EA043549B1 - SGC STIMULANTS - Google Patents

Description

Related applications

The present invention claims priority under 35 USC § 119(e) as of the filing date of US Provisional Application No. 62/607,619, filed December 19, 2017, the contents of which are incorporated herein by reference in their entirety.

Field of technology to which the invention relates

The present invention relates to soluble guanylate cyclase (sGC) stimulators and pharmaceutically acceptable salts thereof. It also applies to pharmaceutical preparations containing them.

BACKGROUND OF THE INVENTION sGC is the major NO receptor in vivo. When bound to sGC, NO activates its catalytic domain and leads to the conversion of guanosine 5'-triphosphate (GTP) into the second messenger cGMP. Elevated cGMP levels, in turn, modulate the activity of downstream effectors, including protein kinases, phosphodiesterases (PDEs), and ion channels. In the body, NO is synthesized from arginine and oxygen through various nitric oxide synthase (NOS) enzymes and through the sequential reduction of inorganic nitrate. Experimental and clinical data indicate that decreased NO concentrations, decreased NO bioavailability, and/or decreased sensitivity to endogenously produced NO contribute to the development of numerous diseases. sGC stimulants are heme-dependent agonists of the sGC enzyme that work synergistically with varying amounts of NO to increase the enzymatic conversion of GTP to cGMP. sGC stimulators are clearly differentiated and structurally unrelated to another class of NO-independent, heme-independent sGC agonists known as sGC activators.

Therapies that improve or restore sGC function offer significant advantages over existing alternative therapies that either target the NO-sGC pathway or otherwise benefit from activation of this pathway. There is a need to develop new sGC stimulators.

The essence of the invention

The present invention relates to compounds of formula IA, formula IB and formula II and their pharmaceutically acceptable salts:

Formula II where: Y is independently N or C;

ring B is phenyl or a 5- or 6-membered heteroaryl ring containing 1 or 2 heteroatoms per ring independently selected from N, O or S;

n is an integer selected from 0-3; and each J ^B independently represents a halogen, -CN, C _1-6 -aliphatic group or -ORB;

wherein each JB that is a C _1-6 aliphatic group is optionally and independently substituted with up to 3 copies of R ³ ;

each RB independently represents hydrogen or a C _1-6 aliphatic group; wherein said RB is optionally and independently substituted with up to 3 copies of R ^3a ;

each R ³ and R ^3a is, in each instance, independently halogen, -CN, C _1-4 alkyl, C 1 ₄ haloalkyl, -O(C _1-4 alkyl) or -O(C _1-4 haloalkyl);

Z is selected from the group consisting of hydrogen, -P(O)(OH) ₂ , -P(O)(OH)O ^- M ⁺ , -P(O)(O ^- )2(M+)2, -P( O)(O ^- )2D ²⁺ and -P(O)(O-Benzyl) ₂ ; where M ⁺ represents a pharmaceutically acceptable monovalent cation and D ²⁺ represents a pharmaceutically acceptable divalent cation;

- 1 043549 m is equal to 0 or 1;

R ¹ represents C _1-4 alkyl, C _1-4 fluoroalkyl, -C(O)NH2 or hydrogen; And

R2 represents C _1-4 alkyl, C _1-4 fluoroalkyl or hydrogen;

both copies of R ⁴ are simultaneously hydrogen, or both copies of R ⁴ together with the carbon atom to which they are attached form a carbonyl group;

J ^D is hydrogen, halogen, methoxy or -CN;

p is 1 or 2; and each ^JC independently represents hydrogen, halogen, a C _1-4 aliphatic group, a C _1-4 alkoxy or -CN; wherein each said C _1-4 aliphatic group and each said C _1-4 alkoxy is optionally and independently substituted with up to 3 units of C _1-4 alkoxy, C _1-4 haloalkoxy, -OH or halogen.

In another embodiment, the present invention relates to pharmaceutical compositions containing a compound of the invention or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient or carrier. In another embodiment, the present invention relates to pharmaceutical dosage forms containing these pharmaceutical compositions.

Detailed Description of the Invention

Reference will now be made in detail to certain embodiments of the present invention, examples of which are illustrated in the accompanying structures and formulas. Although the present invention will be described in connection with the numbered embodiments, it should be understood that they are not intended to limit the present invention to these embodiments. More precisely, the present invention is intended to cover all alternatives, modifications and equivalents that may be included within the scope of the present invention as defined by the claims. The present invention is not limited to the methods and materials described herein, but includes any methods and materials similar or equivalent to those described herein that could be used in practicing the present invention. To the extent that one or more of the included literature references, patents or similar materials differ from or contradict this invention, including, but not limited to, certain terms, usage of a term, methods described, or the like, this invention prevails.

Definitions and general terminology.

For the purposes of this disclosure, chemical elements are defined in accordance with the Periodic Table of the Elements, CAS version and Handbook of Chemistry and Physics, ^75th Ed. 1994. Additionally, general principles of organic chemistry are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito: 1999, and March's Advanced Organic Chemistry, ^5th Ed., Smith, M.B. and March, J., eds. John Wiley & Sons, New York: 2001, which are incorporated herein by reference in their entirety.

Generally, the term substituted refers to the replacement of one or more hydrogen radicals of a given structure with another specified substituent radical other than hydrogen (some non-limiting examples include a hydroxy, phenyl, or alkyl radical). If the structure or moiety is optionally substituted, it may be substituted or unsubstituted. When one or more positions in a given chemical structure may be substituted or optionally substituted by one or more substituents selected from a specified group or list, the substituent or substituents at each position may be independently selected to be identical or the same at each position and for each copy unless otherwise indicated. For example, if phenyl is substituted with two instances of R ¹⁰⁰ and each R ¹⁰⁰ is independently selected from halogen and methyl, this means that each instance of R ¹⁰⁰ is separately selected from halogen or methyl; for example one R ¹⁰⁰ may be fluorine and the other may be methyl, or both may be chlorine, etc.

The choices of substituents and combinations contemplated in this disclosure are only those that result in the formation of stable or chemically feasible compounds. Such selections and combinations will be apparent to those of ordinary skill in the art and can be determined without undue experimentation. As used herein, the term stable refers to compounds that are substantially unchanged when subjected to conditions allowing their production, detection, and, in some embodiments, their regeneration, purification, and use for one or more of the purposes disclosed herein. A chemically feasible compound is a compound that can be prepared by one skilled in the art based on the information disclosed herein, supplemented as necessary by relevant knowledge in the art.

The expression used here up to refers to zero or any integer that is equal to or less than the number following this expression. For example, up to 3 means any of 0, 1, 2 or 3. As described herein, the range of atom or substituent numbers includes any integer. For example, a group of 1-4 atoms may have 1, 2, 3 or 4 atoms. When any variable occurs more than once in any position, its definition in each case is independent of any other case. When a group is replaced with 0 instances of a certain variable, it is

- 2 043549 means that the group is unsubstituted.

Unless one of the isomers is shown or specifically named, the structures depicted herein are intended to also include all stereoisomeric (eg, enantiomeric, diastereomeric, atropisomeric, and cis-trans isomeric) forms of the structure; for example, R and S configurations for each asymmetric center, Ra and Sa configurations for each asymmetric axis, double bond configurations (Z) and (E), and cis and trans isomers. Therefore, individual stereochemical isomers, as well as racemates and mixtures of enantiomers, diastereomers and cis-trans isomers of the compounds of the present invention are included within the scope of the present invention. Unless otherwise indicated, all tautomeric forms of the compounds of the present invention are also included within the scope of the present invention.

In one embodiment, the present invention may include replacing hydrogen with deuterium (i.e., ² H), which may provide certain therapeutic benefits due to greater metabolic stability (eg, increased in vivo half-life or reduced dosage requirements), and therefore, such a replacement may be preferable in some circumstances. The deuterium-labeled compounds of the present invention can generally be prepared using the following procedures similar to those described in the Schemes and/or Examples below, by substituting a deuterated reagent for a non-deuterated reagent.

The term aliphatic, as in an aliphatic group or aliphatic chain, means a straight or branched hydrocarbon (carbon and hydrogen only) chain that is fully saturated or contains one or more units of unsaturation. Suitable aliphatic groups include, but are not limited to, linear or branched, alkyl, alkenyl or alkynyl groups. Specific examples of aliphatic groups include, but are not limited to: methyl, ethyl, propyl, butyl, isopropyl, isobutyl, vinyl, sec-butyl, tert-butyl, butenyl, propargyl, acetylene, and the like. An aliphatic group will be represented by the term Cx.y-aliphatic group; where x and y are the minimum and maximum number of carbon atoms forming an aliphatic chain.

As used herein, the term alkyl, for example in an alkyl chain or alkyl group, refers to a saturated straight-chain or branched monovalent hydrocarbon radical. C _x αlkyl is an alkyl chain containing x carbon atoms, where x is an integer other than 0. C _x . _y alkyl, where x and y are two different integers, both different from 0, is an alkyl chain containing the number of carbon atoms x to y, inclusive. For example, C ₁ . ₆ alkyl is an alkyl as defined above containing any number from 1 to 6 carbon atoms. Examples of alkyl groups include, but are not limited to, methyl (i.e., C ₁ alkyl), ethyl (i.e., C ₂ alkyl), n-propyl (C ₃ alkyl), isopropyl (other C ₃ alkyl), n -butyl, isobutyl, sbutyl, tert-butyl, pentyl, hexyl, heptyl, octyl, etc.

The term alkenyl (as in alkenyl chain or alkenyl group) refers to a straight or branched monovalent hydrocarbon radical with at least one site of unsaturation that is a carbon-carbon double bond in the sp ² hybridization state, the alkenyl radical including radicals having a cis- and trans orientation or, using alternative nomenclature, E and Z orientations. Examples of alkenyls include, but are not limited to, vinyl, allyl, and the like. C _x alkenyl is an alkenyl chain containing x carbon atoms, where x is an integer other than 0. Alternatively, the alkenyl group will be represented by the term C _x . _y αlkenyl; where x and y are the minimum and maximum numbers of carbon atoms forming the alkenyl chain.

The term alkynyl (as in an alkynyl chain or alkynyl group) refers to a straight or branched monovalent hydrocarbon radical with at least one site of unsaturation, which is a carbon-carbon triple bond in sp hybridization. Examples include, but are not limited to, ethynyl, propynyl, and the like. C _x alkynyl is an alkynyl chain containing x carbon atoms, where x is an integer other than 0. Alternatively, an alkynyl group will be represented by the term C _x . _y αlkynyl; where x and y are the minimum and maximum number of carbon atoms forming the alkynyl chain.

The term cycloaliphatic, as in a cycloaliphatic ring or cycloaliphatic group, refers to a ring system formed only of carbon and hydrogen atoms that is fully saturated or contains one or more unsaturation units, but is not aromatic. ^cycloaliphatic is a cycloaliphatic ring containing x carbon atoms, where x is an integer other than 0. Alternatively, a cycloaliphatic ring would be represented by the term C _x . _y cycloaliphatic; where x and y are the minimum and maximum number of carbon atoms forming a cycloaliphatic ring. Suitable cycloaliphatic groups include, but are not limited to, cycloalkyl, cycloalkenyl and cycloalkynyl. Examples of aliphatic groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, norbornyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl and the like. The term cycloaliphatic also includes polycyclic ring systems (for example, bicyclic, tricyclic or tetracyclic). The polycyclic ring system can be a bridged, fused or spiro system.

Bridging ring systems contain two rings that share two non-adjacent ring atoms.

Fused ring systems contain two rings that share two adjacent ring atoms.

Spiro ring systems contain two rings that share one neighboring ring atom.

As used herein, the term cycloalkyl, as in a cycloalkyl ring or cycloalkyl group, refers to a ring system formed only of carbon and hydrogen atoms that is fully saturated. Suitable cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cycloheptenyl, norbornyl, cyclooctyl, cyclononyl, cyclodecyl, cycloundecyl, cyclododecyl, and the like. The cycloalkyl ring will be represented by the term C _x . _y cycloalkyl; where x and y are the minimum and maximum number of carbon atoms forming the cycloalkyl ring. The term cycloalkyl also includes polycyclic ring systems (eg, bicyclic, tricyclic or tetracyclic). The polycyclic ring system can be a bridged, fused or spiro system.

As used herein, the term aryl (as in an aryl ring or aryl group) refers to a ring system formed only of carbon and hydrogen atoms that is aromatic. The term also includes polycyclic ring systems (eg, bicyclic, tricyclic, tetracyclic, etc.). Examples of aryl rings include, but are not limited to, phenyl, naphthyl, indenyl, fluorenyl and anthracenyl.

The term heteroatom refers to one or more of oxygen, sulfur, nitrogen, phosphorus or silicon, including any oxidized form of nitrogen, sulfur, phosphorus or silicon and including the quaternized form of any basic nitrogen.

The term ring atom refers to an atom such as C, N, O, or S that is part of a ring (rings include, for example, a cycloaliphatic ring (e.g., a cycloalkyl ring), a heterocyclic ring, an aryl ring (e.g., a phenyl ring), or a heteroaryl ring .

The term ring heteroatom refers to an atom, such as N, O or S, that is part of a heterocyclic ring or heteroaryl ring.

The replaced ring atom is a ring carbon or nitrogen atom bonded to at least one hydrogen atom. Hydrogen may optionally be replaced with a suitable substituent group. The ring atom being replaced does not include carbon or nitrogen ring atoms in which the structure shows that they are already attached to one or more moieties or substituents other than hydrogen and there are no hydrogens available for replacement. When a particular ring is optionally substituted, it is understood that it may be substituted on one or more or all of its substituted ring atoms, depending on the number of substituents allowed.

