53 (m, 2H), 1.63-1.72 (m, 2H), 3.58 (s, 3H),

10 4.02 (t, J= 7.3, 2H), 4.64 (d, J= 48.9, 1H). ¹³ C-NMR (75 MHz, CDC1 ₃ ): δ 22.30 (d, J= 5.0), 27.76, 29.69, 33.58, 41.16, 90.65 (d, J= 163.9), 141.32, 151.46, 155.29. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 minutes (1.0 mL/min) with 4 minute hold at 95% ACN; Wavelength: 305 nm): retention time: 4.75 min; 98.3% purity.

15 Chiral HPLC (method: Chiralpak AD 25 cm column - isocratic method 93%

hexane/ 7% isopropanol/0.1% diethylamine for 120 minutes at 0.700 mL/min;

Wavelength: 254 nm): retention time: 49.49 min >99.9% ee purity. MS (M+H): 289.2. Elemental Analysis (Ci3Hi ₃ D ₆ FN ₄ 0 ₂ ): Calculated: C=54.16, H=6.64, N=19.43, F=6.59. Found: C=53.96, H=6.28, N=19.13, F=6.90.

20 B: Analytical data for the second eluting enantiomer is as follows:

1H-NMR (300 MHz, CDCI3): δ 1.44-1.53 (m, 2H), 1.64-1.72 (m, 2H), 3.58 (s, 3H), 3.99 (s, 3H), 4.02 (t, J= 7.4, 2H), 4.63 (d, J= 48.7, 1H). ¹³ C-NMR (75 MHz, CDCI ₃ ): δ 22.30 (d, J= 5.0), 27.75, 29.69, 33.58, 41.16, 90.65 (d, J= 163.3), 148.74, 151.46, 155.29. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη

25 C18-RP column - gradient method 5-95%> ACN + 0.1%> formic acid in 14 minutes (1.0 mL/min) with 4 minute hold at 95% ACN; Wavelength: 305 nm): retention time: 4.75 min; 98.8% purity. Chiral HPLC (method: Chiralpak AD 25 cm column - isocratic method 93% hexane/ 7% isopropanol/0.1% diethylamine for 120 minutes at 0.700 mL/min; Wavelength: 254 nm): retention time: 53.25 min >99.9% ee

BOST 1635628.1 - 95 - purity. MS (M+H): 289.2. Elemental Analysis (Ci ₃ Hi ₃ D ₆ FN ₄ 0 ₂ ): Calculated: C=54.16, H=6.64, N=19.43, F=6.59. Found: C=54.19, H=6.68, N=19.27, F=6.73.

The first eluting 501 enantiomer, "Enantiomer A", was treated with K ₂ CO ₃ and 5 water according to General Method D to afford an enantiomer of Compound 503 with the same chirality as the "Enantiomer A" starting material.

1H-NMR (300 MHz, CDCI ₃ ): δ 1.40-1.57 (m, 2H), 1.62-1.74 (m, 2H), 3.58 (s, 3H), 3.99 (s, 3H), 4.02 (t, J= 7.5, 2H), 4.64 (d, J= 48.7, 1H), 7.50 (s, 1H). ¹³ C-NMR (75 MHz, CDCI ₃ ): δ 22.28 (d, J= 5.0), 27.76, 29.67, 33.56, 41.16, 90.60 (d, J= 163.9), 10 107.9, 141.37, 148.76, 151.47, 155.29. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 minutes (1.0 mL/min) with 4 minute hold at 95%> ACN; Wavelength: 305 nm): retention time: 4.74 min; 98.6% purity. MS (M+H): 288.3.

15 The second eluting 501 enantiomer, "Enantiomer B", was treated with K ₂ CO ₃ and water according to General Method D to afford an enantiomer of Compound 503 with the same chirality as the "Enantiomer B" starting material.

1H-NMR (300 MHz, CDCI ₃ ): δ 1.40-1.58 (m, 2H), 1.64-1.72 (m, 2H), 3.58 (s, 3H), 3.99 (d, J= 0.6, 3H), 4.02 (t, J= 7.3, 2H), 4.64 (d, J= 48.7, 1H), 7.50 (d, J= 0.6,

20 1H). ¹³ C-NMR (75 MHz, CDCI3): δ 22.28 (d, J= 5.0), 27.76, 29.67, 33.56, 41.16, 90.60 (d, J= 163.3), 107.9, 141.37, 148.76, 151.47, 155.29. HPLC (method:

Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 minutes (1.0 mL/min) with 4 minute hold at 95% ACN; Wavelength: 305 nm): retention time: 4.76 min; 98.7% purity. MS (M+H):

25 288.3.

As a result of the foregoing two deuterium-to-hydrogen exchange reactions, the f llowing two enantiomers were obtained:

30

BOST 1635628.1 - 96 - Example 35. Synthesis of l-(4,4-^7-6,6,6-^-5,5-Difluorohexyl)-3,7-dimethyl-lH- purine-2,6(3H,7H)-dione (Compound 502)

5 General Method D was followed for the preparation of Compound 502 from

Compound 500 (see Example 32).