As used herein, the term heterocycle (or heterocyclyl, as in a heterocyclic group or heterocyclic ring) refers to a ring system in which one or more ring atoms are an independently selected heteroatom, wherein said ring is fully saturated or contains one or more units of unsaturation, but is not aromatic. Examples of heterocyclic rings include, but are not limited to, the following monocycles: 2-tetrahydrofuranyl, 3-tetrahydrofuranyl, 2-tetrahydrothiophenyl, 3-tetrahydrothiophenyl, 2-morpholino, 3-morpholino, 4-morpholino, 2-thiomorpholino, 3-thiomorpholino, 4-thiomorpholino . inil, 1 -piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 2-thiazolidinyl, 3-thiazolidinyl, 4-thiazolidinyl, 1-imidazolidinyl, 2-imidazolidinyl, 4-imidazolidinyl and 5-imidazolidinyl. Examples of bicyclic heterocyclic ring systems include, but are not limited to: 2-oxa-bicyclo[2.2.2]octyl, 1-aza-bicyclo[2.2.2]octyl. As used herein, the term heterocycle also includes polycyclic ring systems (eg, bicyclic, tricyclic or tetracyclic).

The term heteroaryl (or heteroaromatic, as in heteroaryl group or heteroaryl ring) refers to a ring that is aromatic and contains one or more heteroatoms. Heteroaryl rings include, but are not limited to, the following monocycles: 2-furanyl, 3-furanyl, N-imidazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-oxazolyl, 4- oxazolyl, 5-oxazolyl, N-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, pyridazinyl (e.g. 3-pyridazinyl), 2 -thiazolyl, 4-thiazolyl, 5-thiazolyl, tetrazolyl (for example, 5-tetrazolyl), triazolyl (for example, 2-triazolyl and 5-triazolyl), 2-thienyl, 3-thienyl, pyrazolyl (for example, 2-pyrazolyl), isothiazolyl, 1,2,3-oxadiazolyl, 1,2,5-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,3-triazolyl,

- 4 043549

1,2,3-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, pyrazinyl, 1,3,5-triazinyl. As used herein, the term heteroaryl also includes polycyclic ring systems (eg, bicyclic, tricyclic or tetracyclic). Examples of bicyclic heteroaryl rings include, but are not limited to: indazole, pyrazolopyrimidine, imidazopyridine, etc.

As used herein, the term alkoxy refers to an alkyl group, as previously defined, attached to the molecule through an oxygen atom. An alkoxy group can be represented by -O-(C _x _ _y alkyl), where x and y represent the minimum and maximum number of carbon atoms of the alkyl chain. Examples of alkoxy include, but are not limited to, methoxy (-OMe), ethoxy (-OEt), etc.

As used herein, the term halogen means F, Cl, Br or I.

The terms haloalkyl, haloalkenyl, haloaliphatic and haloalkoxy mean alkyl, alkenyl, aliphatic or alkoxy, as appropriate, substituted with one or more halogen atoms. For example, C _{1_3} -haloalkyl may be, for _{example ,} -CFHCH ₂ CHF ₂ , and C _{1_2} haloalkoxy may be, for example, -OC(Br)HCHF ₂ .

The term fluoroalkyl means alkyl substituted with one or more fluorine atoms. This term includes perfluorinated alkyl groups such as -CF ₃ u-CF ₂ CF ₃ .

The term fluoroalkoxy means alkoxy substituted with one or more fluorine atoms. This term includes perfluorinated alkoxy groups such as -OCF ₃ and -OCF ₂ CF ₃ .

As used herein, the term cyano refers to -CN or -C=N.

As used herein, the term amino group refers to -NH ₂ .

The term hydroxyl or hydroxy refers to -OH.

As used herein, the term carbonyl, used alone or in combination with another group, refers to -C(O)-.

The compounds of the present invention are defined herein by their chemical structures and/or chemical names. If a compound is referred to using both a chemical structure and a chemical name, and the chemical structure and chemical name conflict, the chemical structure is determinative of the identification of the compound.

Substituents, such as, for example, R ¹ , R ² and R ³ , are generally defined when they are introduced and remain so defined throughout the specification and in all independent claims unless otherwise indicated.

When Z is not hydrogen, the compounds of the invention are phosphate ester prodrugs, and pharmaceutically acceptable salts thereof, of compounds of Formula II that are useful as sGC stimulants.

Formula II.

The in vivo biological activity exhibited by the compounds of the invention upon administration is primarily due to the presence of the parent compound of formula II, which results from cleavage of the prodrug after administration.

The term prodrug refers to compounds that are precursors to a drug that, after administration and absorption, release the drug in vivo through some metabolic, enzymatic, hydrolytic, or rapid chemical reaction. Typically, a prodrug has less biological activity than the parent compound on the target itself before being cleaved to the parent drug. A prodrug can improve the physical properties of the parent drug and/or improve the overall effectiveness of the drug, for example, by reducing the toxicity and unwanted side effects of the drug by controlling its absorption, blood levels, metabolic distribution and cellular uptake. Prodrugs may also reduce pharmacokinetic variability between subjects in vivo. The prodrug may also exhibit more desirable pharmaceutical properties and, as such, the prodrug may also enhance the drug composition or facilitate the formulation of the drug into a composition for certain routes of administration.

The term parent drug or parent compound refers to the biologically active substance that is released by metabolic, enzymatic, hydrolytic or rapid chemical transformation after administration of the prodrug. In some embodiments, the parent compound may also be the starting material used to prepare the prodrug.

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Monovalent cations described as M ⁺ include Na ⁺ , K ⁺ or monovalent organic amine cation such as primary, secondary and tertiary amines, cyclic amines, arginine, caffeine, choline, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, N-ethylmorpholine,

N-ethylpiperidine, glucosamine, histidine, isopropylamine, lysine, morpholine, piperidine, triethylamine, trimethylamine, tripropylamine, diisopropylethylamine, etc.

Divalent cations described as D ²⁺ include Ca ²⁺ , Zn ²⁺ , Cs ²⁺ , Mg ²⁺ or a divalent organic amine cation such as N,N1-dibenzylethylenediamine, ethylenediamine, piperazine and the like.

Connection options.

In a first embodiment, a compound of the present invention is selected from:

.

In a second embodiment, the compound of the invention is represented by the following formula:

where: n is an integer selected from 0-3; and each JB independently represents a halogen, -CN, C _{1_6 -aliphatic} group or -ORB;

wherein each JB, which is a C ₁ _ ₆ aliphatic group, is optionally and independently substituted with up to 3 copies of R ³ ;

each RB independently represents hydrogen or a C ₁ _ ₆ aliphatic group; wherein said RB is optionally and independently substituted with up to 3 copies of R ^3a ;

each R ³ and R ^3a is, in each instance, independently halogen, -CN, C ₁ _ ₄ alkyl, C ₁ _ ₄ haloalkyl, -O(C ₁ _ ₄ alkyl) or -O(C ₁ _ ₄ haloalkyl) ;

J ^C independently represents hydrogen, halogen, C _{1_4} aliphatic group, C _{1_4} alkoxy _or -CN _; wherein each said C ₁ _ ₄ aliphatic group and each said C ₁ _ ₄ alkoxy is optionally and independently substituted with up to 3 copies of C ₁ _ ₄ alkoxy, C ₁ _ ₄ haloalkoxy, -OH, or halogen; And

JD is hydrogen, chlorine, fluorine, methoxy or -CN.

In a third embodiment, a compound of the invention is represented by Formula IA, Formula IB or Formula II or a pharmaceutically acceptable salt thereof:

Formula II where: Y is independently N or C;

ring B is phenyl or a 5- or 6-membered heteroaryl ring containing 1 or 2 heteroatoms per ring independently selected from N, O or S;

n is an integer selected from 0-3; and each J ^B independently represents _a halogen, -CN, C _{1_6} -aliphatic group or -ORB;

wherein each JB, which is a C ₁ _ ₆ aliphatic group, is optionally and independently substituted with up to 3 copies of R ³ ;

each RB independently represents hydrogen or a C ₁ _ ₆ aliphatic group; wherein said R ^B is optionally and independently substituted with up to 3 copies of R ^3a ;

each R ³ and _R ^3a represents, in each instance, independently halogen, -CN, C _{1_4} alkyl,

C ₁ _ ₄ haloalkyl, -O(C ₁ _ ₄ alkyl) or -O(C ₁ _ ₄ haloalkyl);

Z is selected from the group consisting of hydrogen, -P(O)(OH)2, -P(O)(OH)OM ⁺ , -P(O)(O-)2(M ⁺ )2, -P(O )(O-) ₂ D ²⁺ and -P(O)(O-benzyl) ₂ ; where M ⁺ represents a pharmaceutically acceptable monovalent cation and D ²⁺ represents a pharmaceutically acceptable divalent cation;

m is 0 or 1;

R ¹ represents C _{1_4} alkyl, C _{1_4 fluoroalkyl} , -C(O)NH2 or hydrogen _; And

R ² represents C _{1_4} alkyl, C _{1_4 fluoroalkyl} or hydrogen _;

both copies of R ⁴ are simultaneously hydrogen, or both copies of R ⁴ together with the carbon atom to which they are attached form a carbonyl group;

JD is hydrogen, halogen, methoxy or -CN, p is 1 or 2; and _{each JC independently represents hydrogen, halogen, a C 1_4 aliphatic} group, a C _1-4 alkoxy or -CN; wherein each said C _{1_4} aliphatic group and _each said C _{1_4} alkoxy is optionally and independently _substituted with up to 3 units of C _1-4 alkoxy, C _{1_4} haloalkoxy, -OH _, or halogen.

In a fourth embodiment, the compound of the invention is represented by Formula IIA or Formula IIB

In a specific embodiment, (i) JD is hydrogen, chlorine, fluorine, methoxy or CN; (ii) JD is methoxy; (iii) JD is hydrogen, chlorine, fluorine or -CN; (iv) JD is hydrogen, chlorine or fluorine; (v) JD is fluorine or hydrogen; (vi) JD represents hydrogen; (vii) JD is fluorine; (viii) JD is -CN; or (ix) JD is chlorine. In another specific embodiment ₍ i) _, each ^JC is independently hydrogen, _{C1_2alkyl} , _{C1_2alkoxy} , _{C1_2fluoroalkyl} , _{C1_2fluoroalkoxy} , halogen, and -CN, _{wherein each} of said C _{1_2} alkyl and C _{1_2 alkoxy are optionally and independently substituted with up to 3 copies of C 1_4 alkoxy , C 1_4} haloalkoxy, -OH or halogen; ( _ii ) each JC is independently hydrogen, _{C1_2alkyl , C1_2alkoxy} , _{C1_2fluoroalkyl} , _{C1_2fluoroalkoxy , halogen} or -CN; (iii) each JC is hydrogen or halogen; (iv) each ^JC is hydrogen; or (v) each ^JC is fluorine or chlorine.

In a fifth embodiment, a compound of the invention is represented by Formula IIIA, Formula IIIA, Formula IVA, Formula IVB, Formula VA, Formula VB, Formula VIA or Formula VIB or a pharmaceutically acceptable salt thereof:

- 7 043549

Formula SHV

Formula IVA

Formula IVB

Formula VA

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Formula VIB where the variable definitions are the same as those described above.

In a sixth embodiment, for compounds of Formula IA, Formula IB, Formula II, Formula IIA, Formula IIB, Formula IIIA, Formula IIIB, Formula IVA, Formula IVB, Formula VA, Formula VB, Formula VIA, or Formula VIB, or a pharmaceutically acceptable salt thereof , (i) ring B is phenyl; (ii) ring B is a 5- or 6-membered heteroaryl ring containing 1 or 2 heteroatoms per ring independently selected from N, O and S; or (iii) ring B is a 6-membered heteroaryl ring containing 1 or 2 ring nitrogen atoms.

In a seventh embodiment, for compounds of Formula IA, Formula IB, Formula II, Formula IIA, Formula IIB, Formula IIIA, Formula IIIB, Formula IVA, Formula IVB, Formula VA, Formula VB, Formula VIA, or Formula VIB, or a pharmaceutically acceptable salt thereof , (i) n is 1, 2 or 3, and each J ^B is independently a halogen or a C1.6 aliphatic group; (ii) n is 1 or 2, and each ^JB is independently halogen or C1. ₆ aliphatic group; (iii) n is 1 or 2 and each J ^B is fluorine; (iv) n is 1; and each J ^B represents fluorine.

In an eighth embodiment, the compound of the present invention is represented by Formula VIIA, Formula VIIIA, Formula VIIB or Formula VIIIB, or a pharmaceutically acceptable salt thereof:

- 9 043549

Formula VIIIB wherein the variables are the same as those described above in the first or sixth embodiment.

In the ninth embodiment, for the compounds of Formula IA, Formula IB, Formula II, Formula IIA, Formula IIB, Formula IIIA, Formula IIIB, Formula IVA, Formula IVB, Formula VA, Formula VB, Formula VIA, Formula VIB, Formula VIIA, Formula VIIB , formula VIIIA or formula VIIIB or a pharmaceutically acceptable salt thereof, (i) R ¹ represents C1.2alkyl, C1.2fluoroalkyl, hydrogen or -C(O)NH2; or (ii) R ¹ is C1. ₂ fluoroalkyl or -C(O)NH2. In another specific embodiment, (i) R ² represents C1.2fluoroalkyl, C1.2alkyl or hydrogen; or (ii) R ² is C1. ₂ fluoroalkyl.

In the tenth embodiment, for the compounds of Formula IA, Formula III, Formula II, Formula IIA, Formula IIB, Formula IIIA, Formula IIIB, Formula IVA, Formula IVB, Formula VA, Formula VB, Formula VIA, Formula VIB, Formula VIIA, Formula VIIB , formula VIIIA or formula VIIIB or a pharmaceutically acceptable salt thereof, (i) R ¹ represents trifluoromethyl or -C(O)NH2; and R ² represents trifluoromethyl; (ii) R ¹ and R ² are both hydrogen or C1-2 alkyl; or one of R ¹ and R ² is hydrogen and the other is C1. ₂ fluoroalkyl; (iii) R ¹ and R ² are both hydrogen; (iv) R ¹ and R ² are both methyl; or (v) one of R ¹ and R ² is hydrogen and the other is trifluoromethyl.

In a fourteenth embodiment, the compound of the present invention is represented by one of the following formulas:

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or a pharmaceutically acceptable salt thereof, wherein the variable definitions are the same as those described in the third or seventh embodiment. In a specific embodiment, J ^D is hydrogen or fluorine. In a specific embodiment, J ^D is fluorine.

In some embodiments, for the compounds described in the third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or fourteenth embodiment, (i) Z is -P(O)(OH)2; (ii) Z is -P(O)(OH)O ^- M ⁺ or -P(O)(O-)2(M ⁺ )2; and M ⁺ represents Na ⁺ , K + , Cs ⁺ or a monovalent organic amine cation; or (iii) Z is -P(O)(O ^- ) ₂ D ²⁺ ; and D ²⁺ is Ca ²⁺ , Zn ²⁺ , Mg ²⁺ or a divalent organic amine cation.

In some embodiments, the compounds of the invention are selected from those listed in Table. I.

- 11 043549

Table I

Hu? _ζ οη _f aJ «l™ 1-1 N'^y ^F nAn ITcf ₃ T PCF ₃ HO JO 1-2 /'ν'^Γ'Γ'νη 'A* IP L· ^H0 &X У 1-3 N^y ^F Λν^ν^νη W ^F, 9 _z OBn AJ /z ^P OBn 1-4 F rN Al \ Ν \ k ^F ° /l ,—L ⁿ n _g YTI OH CF, F 1-5 ^F NA _Λ 1 yu nX У^ои F ^H CF ₃ CF ₃ 1-6 yU n^n ψ^Ν^Η F ^H Me Me 1-7 Г1 a N ^; N AA za/Oh AN y Cl ^H CF ₃ racemic 1-8 €1 I D0 N^N ^OH H 1-9 Λ-Ν Ух N^N ^F V4y< ^OH Cl ^{H CF} 3 ^CF 3 1-10 F NA _Λ A 'ι 4ίη A ^u N ^; N AA ° ^H γ Ν γ □Me ^H CF ₃ racemic 1-11 'Tn T N, 1 _L 1 L ΜΊ Y ^u N^N AA^OH CN ^H CF ₃ CF ₃ 1-12 fl? ^A KY's Y n^n U^on F ^H CF ₃ CF ₃ 1-13 FI OTQ TN Me iAn U-m^i F ^H CF ₃ CF ₃ 1-14 γ-γΆ» A y\ 9 ,oNa _F Aj d' ^p ' ^0Na 1-15 N. Ik A. V iXyl Y N^N O F ^H OH CF ₃ 1-16

Pharmaceutically acceptable salts of the present invention.

Pharmaceutically acceptable salts of the compounds described herein include salts obtained from said compounds when mixed with inorganic or organic acids or a base

- 12043549 niyami. In some embodiments, the salts may be produced in situ during the final isolation and purification of the compounds. In other embodiments, the salts may be prepared from the free form of the compound in a separate synthetic step. The preparation of the pharmaceutically acceptable salts described above and other typical pharmaceutically acceptable salts is more fully described by Berg et al. in Pharmaceutical Salts, J. Pharm. Sci., 1977: 66: 1-19, which is incorporated herein by reference in its entirety.

Pharmaceutically acceptable salts of the compounds of the invention are salts that can be used in medicine. However, salts that are not pharmaceutically acceptable may be used in the preparation of the compound of the invention or pharmaceutically acceptable salts thereof.

When the compound of the invention is acidic, suitable pharmaceutically acceptable salts refer to those derived from pharmaceutically acceptable non-toxic bases, including inorganic and organic bases. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric iron, ferrous iron, lithium, magnesium, manganese trivalent, manganese divalent, potassium, sodium, zinc and the like. Specific embodiments include ammonium, calcium, magnesium, potassium and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, arginine, betaine, caffeine, choline, K,K'-dibenzylethylenediamine, diethylamine, 2 -diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, Nethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, trieth ylamine, trimethylamine, tripropylamine, tromethamine, etc.

When the compound of the invention is basic, salts can be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetate, acetic acid, acid citrate, acid phosphate, ascorbate, benzenesulfonic acid, benzenesulfonate, benzoic acid, benzoate, bromide, bisulfate, bitartrate, camphorsulfonic acid, chloride, citrate, citric acid, ethanesulfonate, ethanesulfonic acid, formate, fumarate, fumaric acid gentisinate, gluconate, glucic acid, glucuroonate, glutamate, glitamic acid, bromistic acid, hydrochloric acid, iodide, ethionic acid, isonicotinate, lactate, lactic acid, maleat, maleic acid, apple acid, almond acid, metatanic acid, metatan acid, metatanic acid, metatanic acid, metatanic acid, metatanic acid, metatanic acid, metatanic acid, metatanic acid, metatan -meters T, mucus acid , nitrate, nitric acid, oleate, oxalate, pamoic acid, pamoate (i.e. 1,1'-methylene-bis-(2-hydroxy-3-naphthoate)), pantothenic acid, pantothenate, phosphate, phosphoric acid, saccharate , salicylate, succinic acid, succinate, sulfuric acid, sulfate, tannate, tartrate, tartaric acid, p-toluenesulfonate, p-toluenesulfonic acid, etc. Specific embodiments include citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric and tartaric acids.

In addition to the compounds described herein, pharmaceutically acceptable salts thereof may also be used in compositions for the treatment or prevention of the diseases described herein.

Pharmaceutical compositions and methods of administration.

The compounds disclosed herein and their pharmaceutically acceptable salts may be formulated as pharmaceutical compositions or preparations.

A typical formulation is prepared by mixing a compound of the invention or a pharmaceutically acceptable salt thereof and a carrier, diluent or excipient. Suitable carriers, diluents and excipients are well known to those skilled in the art and include materials such as carbohydrates, waxes, water-soluble and/or swelling polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water and the like. The particular carrier, diluent, or excipient used will depend on the means and purposes for which the compound of Formula I is formulated. Solvents are generally selected based on solvents generally recognized as safe (GRAS) by those skilled in the art for administration to a mammal. Generally, safe solvents are non-toxic aqueous solvents such as water and other non-toxic solvents that are soluble or miscible with water. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycols (eg PEG400, PEG300), etc. and mixtures thereof. The formulations may also include other types of excipients such as one or more buffers, stabilizers, release agents, surfactants, wetting agents, lubricants, emulsifiers, binders, suspending agents, disintegrating agents, fillers, sorbents, coatings (e.g. enteric or slow release), preservatives, antioxidants, opaque agents, glidants, processing aids, coloring agents, sweeteners, flavoring agents, flavoring agents and other known additives to provide superior presentation of the drug (i.e., the compound of the invention or the pharmaceutical composition thereof ) or assistance in the production of a pharmaceutical product (i.e., a drug).

- 13 043549

Acceptable diluents, carriers, excipients and stabilizers are those that are non-toxic to recipients at the dosages and concentrations used, and include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Active pharmaceutical ingredients can also be enclosed in microcapsules prepared, for example, using coacervation or interfacial polymerization techniques, for example, hydroxymethylcellulose or gelatin microcapsules and polymethylmethacylate microcapsules, respectively; in colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such methods are disclosed in Remington's: The Science and Practice of Pharmacy, ^21st Edition, University of the Sciences in Philadelphia, Eds., 2005 (hereinafter Remington).

The preparations can be prepared using normal dissolution and mixing procedures. As used herein, the term therapeutically effective amount means that amount of an active compound or pharmaceutical agent that produces a biological or therapeutic response in a tissue, system, animal or human being sought by a researcher, veterinarian, physician or other clinician. The therapeutically effective amount of the compound administered will depend on such considerations and is the minimum amount necessary to alleviate, cure or treat the disease or one or more symptoms thereof.

The terms administer, administration, or administering with respect to a compound, composition, or dosage form of the present invention mean introducing the compound into the system of a subject or patient in need of treatment. When a compound of the present invention is provided in combination with one or more other active agents, administration and variations thereof are intended to include simultaneous and/or sequential administration of the compound and other active agents.

The compositions described herein can be administered systemically or topically, for example, orally (including, but not limited to, solid dosage forms, including hard or soft capsules (eg, gelatin capsules), tablets, pills, powders, sublingual tablets, lozenges, lozenges and granules , and liquid dosage forms, including, but not limited to, pharmaceutically acceptable emulsions, microemulsions, aqueous or oily solutions, suspensions, syrups and elixirs, by inhalation (for example, using an aerosol, gas, inhaler, nebulizer, or the like) , in the ear (for example, using ear drops), topically (for example, using creams, gels, inhalation dosage forms, liniments, lotions, ointments, patches, pastes, powders, solutions, sprays, transdermal patches, etc.), ophthalmologically (eg, using eye drops, ophthalmic gels, ophthalmic ointments), rectally (eg, using enemas or suppositories), intranasally, buccally, vaginally (eg, using douches, intrauterine devices, vaginal suppositories, vaginal rings or tablets, etc.) etc.), by ear drops, through an implanted reservoir or the like, or parenterally depending on the severity and type of disease being treated. As used herein, the term parenteral includes, but is not limited to, subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injection or infusion. Preferably, the compositions are administered orally, intraperitoneally or intravenously.

Formulations of the compound for oral administration may be prepared in accordance with any method known in the art for the production of pharmaceutical compositions.

In the case of solid dosage forms, the active compound is mixed with at least one inert pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate, and/or a) excipients or excipients such as starches, lactose, sucrose, glucose, mannitol and silicic acid , b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose and acacia, c) humectants such as glycerin, d) disintegrating agents such as agar, calcium carbonate, potato or cassava starch, alginic acid, some silicates and sodium carbonate, e) dissolution retardants such as paraffin, e) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents, such as kaolin and bentonite clay and i) lubricants such as talc, calcium stearate, magnesium stearate, solid poly

- 14 043549 ethylene glycols, sodium lauryl sulfate and mixtures thereof. Tablets may be uncoated or may be coated by known methods, including microencapsulation, to mask unpleasant taste or to delay disintegration and adsorption in the gastrointestinal tract and thus provide sustained action over a longer period. For example, a retarding material such as glyceryl monostearate or glyceryl distearate may be used, alone or together with a wax. A water-soluble taste-masking material such as hydroxypropyl methylcellulose or hydroxypropylcellulose may be used.

In addition to the active compounds, liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate , propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed, peanut, maize, germ, olive, castor and sesame oils), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycols and sorbitan esters of fatty acids and mixtures thereof. In addition to inert diluents, oral compositions may also include auxiliary agents such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and flavoring agents.

Compositions for oral administration (solid or liquid) may also include excipients and auxiliaries, such as dispersing or wetting agents, such as a naturally occurring phosphatide (eg, lecithin), an alkylene oxide fatty acid condensate (eg, polyoxyethylene stearate), a condensate ethylene oxide with a long chain aliphatic alcohol (eg, heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid, and hexitol anhydride (eg, polyoxyethylene sorbitan monooleate); emulsifiers and suspending agents such as sodium carboxymethylcellulose, croscarmellose, povidone, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; sweeteners, flavorings and flavorings; and/or one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweeteners such as sucrose or saccharin.

Pharmaceutical compositions can also be administered by nasal spray or by inhalation. Such compositions are prepared in accordance with methods well known in the art of pharmaceutical formulation technology and can be formulated as solutions in saline, using benzyl alcohol or other suitable preservatives, absorption promoters to improve bioavailability, fluorocarbons and/or other conventional solubilizers or dispersants. Formulations suitable for intrapulmonary or intranasal administration have particle sizes, for example, in the range of 0.1 to 500 microns (including particles in the range of 0.1 to 500 microns in micron increments, for example, 0.5, 1, 30, 35 microns, etc.), which is carried out by rapid inhalation through the nasal passage or by inhalation through the mouth to reach the alveolar sacs.

The pharmaceutical compositions described herein may also be administered topically, particularly when the target of treatment includes areas or organs readily accessible to topical administration, including diseases of the eyes, ears, skin, or lower intestines. Suitable topical preparations can easily be prepared for each of these areas or organs. The active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservatives or buffers that may be required.

For topical use, the pharmaceutical compositions may be formulated as a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical compositions may be formulated into a suitable lotion or cream containing the active ingredients suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitol monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Alternatively, the active ingredients may be formulated into a cream using an oil-in-water cream base. If desired, the aqueous phase of the cream base may include a polyhydric alcohol, i.e. an alcohol having two or more hydroxyl groups, such as propylene glycol, butane1,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol (including PEG 400) and mixtures thereof. Topical formulations may optionally include a compound that enhances absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogs.

The oil phase of emulsions prepared using a compound of formula I can be formulated from known ingredients in a known manner. Although the phase may only contain emulsion

- 15 043549 gator (otherwise known as an emulsifying agent), desirably includes a mixture of at least one emulsifying agent with a fat or oil or both fat and oil. A hydrophilic emulsifier may be included along with a lipophilic emulsifier, which acts as a stabilizer. In some embodiments, the emulsifier includes both oil and fat. Together, the emulsifier(s), with or without stabilizer(s), constitute the so-called emulsifying wax, and the wax, together with the oil and fat, constitute the so-called emulsifying ointment base, which forms the oil-dispersed phase of the preparations in the form of a cream. Emulsifying agents and emulsion stabilizers suitable for use in formula I formulation include Tween-60, Span™-80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.