1H-NMR (300 MHz, CDC1 ₃ ): δ 1.51-1.56 (m, 2H), 1.66-1.74 (m, 2H), 3.58 (s, 3H), 3.99 (d, J= 0.6, 3H), 4.02 (t, J= 7.4, 2H), 7.51 (d, J=0.5, 1H). ¹³ C-NMR (75 MHz, CDCI ₃ ): δ 19.93 (t, J= 5.0), 27.62, 29.67, 33.56, 40.91, 107.64, 141.42, 148.78,

10 151.45, 155.25. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 minutes (1.0 mL/min) with 4 minute hold at 95% ACN; Wavelength: 305 nm): retention time: 5.01 min; >99.9% purity. MS (M+H): 306.3. Elemental Analysis

(Ci ₃ Hi ₃ D ₅ F ₂ N ₄ 02): Calculated: C=51.14, H=5.94, N=18.35, F=12.54. Found:

15 C=51.01, H=5.96, N=18.32, F=12.63.

Example 36. Synthesis of l-(4,4-^?-6,6,6- 3-5,5-Difluorohexyl)-8- -3-methyl-7- meth l-d3-lH-purine-2,6(3H,7H)-dione (Compound 504).

20 Following General Method A, Compound 504 was prepared from Compound 409 (see Example 2).

1H-NMR (300 MHz, CDCI ₃ ): δ 1.51-1.56 (m, 2H), 1.66-1.74 (m, 2H), 3.58 (s, 3H), 4.02 (t, J= 7.4, 2H). ¹³ C-NMR (75 MHz, CDCI ₃ ): δ 19.94 (t, J= 4.8), 27.62, 29.67, 40.91, 151.46, 155.26. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη CI 8- 25 RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 minutes (1.0 mL/min) with 4 minute hold at 95% ACN; Wavelength: 305 nm): retention time: 5.00 min; 99.2% purity. MS (M+H): 310.2. Elemental Analysis (Ci ₃ H ₉ D ₉ F ₂ N ₄ 02):

BOST 1635628.1 - 97 - Calculated: C=50.48, H=5.87, N=18.11, F=12.54. Found: C=50.30, H=5.79, N=17.76, F=12.28.

Example 37. Synthesis of l-(4,4-^?-6,6,6-^-5,5-Difluorohexyl)-3-methyl-7- 5 meth l-d3-lH-purine-2,6(3H,7H)-dione (Compound 505).

Following General Method D, Compound 505 was prepared from Compound 504 (see Example 36).

1H-NMR (300 MHz, CDC1 ₃ ): δ 1.51-1.56 (m, 2H), 1.66-1.76 (m, 2H), 3.58 (s, 3H), 10 4.02 (t, J= 7.4, 2H), 7.51 (s, 1H). ¹³ C-NMR (75 MHz, CDC1 ₃ ): δ 19.92 (t, J= 4.7), 27.61, 29.67, 40.91, 141.40, 148.78, 151.44, 155.24. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 minutes (1.0 mL/min) with 4 minute hold at 95% ACN; Wavelength: 305 nm): retention time: 5.00 min; >99.9% purity. MS (M+H): 309.3. 15 Elemental Analysis (Ci ₃ Hi ₀ D ₈ F ₂ N ₄ O2): Calculated: C=50.64, H=5.88, N=18.17, F=12.32. Found: C=50.67, H=5.84, N=17.74, F=12.49.

Example 38. Synthesis of l-(4,4-^7-6,6,6- ₃ -5-Fluorohexyl)-8- -3-methyl-7- meth l-d ₃ -lH-purine-2,6(3H,7H)-dione (Compound 506).

Following General Method B, Compound 506 was prepared from Compound 419 (see Example 6).

MS (M+H): 292.2

25 Example 39. Synthesis of (R -l-(4,4- 7-6.6.6- ₃ -5-Fluorohexyn-8- -3-methyl-7- methyl-d ₃ -lH-purine-2,6(3H,7H -dione (Compound 506(R) and (S -l-(4,4-A- 6,6,6-(i ₃ -5-Fluorohexyl)-8-(i-3-methyl-7-methyl-d ₃ -lH-purine-2,6(3H,7H)-dione (Compound 506(S)V

BOST 1635628.1 GO

General Method C was followed for the separation of Compounds 506(R) and

506(S) from racemic Compound 506.

5 A: Analytical data for the first eluting enantiomer is as follows:

1H-NMR (300 MHz, CDC1 ₃ ): δ 1.44-1.50 (m, 2H), 1.64-1.72 (m, 2H), 3.58 (s, 3H), 4.02 (t, J= 7.6, 2H), 4.64 (d, J= 48.6, 1H). ¹³ C-NMR (75 MHz, CDC1 ₃ ): δ 22.28 (d, J= 5.0),|27.76, 29.67, 41.16, 90.60 (d, J= 163.3), 151.48, 155.30. HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95%

10 ACN + 0.1%) formic acid in 14 minutes (1.0 mL/min) with 4 minute hold at 95% ACN; Wavelength: 305 nm): retention time: 4.73 min; 99.4% purity. Chiral HPLC (method: Chiralpak AD 25 cm column - isocratic method 93% hexane/ 7% isopropanol/0.1% diethylamine for 70 minutes at 0.700 mL/min; Wavelength: 254 nm): retention time: 50.54 min >99.9% ee purity. MS (M+H): 292.2. The first

15 eluting enantiomer was found to have an [CXJ D of -6.7 (0.506 g/100 ml CHC1 ₃ ).

B: Analytical data for the second eluting enantiomer is as follows:

HPLC (method: Waters Atlantis T3 2.1 x 50 mm 3 μιη C18-RP column - gradient method 5-95% ACN + 0.1% formic acid in 14 minutes (1.0 mL/min) with 4 minute

20 hold at 95% ACN; Wavelength: 305 nm): retention time: 4.72 min; 99.0% purity.

Chiral HPLC (method: Chiralpak AD 25 cm column - isocratic method 93% hexane/ 7% isopropanol/0.1% diethylamine for 70 minutes at 0.700 mL/min;

Wavelength: 254 nm): retention time: 56.48 min 97% ee purity. MS (M+H): 292.2. The second eluting enantiomer was found to have an [α]ο of +5.5 (0.489 g/100 mL

25 CHC1 ₃ ).

Example 40. Synthesis of (R)-l-(5-hvdroxyhexyl)-3,7-dimethyl-lH-purine- 2.6(3H.7H -dione (Compound 149(R) and (S) -(5-hvdroxyhexyl)-3.7-dimethyl- lH-purine-2,6(3H,7H)-dione (Compound 149(S)V

30

BOST 1635628.1 GO

Step 1. Synthesis of (±)l -(5-hydroxyhexyl)-3,7-dimethyl-lH-purine- 2,6(3H,7H)-dione (60). As outlined in Scheme 26 below, and following the same 5 general method as for the synthesis of Compound 437 in Example 1 1 above,

commercially available 59 was treated with NaBH ₄ in EtOH to afford 2.0 g of 60.

Scheme 26. Pre aration of Compounds 60, 60(R) and 60(S).

Step 2. Separation of Enantiomers of Compound 60. A portion of racemic 60 obtained as described above was separated in the same manner as racemic Compound 437 (see Example 12) to afford separated enantiomers Compound 60(R) (480 mg) and Compound 60(5) (430 mg).

15

Step 3. Synthesis of (R)-l-(5-hvdroxyhexyl)-3,7-dimethyl-lH-purine- 2,6(3H,7H)-dione (Compound 149(R) and (SVl-(5-hvdroxyhexylV3 J-dimethyl- lH-purine-2,6(3H,7H)-dione (Compound 149(SV). Enantiomer 60(R) was subjected to the conditions described in General Method B to afford an enantiomer of 20 Compound 149.

Chiral HPLC (method: Chiralpak AD 25 cm column - isocratic method 93% hexane/ 7% isopropanol/0.1% diethylamine for 70 minutes at 0.700 mL/min;

BOST 1635628.1 - 100 - Wavelength: 254 nm): retention time: 50.48 min (first eluting enantiomer; minor species); 53.90 min (second eluting enantiomer; major species); 90% ee purity. MS (M+H): 283.2.

5 Enantiomer 60(S) was subjected to the conditions described in General Method B to afford an enantiomer of Compound 149.

Chiral HPLC (method: Chiralpak AD 25 cm column - isocratic method 93% hexane/ 7% isopropanol/0.1% diethylamine for 70 minutes at 0.700 mL/min;

Wavelength: 254 nm): retention time: 49.98 min (first eluting enantiomer; major 10 species); 53.67 min (second eluting enantiomer; minor species); 83% ee purity. MS (M+H): 283.2.

As a result of the foregoing two fluorination reactions, the following two

enantiomers were obtained:

Example 41. Synthesis of (S)-l-(4,4,6,6,6-d _s -5-fluorohexyl)-3-methyl-7-methyl-dT lH-purine-2,6(3H,7H)-dione (Compound 507(S) and (RVl-(4.4.6.6.6-d.-5- fluorohexyl)-3-methyl-7-methyl-d^-lH-purine-2,6(3H,7H)-dione (Compound

507 R)V

Step 1. Synthesis of l-(4,4,6.6.6-d 5-fluorohexyn-3-methyl-7-methyl-d lH-

BOST 1635628.1 - 101 - purine-2,6(3H,7H)-dione (Compound 507). Following General Method D, Compound 507 was prepared from Compound 506 (see Example 38).

Step 2. Separation of (S)-l-(4,4,6,6,6-d _s -5-fluorohexyl)-3-methyl-7-methyl- d lH-purine-2,6(3H,7Hydione (Compound 507(SV) and (R)-l-(4A6.6.6-d,-5- 5 fluorohexyl)-3-methyl-7-methyl-d3-lH-purine-2,6(3H,7H)-dione (Compound

507 R)V

General Method C was followed for the separation of Compounds 507(R) and 507(S) from racemic Compound 507.

10 A: Analytical data for the 507(R) is as follows:

1H-NMR (300 MHz, CDC1 ₃ ): δ 1.42-1.53 (m, 2H), 1.64-1.74 (m, 2H), 3.58 (s, 3H), 4.02 (t, J= 7.8, 2H), 4.64 (d, J= 48.6, 1H), 7.50 (s, 1H). ¹³ C-NMR (75 MHz, CDCI ₃ ): δ 22.25, 22.32, 27.76, 29.67, 41.16, 141.35, 148.76, 151.47, 155.29. HPLC (method: Waters Atlantis T3 50 mm - gradient method 5-95% ACN + 0.1% formic

15 acid in 14 min (1.0 mL/min) with 4 min hold at 95% ACN + 0.1% formic acid; wavelength: 305 nm): retention time: 4.68 min; 99.4% purity. Chiral HPLC (method: Chiralpak AD 25 cm - gradient method 93% hexane + 7% IP A (+ 0.1% diethylamine) for 70 min (0.700 mL/min); wavelength: 254 nm): retention time: 55.5 min; >99% ee. MS (M+H): 291.3.