In addition, the present invention provides for the use of transdermal patches, which have the additional advantage of providing controlled delivery of the compound to the body. Such dosage forms can be prepared by dissolving or distributing the compound in a suitable medium. Absorption enhancers can also be used to increase the flux of a compound through the skin. The rate can be controlled either by providing a rate-controlling membrane or by dispersing the compound in a polymer matrix or gel.

For ophthalmic use, the pharmaceutical compositions may be formulated as micronized suspensions in isotonic, sterile pH-adjusted saline, or preferably as solutions in isotonic, sterile, pH-adjusted saline, either with or without a preservative such as benzylalkonium chloride. Alternatively, for ophthalmic applications, the pharmaceutical compositions may be formulated as an ointment such as petroleum jelly. For treating the eyes or other external tissues, such as the mouth and skin, the preparations may be administered as a topical ointment or cream containing the active ingredient(s) in an amount of, for example, 0.075 to 20% by weight. When formulated as an ointment, the active ingredients may be used in an oil, paraffin, or water-miscible ointment base.

Compositions for rectal or vaginal administration are preferably suppositories, which can be prepared by mixing the compounds described herein with suitable non-irritating excipients or carriers, such as cocoa butter, beeswax, polyethylene glycol or suppository wax that is solid at ambient temperature, but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound. Other preparations suitable for vaginal administration may be in the form of pessaries, tampons, creams, gels, pastes, foams or sprays.

Sterile injectable forms of the compositions described herein (eg, for parenteral administration) may be an aqueous or oily suspension. These suspensions can be formulated in accordance with methods known in the art, using suitable dispersants or wetting agents and suspending agents (including those described in the previous paragraph). The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, a solution in 1,3-butanediol. Acceptable carriers and solvents that may be used include water, Ringer's solution, and isotonic sodium chloride solution. Additionally, sterile fatty oils are commonly used as a solvent or suspending medium. Any soft fatty oil can be used for this purpose, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its derivatives in the form of glycerides are useful in the preparation of injectable preparations, as are natural pharmaceutically acceptable oils such as vegetable oil, such as peanut oil, olive oil, sesame oil or coconut oil, especially in their polyoxyethylene variants or in mineral oil such as liquid paraffin. These oily solutions or suspensions may also contain a long chain alcohol diluent or dispersant, such as carboxymethylcellulose or similar dispersants, which are commonly used in the formulation of pharmaceutically acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants such as Tweens, Spans and other emulsifiers or bioavailability enhancers that are commonly used in the manufacture of pharmaceutically acceptable solid, liquid or other dosage forms may also be used for injectable drug purposes. Oily suspensions may contain a thickening agent such as beeswax, paraffin wax or cetyl alcohol. Sweeteners, such as those listed above, and flavoring agents may be added to provide an attractive-tasting oral formulation. These compositions can be preserved by adding an antioxidant such as butylated hydroxyanisole or alphatocopherol.

In another aspect, a compound of the invention or a pharmaceutically acceptable salt thereof can be formulated into a veterinary composition comprising a veterinary carrier. Veterinary carriers are materials useful for the purpose of administering the composition and may be solid, liquid or gaseous materials that are otherwise inert in the veterinary field and compatible with the active ingredient. These veterinary compositions may be administered parenterally, orally, or any other desired route.

Packaging and kits.

The pharmaceutical composition (or drug) for use may be packaged in various ways depending on the method used to administer the drug. Typically, a dispensing article includes a container in which the pharmaceutical preparation is placed in an appropriate form. Suitable containers are well known to those skilled in the art and include materials such as bottles (plastic and glass), sachets, ampoules, plastic bags, cylindrical metal containers, and the like. The container may also include a tamper-resistant unit to prevent unauthorized access to the contents of the package. In addition, a label is placed on the container that describes the contents of the container. The label may also include appropriate warnings.

The compounds and pharmaceuticals described herein may be contained in the kit. The kit may include one or multiple doses of two or more agents, each packaged or formulated individually, or one or multiple doses of two or more agents, packaged or formulated in combination. Thus, one or more agents may be present in the first container, and the set may optionally include one or more agents in the second container. The container or containers are placed in a package, and the package may not necessarily include instructions for use or dosage. The kit may include additional components, such as syringes or other means for administering agents, as well as diluents or other means for preparing the drug. Thus, the kits may contain: a) a pharmaceutical composition containing a compound described herein and a pharmaceutically acceptable carrier, vehicle or diluent; and b) container or package. The kits may optionally include instructions describing a method for using the pharmaceutical compositions in one or more of the methods described herein (eg, preventing or treating one or more of the diseases and disorders described herein). The kit may optionally contain a second pharmaceutical composition containing one or more additional agents described herein for co-therapeutic use, a pharmaceutically acceptable carrier, vehicle or diluent. A pharmaceutical composition containing a compound described herein and a second pharmaceutical composition contained in the kit may optionally be combined in the same pharmaceutical composition.

Examples

All references provided in the examples are incorporated herein by reference. All abbreviations, symbols and conventions used here correspond to those used in modern scientific literature. See, for example, Janet S. Dodd, ed., The ACS Style Guide: A Manual for Authors and Editors, ^2nd Ed., Washington, DC: American Chemical Society, 1997, which is incorporated herein by reference in its entirety.

Common abbreviations used in this invention.___________

Reduction Word or expression Ace Acetyl Ac ₂ O Acetic anhydride ACN Acetonitrile AcOH Acetic acid arr- Visible Vos tert-butoxycarbonyl

- 17 043549

lat. Wide Conc. Concentration DAST Diethylaminosulfur trifluoride DBU 1,8-diazabicyclo(5.4.0)undec-7-ene DCM Dichloromethane DMA N,A-dimethylacetamide DIEA N, A-diisopropylethylamine DMAP 4-A A-dimethylaminopyridine DMF N, A-dimethylformamide DMSO Dimethyl sulfoxide DMSO-d6 Deuterated dimethyl sulfoxide dppa Diphenylphosphoryl azide dppf 1.1 '-bis(diphenylphosphino)ferrocene dba Dibenzylideneacetone ES+ Electrospray ionization with positive ion detection ES- Electrospray ionization with negative ion detection Et Ethyl Et ₂ O Diethyl ether EtOAc Ethyl acetate EtOH Ethanol eq- The equation EQUIV. Equivalent F.A. Formic acid Gen. General 4 watch) HEK Human embryonic kidney HPLC High Performance Liquid Chromatography i-PA Isopropyl alcohol LC-MS Liquid chromatography with mass spectrometry Me Methyl

- 18 043549

MeCN Acetonitrile MeOH Methanol MeOH-d4 Deuterated methanol min minutes ml Milliliter MHz megahertz MS Mass spectrometry MTBE Methyl tert-butyl ether NMP A-methylpyrrolidinone NMR Nuclear magnetic resonance No. Connection number Ph Phenyl h/mil. parts per million Rg I cut through Proc. Procedure Rac Racemic RP-HPLC Reverse phase high performance liquid chromatography RT Ambient temperature pace. Temperature t-BuOH tert-butanol T.F.A. Trifluoroacetic acid THF T ethrahydrofuran TLC Thin layer chromatography TMS-C1 Trimethylsilyl chloride uv UV radiation

Example 1. Synthesis of compounds.

Intermediate-1. 8-(2-fluorobenzyl)imidazo[1,2-a]pyrazine-6-carbonitrile (Int-1)

This intermediate was synthesized according to the procedure of the patent literature (WO 2015/187470A1). 1H NMR (500 MHz, methanol- _d4 ) δ (ppm): 9.09 (s, 1H), 8.14 (s, 1H), 7.91 (s, 1H), 7.35 (t, 1H), 7.28 (d, 1H), 7.10 (m, 2H), 4.60 (s, 2H).

General Procedure A. Intermediate-2 (Int-2).

The title compound was synthesized in 2 steps.

Step 1. Synthesis of 8-(2-fluorobenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide

0 .5 M sodium methanolate solution (5 equiv.) (note: a catalytic amount of sodium methanolate could also be used). After stirring for 1 hour at room temperature, ammonium hydrochloride (10 equiv.) was added and the reaction mixture was allowed to stir overnight. The reaction mixture was concentrated in vacuo, diluted with half-saturated NaHCO ₃ (20 ml) and 1 N NaOH (2 ml) and extracted with 2x20 ml EtOAc. The combined organic phases were dried over sodium sulfate, filtered and concentrated to provide the crude product as a brown solid. It was used in the next step without further purification.

- 19 043549

Step 2. Synthesis of 5-fluoro-2-(8-(2-fluorobenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-ol (Int-2)

Sodium (2)-3-ethoxy-2-fluoro-3-oxoprop was added to a solution of 8-(2-fluorobenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide (1 equiv.) in ethanol (9 ml). -1-ene-1-olate (3 equiv.). The reaction mixture was heated in a closed test tube at 80°C for 3 hours. After cooling, the solvent was removed in vacuo.

The crude material was purified by reverse phase HPLC using a gradient of 595% acetonitrile in water+0.1% TFA to provide the title compound (16 mg, 19% yield) as an off-white solid. 1H NMR (500 MHz, methanol- _d4 ) δ (ppm): 9.48 (s, 1H), 8.28 (s, 1H), 8.09 (d, 1H), 7.98 (s, 1H), 7.38 (s, 1H), 7.29 (d, 1H), 7.12 (m, 2H), 4.71 (s, 2H). When the compound was produced on a larger scale, it was obtained as an off-white solid (57 mg, 33% yield). 1H NMR (500 MHz, DMSO-d6) δ (ppm): 12.5 (br s, 1H), 9.48 (s, 1H), 8.31 (s, 1H), 8. 18 (m, 1H), 7.88 (s, 1H), 7.47 (m, 1H), 7.27 (q, 1H), 7.19 (t, 1H), 7.09 (t, 1H ), 4.59 (s, 2H).

Intermediate-3. 6-(4-chloro-5-fluoropyrimidin-2-yl)-8-(2-fluorobenzyl)imidazo[1,2-a]pyrazine (Int-3)

A solution of intermediate Int-2 (1 equiv.) in POCl ₃ (98 equiv.) was stirred overnight at room temperature. The solvent was removed in vacuo to provide the crude intermediate Int-3 as a brown solid. It was used in the next step without further purification.

Connection I-2

A solution of intermediate Int-3 (1 equiv.) and 2-(aminomethyl)-1,1,1,3,3,3-hexafluoropropan2-ol (3 equiv.) in DMSO (0.5 ml) in a microwavable vial treatment was heated by microwave radiation at 120°C for 2 hours. After cooling, the solution was filtered and the crude material was purified by reverse phase HPLC using a gradient of 30-60% acetonitrile in water + 0.1% formic acid, providing compounds 1-2 (10.2 mg, 42% yield) as a white solid. 1H NMR (500 MHz, DMSO-d ₆ ) δ (ppm) 9.43 (s, 1H), 9.22 (s, 1H), 8.46 (d, 1H), 8.38 ( br s, 1H), 8.28 (s, 1H), 7.84 (s, 1H), 7.53 (t, 1H), 7.23 (b s, 1H), 7.14 (t , 1H), 7.04 (t, 1H), 4.53 (s, 2H), 4.07 (d, 2H).

Compound I-1.

The title compound was synthesized in 2 steps.

Step 1. Synthesis of dibenzyl(1,1,1,3,3,3-hexafluoro-2-(((5-fluoro-2-(8-(2-fluorobenzyl)imidazo[1,2-a]pyrazin6-yl )pyrimidin-4-yl)amino)methyl)propan-2-yl)phosphate (compound I-4)

- 20 043549

To a solution of 1,1,1,3,3,3-hexafluoro-2-(((5-fluoro-2-(8-(2-fluorobenzyl)imidazo[1,2-a]pyrazin-6yl)pyrimindin-4 -yl)amino)methyl)propan-2-ol (compound 1-2) (0.48 g, 1 equiv.) in THF (9 ml) at 0°C was added 1 M LiHMDS in THF (1.2 ml, 1 ,3 equiv.) drop by drop over 5 minutes. The resulting yellow solution was stirred at 0°C for 20 minutes. In a separate flask, a solution of tetrabenzyl diphosphate (0.69 g, 1.4 equiv.) was prepared in THF (5 ml) and cooled to 0°C. The alkoxide solution was added dropwise to the pyrophosphate solution over 15 minutes, and the reaction mixture was allowed to warm to room temperature over 4 hours. It was then stirred at room temperature for 5 days. The reaction mixture was diluted with ethyl acetate (20 ml) and washed with water (10 ml). The organic layer was dried over sodium sulfate, filtered and concentrated to give the crude product as a yellow gum. It was used in the next step without further purification.

Step 2. Synthesis of compound I-1

Connection 1-4 Connection

To a solution of crude dibenzyl(1,1,1,3,3,3-hexafluoro-2-((((5-fluoro-2-(8-(2fluorobenzyl)imidazo[1,2-a]pyrazin-6-yl )pyrimindin-4-yl)amino)methyl)propan-2-yl)phosphate (compound I-4) in ethyl acetate (10 ml) and THF (10 ml) was added 5% palladium on carbon (1 equiv.) The reaction mixture was stirred under hydrogen from a cylinder overnight at room temperature. The mixture was filtered through a pad of celite and washed with methanol (10 ml). The eluent was concentrated to dryness to give a crude yellow solid. The crude material was purified by reverse phase HPLC using a 10-50% gradient acetonitrile in water + 0.1% TFA, providing compound I-1 (18 mg, 13% yield) as a white solid 1H NMR (500 MHz, deuterium oxide) δ (ppm): 8. 81-9.10 (s, 1H), 8.12 (d, 1H), 8.05 (s, 1H), 7.78 (s, 1H), 7.31 (m, 2H), 7.14 (m, 2H), 4.67 (s, 2H), 4.62 (s, 2H).

Intermediate-4 (Int-4).