20 B: Analytical data for 507(S) is as follows:

1H-NMR (300 MHz, CDCI ₃ ): δ 1.40-1.54 (m, 2H), 1.64-1.74 (m, 2H), 3.58 (s, 3H), 4.02 (t, J= 7.6, 2H), 4.64 (d, J= 48.9, 1H), 7.50 (s, 1H). ¹³ C-NMR (75 MHz, CDCI3): δ 22.25, 22.32, 27.76, 29.67, 41.16, 89.52, 91.69, 141.34, 151.47, 155.29. HPLC (method: Waters Atlantis T3 50 mm - gradient method 5-95% ACN + 0.1%

25 formic acid in 14 min (1.0 mL/min) with 4 min hold at 95% ACN + 0.1% formic acid; wavelength: 305 nm): retention time: 4.73 min; 99.5% purity. Chiral HPLC (method: Chiralpak AD 25 cm - gradient method 93% hexane + 7% IP A (+ 0.1% diethylamine) for 70 min (0.700 mL/min); wavelength: 254 nm): retention time: 55.5 min; 95.4% ee. MS (M+H): 291.3. Elemental Analysis (CoHnDgF^C^):

BOST 1635628.1 - 102 - Calculated: C=54.16, H=6.64, N=19.43, F=6.59. Found: C=54.19, H=6.68, N=19.27, F=6.73.

BIOLOGICAL EVALUATION

5 Example 42a. Evaluation of Pharmacokinetics in Dogs Following Oral

Administration. Comparison of Compound 409 and Pentoxifylline

Metabolism of the title compounds were studied following oral administration to male beagle dogs. Blood samples were removed from dosed dogs at various time points and plasma isolated therefrom. The plasma samples were used

10 for the determination of plasma drug levels by LC-MS/MS (liquid chromatography with tandem mass spectrometry) for estimating pharmacokinetic parameters.

Compound 409 and pentoxifylline were dissolved separately in saline to a concentration of 4 mg/mL. A 1 : 1 (v/v) mixture of the two solutions was prepared to yield a solution having a final concentration of 2 mg/mL of both Compound 409 and

15 pentoxifylline.

Two male beagle dogs were fasted overnight and then orally dosed via gavage with 2.5 mg/kg of Compound 409 and pentoxifylline using the mixture described above. Blood samples (1.5 - 2 mL) were collected via the femoral vein at 0 min (pre-dose), 15 min, 30 min, 45 min, 1 hr, 1.5 hr, 2 hr, 3 hr, 4 hr, 6 hr, 8 hr, 10

20 hr, 12 hr, 16 hr and 24 hr post-dose. Blood was stored on ice prior to centrifugation to obtain plasma samples. Centrifugation took place within 1 hour of blood collection to harvest plasma (maximum volume). The plasma was decanted immediately and frozen/stored at -70 °C until analysis.

25 Table 8. Plasma Levels of Compound 409 vs Pentoxifylline in Dogs (Example 41a)

a) % Difference = [(deuterated species)-(nondeuterated

species)] (100)/(nondeuterated species)

Table 8 shows the results of the evaluation described in Example 41a. The average C _max and average AUC for Compound 409, a deuterated version of

BOST 1635628.1 - 103 - pentoxifylline, were significantly greater than for pentoxifylline. The deuterated compound exhibited greater exposure in the dog plasma than pentoxifylline.

Example 42b. Repeat Evaluation of Pharmacokinetics in Dogs Following Oral 5 Administration. Comparison of Compound 409 and Pentoxifylline with Monitoring of Metabolites

Example 41a was repeated with additional monitoring of the pentoxifylline and Compound 409 metabolites. In this experiment Compound 409 and pentoxifylline were dissolved separately in saline to a concentration of 4.4 and 4 10 mg/mL respectively. A 1 : 1 (v/v) mixture of the two solutions was prepared to yield a solution having a final concentration of 2.2 mg/mL of Compound 409 and 2 mg/mL pentoxifylline. Post-dosing data analysis included adjustments to account for the 10% difference in dosing concentration between compound 409 and pentoxifylline.

15 Four beagle dogs (2-3 years of age, and weighed 5 to 8 kg) were fasted overnight and then orally dosed via gavage with 2.75 mg/kg Compound 409 and 2.5 mg/kg pentoxifylline using the mixture described above. Blood samples

(approximately lmL) were collected via femoral vein at 0 min (pre-dose), 5 min, 15 min, 30 min, 45 min, 1 fir, 1.5 hr, 2 hr, 3 hr, 4 hr, and 6 hr post-dose. Blood was

20 stored on ice prior to centrifugation to obtain plasma samples. Centrifugation took place within 15 minutes of blood collection to harvest plasma (maximum volume). The plasma was decanted immediately and frozen/stored at -20 °C until analysis.

Plasma samples were analyzed by LC-MS/MS for the presence of the administered com ound and its corresponding Ml metabolite:

25

pentoxifylline Ml

BOST 1635628.1 - 104 -

Compound 409 (administered) Compound 419 (Ml metabolite)

5

The results from each of the four dogs are shown in FIGS. 1A and IB. The results from one of the four dogs (Dog H, FIG. lb) were inconsistent with that of the other three. That dog showed a 10-fold higher plasma concentration of each of the administered compounds and their respective metabolites at 5 minutes post-

10 administration. In addition, that dog did not show a characteristic increase in plasma concentration of the administered compounds between 5 and 15 minutes post- administration. It was concluded that this dog was most likely improperly gavaged and that the compounds were probably administered through the trachea, rather than into the GI tract as would have been desired. Accordingly, the data from this dog

15 was excluded from the analyses. The summary analysis of the three remaining dogs is shown in Table 9.