This intermediate was prepared in four steps.

Step 1: Cyanidation of intermediate (15) to provide 2-(bromomethyl)-3,3,3trifluoro-2-((trimethylsilyl)oxy)propanenitrile (16)

Trimethylsilanecarbonitrile (153 g, 1.54 mol, 0.97 equiv) and triethylamine (4.44 ml, 3.22 g, 0.032 mol, 0.02 equiv) were added to a suitable reaction vessel equipped with a mechanical stirrer and a digital thermometer . The mixture was cooled to 5°C. 3-Bromo-1,1,1-trifluoropropan-2-one ((15), 304 g, 1.59 mol, 1.0 equiv.) was charged through a dropping funnel over 35 min, maintaining the reaction temperature between 10 and 20 °C.

The mixture was stirred at 20-30°C for 3 hours after addition to obtain intermediate (16) as a thick oil, which was used directly in the next step. 1H NMR (500 MHz, CDCl3) δ (ppm): 3.68 (d, J=11.14 Hz, 1H); 3.57 (d, J=11.14 Hz, 1H), 0.34-0.37 (m, 9H).

Step 2. Conversion of nitrile (16) to amide to provide 2-(bromomethyl)-3,3,3-trifluoro-2hydroxypropanamide (17) ''G H ₂ so _{4 Br} ^no x ^s T ²

O CN —-— fL t (16) (17)

Concentrated sulfuric acid (339 ml, 6.37 mol, 4.0 equiv) was stirred in a suitable reaction vessel equipped with a mechanical stirrer, digital thermometer and addition funnel. Sulfuric acid was heated to 45°C. The above intermediate (16) was added through a dropping funnel over 50 minutes while maintaining the temperature below 75°C. The reaction mixture was stirred at 75°C for 2 hours and then allowed to cool to room temperature. 1H NMR showed that the reaction was complete. The reaction mixture was cooled to -15°C and diluted with ethyl acetate (1824

- 21 043549 ml) through a dropping funnel for 45 min (very exothermic), maintaining the temperature between -15 and 5°C. Water (1520 ml) was added slowly through a dropping funnel over 1 hour 20 minutes (very exothermic) from -10 to 0°C. The layers were separated and the organic layer was washed with 15% aqueous sodium chloride (1520 ml), 25% aqueous sodium carbonate (911 ml), and then 15% aqueous sodium chloride (911 ml). The organic layer was filtered and concentrated under reduced pressure to obtain 348 g of intermediate (17) as a light yellow oil. This oil was dissolved in methanol (1200 ml) and concentrated to give 380 g of intermediate (17) (296 g adjusted weight, 79% yield). 1H NMR (500 MHz, CDCi ₃ ) δ 6.61-6.94 (m, 1H); 5.926.26(m, 1H); 3.93-4.00 (m, 1H); 3.68 (d, J=11.14 Hz, 1H).

Step 3: N-alkylation of compound (17) to provide 2-(aminomethyl)-3,3,3-trifluoro-2hydroxypropanamide (14)

n solution of ammonia in methanol (600 ml, 4.28 mol, 10 equiv.) was charged into a suitable reaction vessel equipped with a mechanical stirrer and a digital thermometer. The solution was cooled to 05°C. Intermediate (17) (102 g, 0.432 mol, 1 equiv) was then added via addition funnel over 30 min at 0 to 5°C. The reaction mixture was heated to 20-25°C for 1 hour and kept for 72 hours. The reaction was completed according to HPLC. The reaction mixture was cooled to 0-5°C and sodium methoxide (78 mL, 5.4 M, 0.421 mol, 0.97 equiv.) was added over 2 min. The reaction mixture was then concentrated under reduced pressure to a volume of 300 ml. 2 L of ethyl acetate was added and concentration was continued under reduced pressure until volume = 700 ml to obtain a suspension. 700 ml of ethyl acetate was added to the suspension to bring the final volume to 1400 ml. 102 ml of water was added and stirred for 2 minutes to obtain a two-phase solution. The layers were separated. The ethyl acetate layer was concentrated under reduced pressure to a volume of 600 ml. The ethyl acetate layer was then heated to >60°C and heptane (600 ml) was added slowly at 55 to 60°C. The mixture was cooled to 15-20°C to obtain a suspension. The suspension was stirred at 15-20°C for 2 hours and filtered. The solids were dried in vacuo at 25°C for 16 hours to give amine (14) as a white solid (48 g, 64% yield). 1H NMR (500 MHz, MeOH-d ₄ ) δ (ppm): 2.94 (d, J=13.73 Hz, 1H); 3.24 (d, J=13.58 Hz, 1H).

Step 4. Chiral solution of amine (14) as (R)-2,2-dimethyl-5-(trifluoromethyl)oxazolidin-5carboxamide (R)-2-hydroxysuccinate (intermediate-4, Int-4)

Amine (14) (105 g, 0.608 mol, 1.0 equiv.), (D)-malic acid (82 g, 0.608 mol, 1.0 equiv.) and acetone (1571 ml) were charged into a suitable reaction vessel equipped with mechanical stirrer and digital thermometer. The reaction mixture was stirred at 20-25°C for 16 hours. The resulting suspension was filtered and the wet cake was washed with acetone (300 ml). The wet precipitate was charged back into the reaction vessel, and acetone (625 ml) was charged. The suspension was heated to 53°C and kept for 6 hours. The suspension was cooled to 20-25°C and kept for 16 hours. The suspension was filtered and the wet cake was washed with acetone (200 ml). The wet residue was dried in vacuo at 40°C for 4 hours to obtain 82.4 g of intermediate Int-4 as a white solid (82.4 g, 39% yield, 97% ee). 1H NMR (500 MHz, D2O) δ (ppm): 4.33 (br s, 1H); 3.61 (width d, J=13.58 Hz, 1H); 3.40-3.47 (m, 1H); 2.76 (width d, J=15.87 Hz, 1H); 2.53-2.63 (m, 1H); 2.16 (broad s, 4H).

Connection I-3

- 22 043549

A solution of intermediates Int-3 and Int-4 and N,N-diisopropylethylamine (3 equiv.) in DMSO (0.4 ml) and water (0.04 ml) was heated by microwave radiation at 120°C in a microwave vial. processing within 2 hours. After cooling, the mixture was filtered and the crude material was purified by reverse phase HPLC using a gradient of 30-60% acetonitrile in water + 0.1% formic acid to provide compound I-3 (13.1 mg, 32% yield) as white solid. 1H NMR (500 MHz, DMSO-d ₆ ) δ (ppm): 9.43 (s, 1H), 8.39 (d, 1H), 8.32 (br s, 1H), 8 .27 (s, 1H), 8.16 (d, 1H), 7.83 (s, 1H), 7.78 (br. s., 1H), 7.60 (b. s., 1H), 7.52 (t, 1H), 7.24 (d, 1H), 7.15 (d, 1H), 7.06 (t, 1H), 4.57 (s, 2H), 4.00 (d , 2H).

Compound of general formula B1.

and I-4, connections to the core

Similar to the synthesis given above for compounds I-1, I-2, I-3 of formula B1 below can be synthesized according to the following scheme:

Compounds of the general formula of the Int-M intermediate can be prepared according to the protocols described in WO 2016081668. The Int-M intermediate can then be further converted using protocols similar to those described above to provide access to compounds I-1 to I -4.

Intermediate-5: 8-(2,5-difluorobenzyl)imidazo[1,2-a]pyrazine-6-carbonitrile (Int-5)

The nitrile intermediate Int-5 was prepared from the starting bromide, 6,8dibromimidazo[1,2-a]pyrazine, using 2,5-difluorobenzyl bromide, according to the patent literature procedure (WO 2015/187470A1) described for the synthesis of Int-1 . MS ES+ m/z (wt/charge) = 271.2 [M+H]+.

Intermediate 6: 6-(4-chloro-5-fluoropyrimidin-2-yl)-8-(2,5-difluorobenzyl)imidazo[1,2-a]pyrazine (Int-6)

Intermediate Int-6 was prepared from intermediate Int-5 following the general procedure described for the synthesis of intermediate Int-3 from Int-1. MS ES+ m/z 376.3 [M+H] ⁺ .

Connection I-5

- 23 043549

A solution of intermediates Int-6 (1 equiv.), Int-4 (3 equiv.), and triethylamine (5 equiv.) in a 3:1 dioxane/water mixture (1.3 ml) was heated at 90°C for 18 hours After cooling to room temperature, the reaction mixture was reduced to half its volume and diluted with 1 N aqueous HCl and ethyl acetate. The layers were separated and the organic layer was washed successively with water, then saturated aqueous sodium chloride, dried (over sodium sulfate) and concentrated in vacuo to give a crude solid. The crude material was purified by reverse phase HPLC using a gradient of 30 to 60% acetonitrile in water (modified with 0.1% formic acid) to provide compound I-5 (20.0 mg, 29% yield) as white solid. 1H NMR (500 MHz, CD ₃ OD) δ (ppm): 9.40 (s, 1H), 8.22-8.25 (m, 1H), 8.18 (s, 1H), 7.83 (s, 1H), 7.15-7.21 (m, 1H), 7.06-7.10 (m, 1H), 6.91-6.98 (m, 1H), 4, 65 (s, 2H), 4.26 (d, 1H), 4.05 (d, 1H). MS ES ⁺ m/z 512.2 [M+H] ⁺ .

Intermediate-7: 8-(2-fluorobenzyl)-[1,2,4]triazolo[1,5-a]pyrazine-6-carbonitrile (Int-7)

A solution of (2-fluorobenzyl)zinc(II) chloride (18.0 ml, 0.5 M in TNF, 9.00 mmol) was added to a mixture of PdCl ₂ (PPh ₃ ) ₂ (0.253 g, 0.360 mmol) and a commercially available intermediate compound Int-7a (2.00 g, 7.20 mmol). The reaction mixture was purged with nitrogen, sealed and heated to 50°C for 1 hour. The reaction mixture was cooled to room temperature and directly purified by silica gel chromatography using a gradient of 0-70% ethyl acetate in hexanes to give intermediate Int-7b ( 1.49 g, 61%). 1H NMR: (500 MHz, DMSO-d ₆ ), δ (ppm): 9.46 (s, 1H), 8.74 (s, 1H), 7.39-7.42 (m, 1H), 7.30-7.34 (m, 1H), 7.14-7.21 (m, 2H), 4.56 (s, 2H). MS ES ⁺ m/z=307.1 [M] ⁺ , 309.0 [M+2] ⁺ .

Stir mixture of Int-7b (1.49 g, 4.85 mmol), zinc(II) cyanide (0.684 g, 5.82 mmol), DPPF (0.538 g, 0.970 mmol), Pd ₂ (dba) ₃ (0.533 g , 0.582 mmol) and zinc (0.162 g, 2.47 mmol) in DMA (8 ml) were heated at 120°C for 2 h under nitrogen atmosphere. Once complete, the mixture was cooled to room temperature, diluted with ethyl acetate:dichloromethane:methanol (2:1:1, 100 ml), filtered through a pad of celite and concentrated. The solid residue was purified by silica gel chromatography using a gradient of 0-85% ethyl acetate in hexanes to give intermediate Int-7 (869 mg, 71%). 1H NMR: (500 MHz, MeOH-d ₄ ), δ (ppm): 9.52 (s, 1H), 8.72 (s, 1H), 7.39-7.42 (m, 1H), 7.28-7.32 (m, 1H) 7.08-7.15 (m, 2H), 4.65 (s, 2H). MS ES ⁺ m/z 254.1 [M+H] ⁺ .

Intermediate 10: 6-(4-chloro-5-fluoropyrimidin-2-yl)-8-(2-fluorobenzyl)-[1,2,4]triazolo[1,5-a]pyrazine (Int-10)

To a stirred room temperature solution of intermediate Int-7 (1.64 g, 6.48 mmol) in MeOH (33 mL) was added a solution of sodium methoxide (4.44 mL, 25% in MeOH, 19.4 mmol). After 1 h, the formation of the imidate ester was confirmed by LC-MS. Solid NH ₄ Cl (1.73 g, 32.4 mmol) was added and the mixture was stirred while the progress of the reaction was monitored by LC-MS analysis. Once complete, the resulting precipitate was filtered and dried in vacuo to provide intermediate Int-8 (1.63 g, 93%). MS ES ⁺ m/z 271.1 [M+H] ⁺ .

To a stirred mixture of intermediate Int-8 (1.63 g, 6.03 mmol) in absolute EtOH (27 ml) at room temperature was added intermediate Int-8a (2.82 g, 18.1 mmol). The mixture was heated at 80°C for 30 min, and the progress of the reaction was monitored by LC-MS analysis. Upon completion, aqueous HCl (18.1 mL, 1 M) was added and the resulting mixture was stirred for 30 min at room temperature, resulting in the formation of a precipitate. The solid was filtered and dried in vacuo. The second batch was removed from the filtrate after standing for 30 minutes. The combined solids were dried under high vacuum to obtain

- 24 043549 niem intermediate compound Int-9 (2.01 g, 98%). MS ES ⁺ m/z 341.0 [M+H] ⁺ .

A stirred mixture of POCl ₃ (1.96 mL, 21.0 mmol) and intermediate Int-9 (358 mg, 1.05 mmol) was heated at 80°C for 30 min while the reaction progress was monitored by LC-MS analysis . Once complete, POCl ₃ was evaporated in vacuo and the residue was partitioned between saturated aqueous sodium bicarbonate (20 ml) and dichloromethane (40 ml). The aqueous layer was extracted with dichloromethane (2x40 ml), and the combined organic layers were dried (MgSO ₄ ), filtered and concentrated to provide a crude residue. The crude product was purified by silica gel chromatography using a gradient of 0-50% ethyl acetate in hexanes to give intermediate Int-10 (200 mg, 53.0%) as a solid. ^1H NMR (500 MHz, DMSOd ₆ ) δ (ppm): 9.65 (s, 1H), 9.14 (s, 1H), 8.82 (s, 1H), 7.40 ( m, 1H), 7.34-7.26 (m, 1H), 7.23 7.15 (m, 1H), 7.157.08 (m, 1H), 4.66 (s, 2H). MS ES ⁺ m/z 359.0 [M+H] ⁺ .

Connection I-6

A solution of intermediate Int-10 (1 equiv.), 2-(aminomethyl)-1,1,1,3,3,3-hexafluoropropan2-ol (3 equiv.) and N-ethyl-N-isopropylpropan-2-amine in DMSO (0.8 ml) was heated at 90°C for 1 hour, then 70°C for another 12 hours. After cooling to room temperature, the reaction mixture was diluted with 1 N aqueous HCl to pH=3, which resulted in the formation of orange precipitate, which was filtered and dried. Purification of the crude material was accomplished by silica gel chromatography using a gradient of 0 to 20% 7:1 acetonitrile/methanol in dichloromethane over 25 minutes followed by reverse phase HPLC using a gradient of 20 to 70% acetonitrile in water ( modified with 0.1% trifluoroacetic acid) for 25 minutes to obtain compound I-6 (12.2 mg, 17% yield) as a light yellow solid. 1H NMR: (500 MHz, DMSO- _d6 ), δ (ppm): 9.57 (s, 1H), 8.83 (s, 1H), 8.80 (s, 1H), 8 .49 (d, 1H), 8.31 (m, 1H), 7.48-7.51 (t, 1H), 7.26-7.30 (m, 1H), 7.14-7.18 (t, 1H), 7.07-7.10 (t, 1H), 4.60 (s, 2H), 4.16 (d, 2H). MS ES ⁺ m/z=520.3.

Intermediate 11: 3-chloro-6-(4-chloro-5-fluoropyrimidin-2-yl)-8-(2-fluorobenzyl)imidazo[1,2-a]pyrazine (Int-11)

To a solution of intermediate Int-2 (103 mg, 0.304 mmol) in MeOH (5 ml) at 0°C was added N-chlorosuccinimide (NCS) (44.6 mg, 0.344 mmol). After 30 minutes, the reaction mixture was heated to ambient temperature for 17 hours and then to 50°C for 2.5 hours. The reaction mixture was concentrated and the resulting residue was purified by silica gel chromatography using a 0-20% gradient. acetonitrile/methanol (7:1) in dichloromethane to give intermediate Int-2a (99.6 mg, 85%). MS ES ⁺ m/z 374.2 [M+H] ⁺ .

A mixture of intermediate Int-2a (47.9 mg, 0.128 mmol) in POCl ₃ (0.836 ml, 8.97 mmol) was stirred for 2.5 h at 60°C, after which analysis by LC-MS showed that the reaction is complete. The mixture was concentrated to dryness. The residual amount of POCl3 was removed by squeezing with toluene (2x1 ml) and subsequent drying in vacuum. The resulting crude substance Int-11 was used without purification. MS ES ⁺ m/z 392.2 [M+H] ⁺ .

Connection I-7

- 25 043549

A suspension of intermediate Int-11 (1.0 equiv.), N-ethyl-N-isopropylpropan-2-amine (6.0 equiv.) and 1-amino-2-methylpropan-2-ol (1.8 equiv.) ) in DMSO (1.0 ml) was heated to 90°C for 16 hours. After cooling to room temperature, the reaction mixture was purified directly by silica gel chromatography using a 7:1 acetonitrile/methanol gradient in dichloromethane over 75 minutes , giving compound I-7 (19.4 mg, 35% yield) as an off-white solid. 1H NMR: (500 MHz, DMSO- _d6 ), δ (ppm): 8.90 (s, 1H), 8.28 (d, 1H), 8.00 (s, 1H), 7 .55-7.57 (m, 1H), 7.44-7.47 (m, 1H), 7.25-7.30 (m, 1H), 7.15-7.19 (m, 1H) , 7.09-7.12 (m, 1H), 4.73 (s, 1H) 4.58 (s, 2H), 3.49 (d, 2H), 1.15 (s, 6H). MS ES ⁺ m/z=445.2.

Intermediate 13: 6-(4,5-dichloropyrimidin-2-yl)-8-(2-fluorobenzyl)imidazo[1,2-a]pyrazine (Int-13)

Cl Cl

Int-12 Int-13

To a stirred mixture of 8-(2-fluorobenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide (5.00 g, 18.6 mmol) and Int-12a (4.19 g, 27.9 mmol) in anhydrous MeOH (100 mL) at room temperature, a solution of NaOMe (8.49 mL, 25 wt% in MeOH, 37.1 mmol) in anhydrous MeOH (24 mL) was added over 5 min. The resulting solution was heated under reflux. After 2 hours the reaction was incomplete; accordingly, more Int-12a (900 mg, 5.98 mmol) was added. After heating for an additional 2 hours, the mixture was cooled to room temperature and stirred overnight. The mixture was concentrated and the resulting residue was stirred in H2O (100 ml). The pH was adjusted to 3 with aqueous HCl (2 mL, 6 M), the mixture was stirred for 30 min and filtered. The solids were washed with H2O (2x30 ml) and dried under high vacuum to provide intermediate Int-12 (5.20 g, 79%). MS ES ⁺ m/z 356.2 [M+H] ⁺ .

A stirred mixture of Int-12 (5.20 g, 14.6 mmol) and DMAP (179 mg, 1.46 mmol) in POCl ₃ (27.0 mL, 292 mmol) was heated at 90°C for 3 hours. The mixture concentrated and the remaining POCl ₃ azeotropically removed using toluene (100 ml). The residue was redissolved in dichloromethane (200 ml), cooled in an ice bath and neutralized with 10% aqueous NaHCO3 (150 ml). The mixture was diluted with DCM (100 ml) and the phases were separated. The organic layer was washed with H2O (50 ml) and saturated aqueous NaHCO ₃ (2x50 ml), dried over Na ₂ SO ₄ , filtered and concentrated. The residue was purified by silica gel chromatography using a gradient of 20-80% ethyl acetate in hexanes to give intermediate Int-13 (2.50 g). 1H NMR (500 MHz, CDCl ₃ ) δ (ppm): 9.18 (s, 1H), 8.77 (s, 1H), 7.76 (d, 1H), 7.69-7 .73 (m, 1H), 7.30 (m, 1H), 7.08-7.12 (m, 1H), 6.89-7.01 (m, 2H), 4.71 (s, 2H ). MS ES ⁺ m/z 374.1 [M+H] ⁺ .

Connection I-8

Int-13 Connection 1-8

To a solution of intermediate Int-13 (1.0 equiv.) and racemic-3-amino-1,1,1trifluoropropan-2-ol (3.0 equiv.) in a mixture of dioxane/water in a ratio of 3:1 (1.0 equiv. 3 ml) triethylamine (5.0 equiv.) was added. The reaction mixture was stirred at 90°C for 18 hours. After cooling to room temperature, the reaction mixture was reduced to half its volume and diluted with 1 N aqueous HCl and ethyl acetate. The layers were separated and the organic layer was washed successively with water, then saturated aqueous sodium chloride, dried (over sodium sulfate) and concentrated in vacuo to give a crude solid. The crude material was purified by reverse phase HPLC using a gradient of 30 to 60% acetonitrile in water (modified with 0.1% formic acid) to provide racemic compound I-8 (34.5 mg, 55% yield) as pale yellow solid. 1H NMR (500 MHz, CD3OD) δ (ppm): 9.35 (s, 1H), 8.32 (s, 1H), 8.12 (s, 1H), 7.80 (s, 1H), 7.25-7.30 (m, 1H), 7.17-7.24 (m, 1H), 6.99-7.02 (m, 1H), 7.02-7.07 ( m, 1H), 4.66 (s, 2H), 4.33-4.41 (m, 1H), 4.03 (dd, 1H), 3.76 (dd, 1H). MS ES ⁺ m/z=467.2

- 26 043549

Intermediate 15: 2-(8-(2-fluorobenzyl)imidazo[1,2-a]pyrazin-6-yl)pyrimidin-4-ol (Int 15)

A solution of 8-(2-fluorobenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide (1.00 g, 3.71 mmol), intermediate Int-14a (1.21 mL, 11.1 mmol) and N-ethyl-N-isopropylpropan-2-αminα (1.95 ml, 11.1 mmol) in ethanol (14.9 ml) was stirred at 80°C for 12 hours. The reaction mixture was cooled to room temperature, to which was added ammonium chloride (0.6 g), and the resulting mixture was stirred at room temperature for 6 hours. The reaction mixture was then heated to 80°C for another 20 hours, at which time LC-MS indicated that the reaction was complete. The reaction mixture was cooled to room temperature, which led to the formation of a precipitate. This solid was filtered and dried to give a yellow solid (350 mg). The filtrate was concentrated to dryness, diluted with ethyl acetate (100 ml) and water (100 ml). The layers were separated and the aqueous layer was extracted with ethyl acetate (2x100 ml). The combined organic layers were washed with saturated sodium chloride solution, dried (sodium sulfate), filtered and concentrated to obtain an additional 380 mg of the desired product. The solid batches were combined to give Int-14 (730 mg, 58% yield) as a dark yellow solid. MS ES ⁺ m/z 322.1 [M+H] ⁺ .

A mixture of intermediate Int-14 (350 mg, 1.09 mmol) and POCl ₃ (2.00 mL, 21.8 mmol) was stirred at 90°C for 3 h, at which time LC-MS indicated complete conversion. POCl ₃ was removed in vacuo and the resulting residue was partitioned between saturated aqueous sodium bicarbonate (25 ml) and dichloromethane (50 ml). The aqueous layer was extracted with dichloromethane (2x25 ml) and the combined organic extracts were dried (MgSO ₄ ), filtered and concentrated under reduced pressure to give crude Int-15 (380 mg, 103% yield). In the next stage it was used without purification. MS ES ⁺ m/z 340.1 [M+H] ⁺ .

Connection I-9

Triethylamine (5.0 equiv) was added to a solution of intermediate Int-15 (1.0 equiv) and 2-aminoethan-1-ol (3.0 equiv) in DMSO (1 ml), heated to 90°C for 18 hours. The reaction mixture was cooled to room temperature, which resulted in the formation of a precipitate, which was collected. Purification of the crude solid was achieved by reverse phase HPLC using a gradient of 20-50% acetonitrile in water (modified with 0.1% formic acid) to give compound I-9 as a clear glass. 1H NMR (500 MHz, CD3OD) δ (ppm): 9.58 (br s, 1H), 8.26 (s, 2H), 8.21 (s, 1H), 7.87 ( s, 1H), 7.18-7.30 (m, 2H), 7.04-7.08 (m, 1H), 7.00-7.04 (m, 1H), 4.67 (s, 2H), 3.81 (br s, 4H). MS ES ⁺ m/z 366.1 [M+H] ⁺ (secondary protonation observed).

Intermediate 16: 8-(3,5-difluoro-4-methylbenzyl)imidazo[1,2-a]pyrazine-6-carbonitrile (Int-16)

The nitrile intermediate Int-16 was prepared from the starting bromide, 6,8 dibromimidazo[1,2-a]pyrazine, using 3,5-difluoro-4-methylbenzyl bromide, according to the patent literature procedure (WO 2015/187470A1) described for the synthesis of intermediate Int-1. MS ES ⁺ m/z=285.2 [M+H] ⁺ .

- 27 043549

Intermediate-19: 6-(4,5-dichloropyrimidin-2-yl)-8-(3,5-difluoro-4methylbenzyl)imidazo[1,2-a]pyrazine (Int-19)

The amidine intermediate Int-17 was prepared from Int-16 as described for the synthesis of Int-2 from Int-1. MS ES ⁺ m/z=302.2 [M+H] ⁺ .

Intermediate Int-19 was prepared from Int-17 as described in the procedure for preparing Int-13 from 8-(2-fluorobenzyl)imidazo[1,2-a]pyrazine-6-carboximidamide. ^1H NMR (500 MHz, CD ₃ OD) δ (ppm): 9.88 (s, 1H), 8.79-8.90 (m, 1H), 8.58 (d, 1H) , 8.36(d, 1H), 7.06-7.20. (m, 2H), 4.71 (s, 2H), 2.15 (s, 3H). MS ES ⁺ m/z 406.1 [M+H] ⁺ .

Connection I-10

To a solution of Int-19 (1.0 equiv.) and 2-(aminomethyl)-1,1,1,3,3,3-hexafluoropropan-2-ol (3.0 equiv.) in a dioxane/water mixture in the ratio Triethylamine (5.0 equiv.) was added 3:1 (1.3 mL). The reaction mixture was stirred at 90°C for 18 hours. After cooling to room temperature, the reaction mixture was reduced to half its volume and diluted with 1 N aqueous HCl and ethyl acetate. The layers were separated and the organic layer was washed successively with water, then saturated aqueous sodium chloride, dried (over sodium sulfate) and concentrated in vacuo to give a crude solid. The crude material was purified by reverse phase HPLC using a gradient of 30 to 60% acetonitrile in water (modified with 0.1% formic acid), providing compound I-10 (9.7 mg, 14% yield) as completely white solid. ^1H NMR (500 MHz, CD ₃ OD) δ (ppm): 9.38 (s, 1H), 8.41 (s, 1H), 8.14 (s, 1H), 7.86 (s, 1H), 7.23 (d, 2H), 4.49 (s, 2H), 4.17 (s, 2H), 2.08 (s, 3H). MSC ES ⁺ m/z 567.2 [M+H] ⁺ .