Table 9. Plasma Levels of Compound 409 vs Pentoxifylline in Dogs (Example 41b)

a) The dosing concentration of compound 409 was 10% higher than that for

20 pentoxifylline and thus the numbers reported here reflect the adjustment for that 10%) increase.

b) % Difference = [(deuterated species)-(nondeuterated

species)] (100)/(nondeuterated species)

25

BOST 1635628.1 - 105 - As can be seen in Table 9, higher levels of Compound 409 in terms of C _max and AUC were observed when compared to pentoxifylline co-dosed at the same level. FIG. 1 demonstrates that Compound 409 was more slowly cleared from the plasma than pentoxifylline in the three dogs that were orally dosed. FIG. la and lb demonstrate that Compound 409 was more slowly cleared from the plasma than pentoxifylline in the three dogs that were orally dosed. FIGS, la and lb also show that overall systemic exposure to Compound 419 (the deuterated Ml metabolite of 409) following dosing of Compound 409 was greater than that of the Ml metabolite following dosing of pentoxifylline.

Example 42c. Evaluation of Pharmacokinetics in Dogs Following Oral

Administration. Comparison of Compound 413 and Pentoxifylline.

This study was similar to those described in Examples 41a and 41b, except that Compound 413 was evaluated. Four male beagle dogs were orally dosed by gavage with a mixture containing 2 mg/mL each of pentoxifylline and Compound 413 in saline. Blood samples were taken as in Example 41b.

Table 10. Plasma Levels of Compound 413 vs Pentoxifylline in Dogs (Example

20 a) % Difference = [(deuterated species)-(nondeuterated

species)] (100)/(nondeuterated species)

The results of this study are summarized in Table 10 above. The table depicts the plasma levels of Compound 413 compared to pentoxifylline following 25 oral dosing. Higher levels of Compound 413 in terms of C _max and AUC were

observed when compared to pentoxifylline co-dosed at the same level.

Example 43. Evaluation of the Stability of Compounds in Rat Whole Blood. Comparison of Compounds 409, 435(5), 435(R) and Pentoxifylline and its M-l 30 Metabolites.

This study was performed to evaluate the stability of the title compounds in

BOST 1635628.1 ] ( f. rat whole blood. Because the ketone (or keto-compound; either pentoxifylline or 409) and its corresponding M-l alcohol metabolite interconvert, levels of these components were measured after either the keto-compound was added to the blood or the M-l was added. In other words, in some tests the keto-compound was the 5 starting test compound and in other tests an M-l metabolite was the starting test compound.

Fresh rat whole blood was obtained from ViviSource Laboratories, Waltham, MA. Stock solutions (7.5 millimolar (mM)) of test compounds were prepared in dimethyl sulfoxide (DMSO). The 7.5 mM stock solutions were diluted to 500

10 micromolar (μΜ) in acetonitrile (ACN). To 990 microliters (μί) of blood pre- warmed to 37 °C for 7 minutes was added 10 of 500 μΜ test compound to a final concentration of 5 μΜ. The test compounds were pentoxifylline, (5)-Ml metabolite of pentoxifylline, (i?)-Ml metabolite of pentoxifylline, Compound 409, Compound 435(5), and Compound 435(R). The latter two test compounds are deuterated (S)-

15 Ml and (i?)-Ml metabolites, respectively, of Compound 409. The reaction mixture was incubated at 37 °C. Aliquots (50 μί) were removed at 0 min, 5 min, 15 min, 30 min, 1 hour and 2 hours following the addition of test compound and added to 96- well plates containing 150 μί of ice cold acetonitrile with an internal standard to stop the reaction. The plates were stored at -20 °C for 20 minutes after which 100

20 μΐ, of 50% acetonitrile/water was added to the wells of the plate prior to

centrifugation to pellet precipitated proteins. A 200-μί aliquot of each supernatant was transferred to another 96-well plate and analyzed by LC-MS/MS using an Applied Bio-systems API 4000 mass spectrometer for amounts of the administered compound and its specific metabolite listed in Table 1 1 below.

25

Table 1 1. Compound-Metabolite Pairs Analyzed in Rat Whole Blood. (Experiments

BOST 1635628.1 - 107 - Experiment Pair Compound Incubated with Metabolite Analyzed

Blood

F Compound 435(R) Compound 409

a) Mass observed via LC-MS/MS. Stereochemistry presumed to be >95% (S) based on published pentoxifylline metabolism reports.

The results of this study are depicted in FIGS. 2 and 3. The time course of 5 metabolite formation is shown in FIG. 2. The relative amount of metabolite formed, as shown in FIG. 3, was calculated based on the amount present at 2 hr relative to the earliest time point at which it was detected in the incubation mixture, 5 minutes for A and B, and 15 minutes for C.

As seen in FIG. 3, after approximately 2 hours the amount of (5)-Ml formed

10 in rat whole blood incubated with pentoxifylline (Fig 3, column A) was similar to the amount of Compound 419(5) formed in rat whole blood incubated with

Compound 409 (Fig 3, column B). Thus, the deuterium substitution in Compound 409 had no discernable effect on the relative level of deuterated (5)-Ml metabolite (Compound 419(5)) formed as compared to the relative level of undeuterated (S)-

15 Ml formed from undeuterated pentoxifylline.

For the reverse reaction, (5)-Ml to the keto-compound, deuteration did have a significant effect. Column C in FIG. 3 shows an appreciable amount of pentoxifylline present after addition of (5)-Ml . By contrast, 2 hours after addition of Compound 435 (5), Compound 409 was not detected (FIG. 3, column D). Under

20 these conditions, the deuterium substitution in Compound 435 (5) impedes the

conversion of this compound to the corresponding ketone. Such an effect is particularly beneficial for enhancing the plasma levels of the desired M-l metabolite.

No metabolism of (i?)-Ml to pentoxifylline was detected in this assay.