Intermediate-21: 6-(4-chloro-5-methoxypyrimidin-2-yl)-8-(2-fluorobeyyl)-[1,2,4]triazolo[1,5-a]pyrazine (Int-21)

A mixture of ethyl methoxyacetate (1.0 equiv.) and tert-butoxybis(dimethylamino)methane (2.0 equiv.) was heated at 110°C for 21 h, after which LC-MS showed conversion to the product. The crude reaction mixture was directly purified by silica gel chromatography using a gradient

- 28 043549 from 0 to 60% ethyl acetate in hexanes for 30 minutes, obtaining ethyl 3-(dimethylamino)-2methoxyacrylate (Int-20a) as a light yellow oil. MS ES ⁺ m/z 174.0 [M+H] ⁺ .

A solution of intermediates Int-8 (170 mg, 0.629 mmol), Int-20a (327 mg, 1.89 mmol) and DBU (0.285 ml, 1.89 mmol) in absolute ethanol (1.5 ml) was heated at 90° C for 1 h, after which analysis by LC-MS showed that the transformation was complete. After cooling to room temperature, the reaction mixture was acidified to pH 2-3 with 2.5 M HCl in ethanol, which resulted in the formation of a fine precipitate. The solid was collected and dried to yield intermediate Int-20 (109 mg, 49% yield) as a brown solid. 1H NMR (500

MHz, DMSO-d6) δ (ppm): 12.30 (s, 1H), 9.55 (s, 1H), 8.82 (s, 1H), 7.73 (s, 1H), 7.48-7.52 (m, 1H), 7.26-7.34 (m, 1H), 7.18-7.24 (m, 1H), 7.11-7.14 ( m, 1H), 4.63 (s, 2H), 3.83 (s, 3H). MS ES ⁺ m/z 353.1 [M+H] ⁺ .

A mixture of intermediate Int-20 (108.5 mg, 0.308 mmol) in POCl ₃ (574 μl, 6.16 mmol) was heated to 90°C for one hour, after which LC-MS analysis indicated that the conversion was complete. After cooling the reaction mixture to room temperature, POCl ₃ was removed in vacuo and the resulting residue was suspended in ice water and neutralized with saturated sodium bicarbonate solution. The aqueous mixture was extracted with 5:1 dichloromethane:ipopropanol (3 x 20 mL), dried (sodium sulfate), filtered and concentrated to give intermediate Int-21 (94.9 mg, 83% yield) as a brown solid. This material was used in the subsequent step without purification. MS ES ⁺ m/z 371.1 [M+H] ⁺ .

Connection I-11

lnt-21 Connection 1-11

A suspension of intermediate Int-21 (1.0 equiv.) and racemic-3-amino-1,1,1trifluoropropan-2-ol (3.0 equiv.) and triethylamine (5.0 equiv.) in dioxane/water. in a 3:1 ratio (1.3 ml) was heated at 90°C for 12 h, after which LC-MS analysis showed that the transformation was complete. The reaction mixture was diluted with water and adjusted to pH 6-7 with 1.0 N aqueous hydrochloric acid, which resulted in the formation of an orange precipitate. The solid was filtered and dried. Purification of the crude solid was accomplished by reverse phase HPLC using a gradient of 10-40% acetonitrile in water (modified with 0.1% trifluoroacetic acid) over 20 min to give racemic compound I-11 (57.4 mg, yield 48%) as an off-white solid. 1H NMR: (500 MHz, CD3OD), δ (ppm): 9.63 (s, 1H), 8.61 (s, 1H), 7.74 (s, 1H), 7.30- 7.33 (m, 1H), 7.15-7.20 (m, 1H), 6.97-7.01 (m, 2H, overlapping shifts), 4.67 (s, 2H), 4.28 -4.32 (m, 1H), 3.984.02 (m, 4H, overlapping shifts), 3.86-3.91 (m, 1H). MS ES ⁺ m/z 464.2 [M+H] ⁺ .

Intermediate-23: 4-chloro-2-(8-(2-fluorobenzyl)-[1,2,4]triazolo[1,5-a]pyrazin-6yl)pyrimidine-5-carbonitrile (Int-23)

int-23

A solution of ethyl(ethoxymethylene)cyanoacetate (1.72 g, 10.2 mmol), Int-8 (0.917 g, 3.39 mmol) and DBU (1.53 mL, 10.2 mmol) in ethanol (6.8 mL ) was heated to 100°C for 24 h, after which LC-MS analysis showed a mixture of products. The reaction mixture was then cooled to room temperature and acidified by adding 2.5 N HCl in ethanol (4.07 mL, 10.2 mmol). The reaction mixture was concentrated to dryness and then purified by silica gel chromatography using a gradient of 0 to 100% 7:1 acetonitrile/methanol in dichloromethane over 75 min. A second purification was required using silica gel chromatography using a gradient of 0 to 70% 7:1 acetonitrile/methanol in dichloromethane over 75 min, providing Int-22a

- 29 043549 (214 mg, 16% yield) as a brown solid (90% purity). Int-22b was not isolated.

1H NMR (Int-22a): (500 MHz, DMSO-d6), δ (ppm): 13.43 (br s, 1H), 9.78 (s, 1H), 8.88 ( s, 1H), 8.80 (broad.

s, 1H), 7.44-7.53 (bw, 1H), 7.28-7.32 (m, 1H), 7.19-7.22 (m, 1H), 7.09- 7.12 (m, 1H) 4.64 (s, 2H). MS ES ⁺ m/z 348.1 [M+H] ⁺ .

A suspension of Int-22a (211 mg, 0.608 mmol) was heated in POCl ₃ (1.42 ml, 15.2 mmol) at 80°C. After 3 h, LC-MS analysis indicated that the conversion was complete. The reaction mixture was concentrated to dryness to give crude Int-23 (331 mg) as a brown solid. ^1H NMR: (500 MHz, DMSO-d6), δ (ppm): 9.84 (s, 1H), 9.49 (s, 1H), 8.88 (s, 1H), 7 .40-7.43 (m, 1H), 7.28-7.33 (m, 1H), 7.18-7.21 (m, 1H), 7.11-7.14 (m, 1H) , 4.67 (s, 2H). MS ES ⁺ m/z 366.1 [M+H].

Connection I-12

Connection 1-12

A solution of intermediate Int-23 (1.0 equiv.), N-ethyl-N-isonropylpropan-2-amine (10.0 equiv.) and 2-(aminomethyl)-1,1,1,3,3,3 -hexafluoropropan-2-ol (2.5 equiv.) in dimethyl sulfoxide (1.2 ml) was heated to 95°C for 24 hours. After cooling to room temperature, the reaction mixture was purified directly by reverse phase HPLC using a gradient from 20 to 60% acetonitrile in water (modified with 0.1% formic acid) for 10 minutes to give compound I-12 (8.6 mg, 8% yield) as a gold-colored solid. 1H NMR: (500 MHz, DMSO- _d6 ), δ (ppm): 9.71 (s, 1H), 8.94 (s, 1H), 8.85 (s, 1H), 8 .62 (broad s, 1H), 8.19 (s, 1H), 7.46-7.49 (m, 1H), 7.26-7.30 (m, 1H), 7.15-7 .18 (m, 1H), 7.07-7.10 (m, 1H) 4.61 (s, 2H), 4.23-4.26 (m, 2H). MS ES ⁺ m/z 525.4 [MH] ⁺ .

Intermediate 24: 8-(thiophen-3-ylmethyl)imidazo[1,2-a]pyrazine-6-carbonitrile (Int-24)

Int-24 as a nitrile was prepared from the starting bromide, 6,8 Dibromimidazo[1,2-a]pyrazine intermediate, using 3-(chloromethyl)thiophene, following the patent literature procedure (WO 2015/187470A1) described for the synthesis of the intermediate compound-1, as well as a reference to J. Org. Chem. 2018, 83, 11437-11445 for the preparation of (thiophen-3-ylmethyl)zinc(II) chloride. Intermediate Int-24a was isolated as an off-white solid (68% yield), and Int-24 was isolated as a yellow solid (36% yield). Int-24a: MS ES ⁺ m/z 295.9 [M+H] ⁺ . Int-24: MS ES ⁺ m/z 241.0 [M+H] ⁺ .

Intermediate-26: 6-(4-chloro-5-fluoropyrimidin-2-yl)-8-(thiophen-3-ylmethyl)imidazo[1,2-a]pyrazine (Int-26)

Intermediate Int-26 was prepared using the same procedure described for preparing Int-3 from Int-1. Int 25a: MS ES ⁺ m/z 258.0 [M+H] ⁺ . Int 25: 1H NMR: (500 MHz, DMSO-d6), δ (ppm): 12.82 (br s, 1H), 9.46 (br s, 1H), 8.30 ( s, 1H), 8.22 (br s, 1H), 7.90 (s, 1H), 7.43-7.46 (m, 2H, overlapping shifts), 7.30 (br s, 1H ), 4.54 (s, 2H). MS ES ⁺ m/z 328.0 [M+H] ⁺ . Int 26: MS ES ⁺ m/z 346.0 [M+H] ⁺ .

- 30 043549

Connection I-13

A solution of intermediate Int-26 (1.0 equiv.) and 2-(aminomethyl)-1,1,1,3,3,3hexafluoropropan-2-ol (3.0 equiv.) and triethylamine (5.0 equiv. ) in a 2:1 dioxane/water mixture (0.6 ml) was heated at 90°C for 22 hours, after which the reaction was completed. The reaction mixture was diluted with water and extracted with dichloromethane:isopropanol (5:1) (3x25 ml). The combined organic layers were dried (sodium sulfate), filtered and concentrated to a brown residue. Purification was accomplished by chromatography on silica gel using a gradient of 0 to 40% acetonitrile/methanol 7:1 in dichloromethane over 12 minutes to give compound I-13 (15 mg, 25% yield) as an off-white solid substances. 1H NMR: (500 MHz, DMSO-d ₆ ), δ (ppm): 9.57 (s, 1H), 9.42 (s, 1H), 8.48-8.51 (m, 2H, overlapping shifts), 8.28 (s, 1H), 7.87 (s, 1H), 7.42 (lat s, 1H), 7.38 (lat s, 1H), 7.26 -7.27 (m, 1H), 4.42 (s, 2H), 4.08-4.09 (m, 2H). MS ES ⁺ m/z 507.1 [M+H] ⁺ .

Intermediate-27: 8-((2-methylpyrimidin-5-yl)methyl)imidazo[1,2-a]pyrazine-6carbonitrile (Int-27)

Intermediate Int-27a is prepared by lithiation of the starting bromide, 6,8dibromimidazo[1,2-d]pyrazine, and quenching with 2-methylpyrimidine-5-carbaldehyde according to Journal of the American Chemical Society, 2003, 125, 8082-8083.

Intermediate Int-27b is prepared by standard reduction using trifluoroacetic acid and triethylsilane. Conversion of Int-27b to the cyano intermediate Int-27 is achieved using the procedure described for the preparation of Int-1.

Intermediate 28: 6-(4-chloro-5-fluoropyrimidin-2-yl)-8-(2,5-difluorobenzyl)imidazo[1,2-a]pyrazine (Int-28)

Intermediate Int-28 is prepared from Int-27 following the general procedure described for the synthesis of Int-3 from Int-1.

Connection I-14

Compound I-14 is prepared using the same conditions as described for the preparation of compound 1 -2 from Int-3.

Connection I-15.

The disodium salt of prodrug I-1 was prepared using NaOMe and following the procedure described in the experimental part of J. Med. Chem. 2008, 51, 1111-1114.

- 31 043549

Connection I-16

A solution of intermediate Int-10 (1 equiv.), intermediate Int-4 (3 equiv.), and triethylamine (6.0 equiv.) in 1:1 dioxane/water (1.2 ml) was heated at 80 °C for 24 hours. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate and the resulting organic layer was dried (sodium sulfate), filtered and concentrated to give an oil. Purification of the crude product was achieved by silica gel chromatography using a gradient of 0 to 100% ethyl acetate in hexanes to give compound I-16 (64.2 mg, 56% yield) as a light yellow solid. 1H NMR (500 MHz, CDCl ₃ ) δ (ppm): 9.52 (s, 1H), 8.56 (s, 1H), 8.26 (s, 1H), 7.64 (visible 1H), 7.20-7.25 (m, 2H), 7.02-7.14 (m, 2H), 4.77 (s, 2H), 4.22-4.28 (m, 2H) [2 acidic protons not observed due to exchange with CDCl ₃ ]. Compound I-16: MS ES+ m/z 495.1 [M+H] ⁺ .

Example 2: Measuring biological activity using a cGMPGloSensor-based cell-based assay in a 384-well format.

Human embryonic kidney cells (HEK293) expressing cGMP biosensors GloSensor™ 40F (Part No.: CS 182801, Promega) were used to evaluate the activity of test compounds. Luminescent biosensors (engineered luciferases) that were incorporated into these cells detect cGMP produced by sGC enzyme-stimulating compounds and emit luminescence.

Cells expressing cGMP-GloSensor were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with fetal bovine serum (FBS, 10% final concentration) and hygromycin (200 μg/ml). The day before analysis, cells were seeded in DMEM with 10% FBS in a volume of 50 μl at a density of 1.5 x 104 cells/well in a 384-well flat white bottom polyD-lysine coated plate (Corning catalog no. 35661). Cells were incubated overnight at 37°C in a humidified chamber with 5% CO2. The next day, the medium was removed and the cells were placed in 40 μl/well of GloSensor™ substrate, 2 mM (Promega catalog no. E1291). Cells were treated for 90 min at 25°C to equilibrate the substrate within the cells. Test compounds and diethylenetriamine NONOate (DETANONOate) were diluted to 3 mM (20x) in serum-free, CO2-free medium and serially diluted in 4-fold dilutions to generate a dose-response curve in 5X quantities, of which 10 μl (x µM concentration for the test compound solution and 10 µM concentration for the DETA-NONOat solution, where x represents one of the following final concentrations): 30,000; 7500; 1875; 468.75; 117.19; 29.29; 7.32; 1.83; 0.46; 0.114; 0.029 nM.