25 Similarly, Compound 409 was not detected after addition of Compound 435 (R) to the rat blood. Thus, no conclusions could be made concerning the effect of deuteration on the conversion of (i?)-Ml to pentoxifylline. FIG. 2 shows the time course of the specific metabolite produced during incubation of the administered compound with rat whole blood.

BOST 1635628.1 - 108 - Example 44. Evaluation of Compound Stability in Human Liver Microsomes. Comparison of Compounds 409, 435(5), 435(R) and Pentoxifylline.

Example 43 is similar to Example 42 in design, except that human liver 5 microsomes were used instead of rat whole blood to study the metabolism of the compounds. Table 11 above shows each pair of test compound and metabolite that was analyzed in this Example 43.

Human liver microsomes (20 mg/mL) were obtained from Xenotech, LLC (Lenexa, KS). β -nicotinamide adenine dinucleotide phosphate, reduced form

10 (NADPH), magnesium chloride (MgCl ₂ ), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich.

Stock solutions containing 7.5 mM of test compounds (pentoxifylline, (S)- Ml metabolite, (i?)-Ml metabolite, Compound 409, Compound 435(5), and

Compound 435(R)) were prepared in DMSO. The 7.5-mM stock solutions were

15 diluted to 250 μΜ in acetonitrile (ACN). The human liver microsomes were diluted to 2.5 mg/mL in 0.1 M potassium phosphate buffer, pH 7.4, containing 3 mM MgCl ₂ . The diluted microsomes were added to wells of a 96-well deep-well polypropylene plate in triplicate. 10 μΐ, οΐ the 250 μΜ test compound was added to the microsomes and the mixture was pre-warmed to 37 °C for 10 minutes.

20 Reactions were initiated by addition of pre-warmed NADPH solution. The final reaction volume was 0.5 mL and contained 2.0 mg/mL human liver microsomes, 5 μΜ test compound, and 2 mM NADPH in 0.1 M potassium phosphate buffer, pH 7.4, and 3 mM MgCl ₂ . The reaction mixtures were incubated at 37 °C, and 50-μί aliquots were removed at 0, 5, 10, 20, and 30 minutes and added to shallow-well 96-

25 well plates which contained 50 μί of ice-cold acetonitrile with internal standard to stop the reactions. The plates were stored at 4 °C for 20 minutes after which 100 μΐ _^ of water was added to the wells of the plate before centrifugation to pellet precipitated proteins. Supernatants were transferred to another 96-well plate and analyzed for the amount of the administered compound and its specific metabolite

30 (listed in Table 11 above) by LC-MS/MS using an Applied Bio-systems API 4000 mass spectrometer.

The results of this study are depicted in FIGS. 4 and 5. The time course of metabolite formation is shown in FIG. 4. The relative amount of metabolite formed,

BOST 1635628.1 - 109 - as shown in FIG. 5, was calculated based on the amount present at 30 minutes relative to the earliest time point at which it was detected in the incubation mixture, 0 minutes for A, B, C and E, 5 minutes for D, and 10 minutes for F. The amount of (iS)-Ml formed in human liver microsomes incubated with pentoxifylline (FIG. 5, 5 column A) after 30 minutes was similar to the amount Compound 419(5) formed in human liver microsomes incubated with Compound 409 (FIG. 5, column B). Thus, deuteration of pentoxifylline as embodied by Compound 409 had no discernable effect on the relative level of deuterated (5)-Ml metabolite (Compound 419(5)) formed as compared to the relative level of undeuterated (S)-M1 formed from 10 undeuterated pentoxifylline. These results in human liver microsomes were

consistent with those seen using rat whole blood.

For the reverse reaction, (5)-Ml to the keto-compound, deuteration did have an appreciable effect. Column C in FIG. 5 shows a significant amount of pentoxifylline present 30 minutes after addition of (5)-Ml . By contrast, after

15 addition of Compound 435 (5), the level of Compound 409 that was detected after 30 minutes was less than the level of (S)-M1 (Fig 5, column D). Approximately 30% more pentoxifylline was produced from (S)-M1 than Compound 409 produced from Compound 435 (5). Under these conditions, the deuterium substitution in Compound 435 (5) impedes the conversion of this compound to the corresponding

20 ketone. While deuterium had a greater effect in rat blood, the results are consistent.

A dramatic deuterium effect on the metabolism of (i?)-Ml metabolite was observed in human liver microsomes. Deuteration of (i?)-Ml (Compound 435(R)) reduced by almost 5 -fold the amount of deuterated pentoxifylline formed

(Compound 409) after 30 minute incubation with human liver microsomes as

25 compared to the amount of undeuterated pentoxifylline formed from undeuterated (i?)-Ml (comparing columns E and F in FIG. 5). FIG. 4 shows the time course of the specific metabolite produced during incubation of the administered compound with human liver microsomes.

30 Example 45. Pharmacokinetic Study in Rats of (5)-Ml and Compound

435(5) After Oral and Intravenous Dosing.

(iS)-Ml and Compound 435(5) (a deuterated form of (5)-Ml) were separately dissolved in saline at a concentration of 10 mg/mL. A 1 : 1 mixture of the two

BOST 1635628.1 - 1 10 - compounds was then prepared containing a final concentration of 5 mg/mL of each compound, which was used for intravenous administration. For oral administration the mixture was further diluted in saline to a final concentration of 1 mg/mL for each compound.