For kinetic studies, luminescence was measured immediately for 0.2 s per well using Envision (Perkin Elmer). For endpoint screening using SAR analysis, data were collected after 55 min of incubation at room temperature.

Data were driven to high point control using the following equation: 100 * (sample - high point control)/(high point control - low point control) where low point control is the average of sixteen data points for DMSO (1%) as control, and the high point control is the average of sixteen data points for compound Y (30 µM), where compound Y is depicted below. Data were fit using a 4-parameter fit (log(agonist) vs. response - variable slope) using GraphPad Prism v.5 software. n=2 for all connections. The absolute EC ₅₀ was interpolated from the curve fit and is defined as the concentration at which a given compound produces 50% of the response when controlling for the high point after adjusting the data as above. Compounds that do not produce a minimum response=50% are reported as >30 µM or ND (not determined). For compounds tested in duplicate, or when n is greater than 2, the result reported here is the geometric mean of several results obtained. In table II summarizes the results obtained for selected compounds from Table. I, respectively.

- 32 043549

Connection Y.

Table II

Whole Cell Activity in GloSensor Cell Based Assay, 384 Well Format (Example 2, Compounds in Table I)

Compound Absolute EC50 (nM) in GlosGC assay 1-2 A 1-3 A 1-5 A 1-6 IN 1-7 IN 1-8 IN 1-9 V 1-10 With 1-11 With 1-12 V 1-15 With 1-16 V

Values of sGC enzymatic activity in HEK cells determined using the GloSensor assay. (~) Definitions of codes for sGC enzyme activity values expressed as absolute EC ₅ o, which is defined as the concentration at which a given compound produces 50% of the response in the case of the high point control (compound Y) after reporting the data. EC ₅₀ OD<100 nM=A; 100 nM <EC ₅₀ OP<SOOO nM=V; 1000 nM < EC ₅₀ OD <30 µM=C; 30 µM < EC ₅₀ On=D. Compounds that did not produce a minimal response less than 50% of the high point control were reported as >30 μM (and fell into category D).

Example 3 Blood Pressure Changes in Sprague-Dawley Rats.

Male rats (250-350 g body weight, available from Harlan Laboratories) were anesthetized with ketamine/xylazine, and a heparinized saline-filled catheter was implanted into the right femoral artery. The catheter was withdrawn between the shoulder blades, capped, and the animal was allowed to recover for at least 7 days postoperatively before any testing of connections. Prior to testing, animals were maintained on a normal diet with free access to drinking water using a 12-hour light-dark cycle.

On the day of the experiment, under inhalational isoflurane anesthesia, the catheter was opened and connected to a connector (Instech Labs) and a pressure transducer (Harvard Apparatus). Subsequently, blood pressure and heart rate were measured and analyzed using a dedicated data acquisition system (PowerLab, ADInstruments). The data sampling rate was set to 1 cycle per second. After connection, each rat was allowed to recover from anesthesia, and baseline blood pressure and heart rate values were determined in these conscious, freely moving animals. After baseline determination, either vehicle (0.5% methylcellulose or 100% PEG400) or test compound was administered orally PO, 10 mg/kg, and effects on blood pressure and heart rate were monitored for up to 24 hours.

The sodium salt (compound 1-15) of the phosphorus-containing prodrug (compound 1-1) compound 1-2 was administered orally in water at a dose of 10 mg/kg to determine the effects of compound 1-2 on blood pressure. One hour after dosing, 97.5% of the administered compound 1-15 was converted to compound 1-2. At 160 minutes post-dose, Compound 1-2 reduced MAP (mean blood pressure) by approximately mmHg. below the original level.

Although exemplary embodiments have been set forth for purposes of illustration, the foregoing descriptions and examples should not be construed as limiting the scope of the present invention. Accordingly, various modifications, adaptations and alternatives may come to mind to one skilled in the art without departing from the spirit and scope of the present invention.

Claims (25)

CLAIM 1. Connection selected from: 2. A compound represented by the following formula: where: n is an integer selected from 0-3; and each J ^B independently represents a halogen, -CN, C _{1-6 -α} -liphatic group or -OR ^B ; wherein each J ^B that is a C _1-6 -α-liphatic group is optionally and independently substituted with up to 3 copies of R ³ ; each R ^B independently represents hydrogen or a C _1-6 aliphatic group; wherein said R ^B is optionally and independently substituted with up to 3 copies of R ^3a ; each R ³ and R ^3a represents, in each instance, independently halogen, -CN, C _1-4 alkyl, C _1-4 haloalkyl, -O(C _1-4 alkyl) or -O(C _1-4 haloalkyl); J ^C independently represents hydrogen, halogen, C _1-4 aliphatic group, C _1-4 alkoxy or -CN; wherein each specified C _{1-4 -α} -liphatic group and each specified C _1-4 alkoxy is optionally and independently substituted with up to 3 copies of C _1-4 alkoxy, C _1-4 haloalkoxy, -OH or halogen; And J ^D is hydrogen, chlorine, fluorine, methoxy or -CN. 3. A compound according to claim 2, wherein J ^D is fluorine. 4. A compound according to claim 2 or 3, wherein (i) J ^C is independently hydrogen, C _1-2 alkyl, C _1-2 alkoxy, C _1-2 fluoroalkyl, C _1-2 fluoroalkoxy, halogen and -CN wherein each of said C _1-2 alkyl and C _1-2 alkoxy is optionally and independently substituted with up to 3 copies of C _1-4 alkoxy, C _1-4 haloalkoxy, -OH or halogen; (ii) ^JC is hydrogen or halogen; or (iii) ^JC is hydrogen. 5. A compound according to any one of claims 2 to 4, wherein (i) n is 1, 2 or 3, and each J ^B independently represents a halogen or a C1-6 aliphatic group; (ii) n is 1 or 2, and each J ^B independently represents a halogen or a C1-6 aliphatic group; (iii) n is 1 or 2 and each J ^B is fluorine; or (iv) n is 1 and J ^B is fluorine. 6. A compound of formula IA, formula IB or formula II, or a pharmaceutically acceptable salt thereof, - 34 043549 where Y is independently N or C; ring B is phenyl or a 5- or 6-membered heteroaryl ring containing 1 or 2 heteroatoms per ring independently selected from N, O or S; n is an integer selected from 0-3; and each JB is independently halogen, -CN, C1. ₆ -aliphatic group or -ORB; with each JB which represents C ₁ . ₆ -aliphatic group, optionally and independently substituted with up to 3 copies of R ³ ; each RB independently represents hydrogen or C ₁ . ₆ -aliphatic group; wherein said RB is optionally and independently substituted with up to 3 copies of R ^3a ; R ³ and R ^3a are each, in each instance, independently halogen, -CN, C ₁ . ₄ alkyl, C ₁ . ₄ haloalkyl, -O(C ₁ . ₄ alkyl) or -O(C ₁ . ₄ haloalkyl); Z is selected from the group consisting of hydrogen, -P(O)(OH) ₂ , -P(O)(OH)O'M ⁺ , -P(O)(O')2(M+)2, -P( O)(O')2D ²⁺ and -P(O)(O-benzyl)2; where M ⁺ represents a pharmaceutically acceptable monovalent cation and D ²⁺ represents a pharmaceutically acceptable divalent cation; m is 0 or 1; R ¹ represents C ₁ . ₄ alkyl, C ₁ . ₄ fluoroalkyl, -C(O)NH ₂ or hydrogen; And R ² represents C ₁ . ₄ alkyl, C ₁ . ₄ fluoroalkyl or hydrogen; both copies of R ⁴ are simultaneously hydrogen, or both copies of R ⁴ together with the carbon atom to which they are attached form a carbonyl group; J ^D is hydrogen, halogen, methoxy or -CN, p is 1 or 2; and each JC independently represents hydrogen, halogen, C ₁ . ₄ -aliphatic group, C ₁ . ₄ alkoxy or -CN; and each specified C ₁ . ₄ -aliphatic group and each specified C ₁ . ₄ alkoxy is optionally and independently substituted with up to 3 copies of C ₁ . ₄ alkoxy, C ₁ . ₄ haloalkoxy, -OH or halogen. 7. The compound according to claim 6 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by 8. The compound according to claim 6 or 7, in which (i) J ^D represents hydrogen, chlorine, fluorine, methoxy or -CN; (ii) J ^D is methoxy; (iii) J ^D is hydrogen, chlorine, fluorine or -CN; (iv) J ^D is hydrogen, chlorine or fluorine; (v) J ^D is fluorine or hydrogen; (vi) J ^D represents hydrogen; (vii) J ^D is fluorine; (viii) J ^D is -CN; or (ix) J ^D is chlorine. 9. A compound according to any one of claims 6 to 8, wherein (i) each JC is independently aqueous - 35 043549 genus, C1-2alkyl, C1-2alkoxy, C1-2fluoroalkyl, C1-2fluoroalkoxy, halogen and -CN, each of the above C _1-2 alkyl and C _1-2 alkoxy are optionally and independently substituted with up to 3 copies of C _1-4 alkoxy, C _1-4 haloalkoxy, -OH or halogen; (ii) each J ^C is independently hydrogen, C _1-2 alkyl, C _1-2 alkoxy, C _1-2 fluoroalkyl, C _1-2 fluoroalkoxy, halogen or -CN; (iii) each ^JC is hydrogen or halogen; (iv) each JC is hydrogen; or (v) each JC is fluorine or chlorine. 10. A compound according to any one of claims 6 to 9, which is a compound of formula IIIA, formula IIIB, formula IVA, formula IVB, formula VA, formula VIA, formula VB or formula VIB: Formula VB Formula VIB or a pharmaceutically acceptable salt thereof. 11. The compound according to any one of claims 6 to 10, in which (i) ring B represents phenyl; (ii) ring B is a 5- or 6-membered heteroaryl ring containing 1 or 2 heteroatoms per ring independently selected from N, O and S; or (iii) ring B is a 6-membered heteroaryl ring containing 1 or 2 ring nitrogen atoms. 12. A compound according to any one of claims 6 to 11, wherein (i) n is 1, 2 or 3, and each JB independently represents a halogen or a C _1-6 aliphatic group; (ii) n is 1 or 2, and each JB independently represents a halogen or a C _1-6 aliphatic group; (iii) n is 1 or 2 and each JB is fluorine; (iv) n is 1; and each JB represents fluorine. 13. A compound according to claim 11, which is a compound of formula VIIA, formula VIIIA, formula VIIB or formula VIIIB: - 36 043549 Formula VIIB Formula VIIIB or a pharmaceutically acceptable salt thereof. 14. A compound according to any one of claims 6 to 13, in which (i) R ¹ represents C1_2alkyl, C1-2fluoroalkyl, hydrogen or -C(O)NH2; or (ii) R ¹ is C1-2fluoroalkyl or -C(O)NH2. 15. A compound according to any one of claims 6 to 14, in which (i) R ² represents C1-2fluoroalkyl, C1-2alkyl or hydrogen; or (ii) R ² is a C1-2fluoroalkyl. 16. The compound according to claim 14 or 15, in which (i) R ¹ represents trifluoromethyl or -C(O)NH2; and R ² represents trifluoromethyl; (ii) R ¹ and R ² are both hydrogen or C1-2 alkyl; or one of R ¹ and R ² is hydrogen and the other is C1-2fluoroalkyl; (iii) R ¹ and R ² are both hydrogen; (iv) R ¹ and R ² are both methyl; or (v) one of R ¹ and R ² is hydrogen and the other is trifluoromethyl. 17. Connection according to claim 6 or 16, selected from: - 37 043549 where J ^D is selected from hydrogen or fluorine. 18. The compound according to claim 17, wherein J ^D is fluorine. 19. A compound according to any one of claims 6 to 18, in which (i) Z represents -P(O)(OH) ₂ ; (ii) Z is -P(O)(OH)OM ⁺ or -P(O)(O-)2(M ⁺ )2; and M ⁺ represents Na ⁺ , K ⁺ , Cs ⁺ or a monovalent organic amine cation; or (iii) Z is -P(O)(O-) ₂ D ²⁺ ; and D ²⁺ is Ca ²⁺ , Zn ²⁺ , Mg ²⁺ or a divalent organic amine cation. 20. The connection according to claim 17, selected from: 21. The compound according to claim 20, represented by the formula: 22. A compound according to claim 6, selected from the group consisting of - 38 043549 сг-\ X 1 n^n 4Λ _Ν -χ ^ΟΗ F ^Н Me Me 1-7 Cl I ^ΊΓ'Ρ Y - N'N L JI he γ Cl ^H CF ₃ racemic 1-8 Cl I Ύ n^n H 1-9 Yx N^N ^F L 1 .on γ^Ν χ _C | ^H CF ₃ CF ₃ 1-10 g-NV 1 ηή I ^u N ^: N yyv ^b OMe CF ₃ racemic 1-11 ts A _L 1 L CHI Y ^u N^N n γ CN ^H CF ₃ CF ₃ 1-12 Cl I ¹ iCY^s Y ^u n^n V'N'- _X ° ^H F ^H CF ₃ CF ₃ 1-13 Cl y ^N ^lC _Me N^N Υ _Ν -χ ^0Η F ^H CF ₃ CF ₃ 1- 14 /N'^Y^N^NH 7% ΥΥ 9 ,ONa X JI gР-ONa F^^ 0 1-15 N, X. 1 iOj Y ^u F ^M OH CF ₃ 1-16 23. The connection according to claim 22, represented by: 24. The connection according to claim 20, presented 25. A pharmaceutical composition containing a compound according to any one of claims 6 to 24 and at least one pharmaceutically acceptable excipient or carrier. Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky lane, 2