5 Three male Sprague-Dawley rats were used in each of the oral and

intravenous studies. Animals were fasted overnight prior to administration of compounds. Intravenous administration was achieved by bolus injection of a single 5 mg/kg dose of the 1 : 1 combination into the cannulated jugular vein of the rats. Cannulation was achieved the day prior to dosing on rats that had been placed under

10 anesthesia using ketamine (IM 30 mg/kg). Oral administration was achieved by oral gavage of a single 5 mg/kg dose. Blood samples (250 μί) were collected from the dosed rats at various times post-dosing (2 min, 5 min, 10 min, 20 min, 30 min, 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr) by retro-orbital sampling of the rats temporarily anesthetized with isoflurane. Blood samples were placed in tubes containing K ₂ -

15 EDTA and stored on ice until centrifuged. Within 30 minutes of collection, plasma was isolated by centrifugation. A 100-μΙ, aliquot was removed, mixed with 200 μΙ_, of acetonitrile and stored at -20 °C until further analysis by LC-MS/MS using an Applied Bio-systems API 4000 mass spectrometer.

Samples were analyzed for the presence of the administered compound, the

20 corresponding ketone (pentoxifylline and Compound 409) and the corresponding M5 metabolite. Samples (10 μί) were injected into a Zorbax SB-C8 (Rapid

Resolution) column (2.1 x 30 mm, 3.5 μιη). The initial mobile phase condition was 100% A (10 mM ammonium acetate in water) and 0% B (methanol) with a flow rate at 0.5 mL/min. Mobile phase B was allowed to reach 55% in 3 minutes and from

25 55% to 90% in 1 minute before ramping back to 0% in another minute. The overall run time was 5 minutes. For pentoxifylline and its Ml and M5 metabolites, the precursor/product ion pairs were set at m/z 281/193 (Ml), m/z 279/181

(pentoxifylline), and m/z 267/221 (M5).

For Compound 435(5) and Compound 409 more than one ion pair was set up

30 for to detect species that arose from loss of deuterium. It was found that some

degree of deuterium loss occurs on those compounds of the invention, such as Compound 409, which have deuterium on the side chain at positions adjacent to the carbonyl carbon. This loss of deuterium appears to occur both in vivo and ex vivo

BOST 1635628.1 . 1 1 1 . by an unknown mechanism. The addition of acetonitrile to serum samples was used to stop any additional ex vivo deuterium loss prior to analysis. Typically, no more than 2 deuterium atoms were replaced by hydrogen. For Compound 435(5), there is a deuterium at the methinyl position which was lost upon oxidation to the keto- 5 compound 409. Reduction of 409 to an Ml metabolite introduced a proton at the methinyl position. When serum from animals dosed with 435(5) were analyzed to quantitate administered compound and metabolites, compound species were included with one and two less side chain deuteriums in the total amounts (referred to hereinafter as the "-1D" and the "-2D" species). Thus, for Compound 435(5) and

10 Compound 409 separate ion pairs were set up to detect the compound and its

corresponding - ID and -2D species. For Compound 435(5) three ion pairs were detected: m/z 291/197, 290/197, and 189/197. For Compound 409 ion pairs of m/z 288/186, 287/186 and 286/186 were monitored. Inclusion of -ID and -2D species in the measurements of Compound 409 and Compound 435(5) more accurately

15 quantitates the total active species and is reasonable based on what is known about the metabolism and activities of pentoxifylline and its M-l metabolites. Increased plasma exposure to Compound 409 or any M-l metabolites of 409 would be desirable. This includes the - ID and -2D species.

For the corresponding deuterated M5 metabolite (M5a):

20 (M5a), which has no deuterium on its acid side chain, only one ion pair was used at m/z 271/225. The internal standard for the analysis was indiplon.

BOST 1635628.1 - 112 - Table 12. Pharmacokinetic Results After Oral Administration of 435(S) and (S)-Ml in Rats.

a) Mass observed via LC-MS/MS. Stereochemistry presumed to be >95%> (S) based on published pentoxifylline metabolism reports.

5 b) % Difference = [(deuterated species)-(nondeuterated

species)] (100)/(nondeuterated species)

The results of the oral administration in rats are shown in Table 12. The deuterated Compound 435(5) demonstrated a significantly higher AUCo- _∞ and C _max

10 than its undeuterated counterpart (5)-Ml . Because there is a significant serum

interconversion between S)-M1 and pentoxifylline and both species are

therapeutically active, we also quantitated AUCo- _∞ and C _max for S)-M1 together with pentoxifylline, and for Compound 435(5) together with Compound 409.

Compound 435(5) together with Compound 409 demonstrated a significantly higher

15 AUCo-oo and C _max than did S)-M1 together with pentoxifylline after the oral

administration of (5)-Ml and 435(5) respectively.

The AUCo-oo was also measured for the M-5 and M5a metabolites arising from the oral administration of (5)-Ml and 435(5), respectively. The M-5 metabolite may be associated with toxicity in certain patients and is considered

20 undesirable. Table 12 shows that oral administration of Compound 435(5) provides considerably less M5a compared to the level of M5 obtained after administration of non-deuterated (5)-Ml . The ratio of active species to M5 metabolite was much more favorable for the deuterated compounds than for the non-deuterated compounds. The ratio of (Compound 435(5) + Compound 409) to M5a was 7.0, which was much

25 better than the ratio of 1.6 for ((5)-M 1 + pentoxifylline) to M5.

BOST 1635628.1 - 1 13 - Table 13. Pharmacokinetic Results After Intravenous Administration in Rats.

a) Mass observed via LC-MS/MS. Stereochemistry presumed to be >95%> (S) based on published pentoxifylline metabolism reports.

5 b) % Difference = [(deuterated species)-(nondeuterated

species)] (100)/(nondeuterated species)

Table 13 shows the results following intravenous administration in rats. The results for intravenous administration were similar to those for oral administration.

10 Compound 435(5) had an average AUCo_ _∞ that was 1 10% greater than its

undeuterated counterpart (5)-Ml after intravenous administration. Compound

435(5) together with Compound 409 had an average AUCo_ _∞ that was 79% greater than (5)-Ml together with pentoxifylline after intravenous administration.

Intravenous administration of Compound 435(5) provides an amount of M5a

15 metabolite that is 15% less than the amount of M5 metabolite than is provided by intravenous administration of (5)-Ml . The ratio of active species to the

corresponding M5 metabolite in rats that were intravenously administered

Compound 435(5) was 7.4 as compared to 3.5 for rats that were intravenously administered (S)-M 1.

20

Example 46. Pharmacokinetic Study of Pentoxifylline and Compound 435(S) in Chimps After Oral and Intravenous Dosing.

Pentoxifylline and Compound 435(5) were separately dissolved in warm (65 °C) saline at a concentration at 10 mg/mL. A 1 : 1 mixture of the two compounds 25 was then prepared containing a final concentration of 5 mg/mL of each compound

BOST 1635628.1 - 1 14 - and the mixture was then sterile filtered through a 0.2-μιη filter.

Two chimps (one male and one female) were used in each of the oral and intravenous studies. Animals were fasted overnight prior to administration of compounds. All animals were sedated with ketamine (approximately 10 mg/kg) 5 and/or telazol (approximately 5 mg/kg) prior to dosing. Intravenous administration was achieved by IV infusion of 75 mg of each compound (15 mL total dosing solution) over 10 minutes. Oral administration was achieved by oral gavage of a single 75 mg dose of each compound (15 mL total dosing solution). Blood samples (6 mL) were collected from the dosed chimps at various times prior to and after

10 dosing. For intravenous administrations blood samples were collected at 0 min (preinfusion), 5 min, 9.5 min (immediately before the end of the infusion), then 6, 15, 30 and 45 min, and 1, 2, 4, 6, 8, 10 and 12 hr after the infusion is stopped. For oral administrations, blood samples were collected at 0 min (predose), 15 and 30 min, and 1, 1.5, 2, 4, 6, 8, 10 and 12 hr postdose.

15 Blood samples were placed in tubes containing sodium heparin, mixed and stored on ice until centrifuged. Within 30 minutes of collection, plasma was isolated by centrifuging the blood samples and removing an aliquot (200 μί) of the resulting plasma. Each 200-μί aliquot of plasma was mixed with 400 μΐ _^ acetonitrile and stored at -70 °C until further analysis by LC-MS/MS using an Applied Bio-systems

20 API 4000 mass spectrometer.

The analysis of all samples by LC-MS/MS was performed as described above for the rat plasma samples in Example 44.

BOST 1635628.1 Table 14. Pharmacokinetic Results Following Oral Administration in Chimps.

a) Mass observed via LC-MS/MS. Stereochemistry presumed to be >95%> (S) based on published pentoxifylline metabolism reports.

b) % Difference = [(deuterated species)-(nondeuterated

species)] (100)/(nondeuterated species)

Table 14 shows the results of oral administration of 435(5) and pentoxifylline in chimps. Following oral administration of a 1 : 1 combination of Compound 435(5) and pentoxifylline, both Compound 435(5) and its corresponding ketone Compound 409 demonstrated significantly higher average AUCo_ _∞ values than the corresponding undeuterated counterparts, S)-M1 and pentoxifylline. The average AUCo_ _∞ for Compound 435(5) together with Compound 409 was significantly higher than the average AUCo_ _∞ for (5)-Ml together with

pentoxifylline. In addition, the average AUCo_ _∞ for the undesired deuterated M-5 metabolite (M5a) was significantly lower than that of the undeuterated M-5.

Finally, the ratio of active species to M5 metabolite for the deuterated compounds {(435(5) + 409) : (deuterated M5)} was approximately 8-fold higher than the corresponding ratio for the undeuterated species {( S)-M1 + pentoxifylline) : M5} .

BOST 1635628.1 - 1 16 - Table 15. Pharmacokinetic Results Following Intravenous Administration in Chimps.

on published pentoxifylline metabolism reports.

5

b) % Difference = [(deuterated species)-(nondeuterated

species)] (100)/(nondeuterated species)

Table 15 shows the results of intravenous administration of 435(5) and 10 pentoxifylline in chimps. The results following intravenous administration showed favorable differentiation of the deuterated compounds, though not as pronounced as those observed following oral administration. Compared to administration of pentoxifylline, the amounts of active species produced from the administration of Compound 435(5) were between 40 and 57% higher, while the amounts of M5 15 metabolite produced decreased by between 60 and 65%. The ratio of active species to M5 metabolite in chimps that were intravenously administered Compound 435(5) was approximately 4-fold higher than in chimps administered pentoxifylline.

The above results show that compounds of this invention provide significantly greater plasma exposure of desired active species than the

20 corresponding non-deuterated compounds. Moreover, deuterium substitution in the present compounds was shown to reduce levels of the M5 metabolite, which may be associated with intolerability in renally-impaired patients.

Without further description, it is believed that one of ordinary skill in the art can,

BOST 1635628.1 - 1 17 - using the preceding description and the illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. It should be understood that the foregoing discussion and examples merely present a detailed description of certain preferred embodiments. It will be apparent to those of 5 ordinary skill in the art that various modifications and equivalents can be made without departing from the spirit and scope of the invention.

BOST 1635628.1 - 118 -