TWI639613B - Hcv polymerase inhibitors - Google Patents

Abstract

The present invention provides a compound of the formula:

Wherein B is selected from the nucleobases of groups (a) to (d): And other variables are used to treat or prevent hepatitis C virus infection, and related aspects, as defined in the scope of the patent application.

Description

HCV polymerase inhibitor

The present invention relates to nucleoside derivatives which are inhibitors of the polymerase of hepatitis C virus (HCV). The invention further relates to prodrugs of nucleoside derivatives, compositions comprising the same, and methods for treating or preventing HCV infection.

HCV is a single-stranded, forward RNA virus of the Flaviviridae family of viruses in the genus Hepatitis. The NS5B region of the RNA polygene encodes an RNA-dependent RNA polymerase (RdRp), which is required for viral replication. After the initial acute infection, most infected individuals develop chronic hepatitis because HCV preferentially replicates in hepatocytes but is not directly cytopathic. In particular, the high tendency to be free of severe T-lymphocyte responses and viral mutations appears to promote a high rate of chronic infection. Chronic hepatitis can progress to liver fibrosis, leading to cirrhosis, advanced liver disease, and HCC (hepatocellular carcinoma), making it the leading cause of liver transplantation.

There are six major HCV genotypes and more than 50 subtypes that are geographically distinctly distributed. HCV genotype 1 is the major genotype in Europe and the United States. The extensive genetic heterogeneity of HCV has important diagnostic and clinical implications, which may explain the difficulty of vaccine development and non-responsiveness to current therapies.

The spread of HCV can occur via contact with contaminated blood or blood products after, for example, blood transfusion or intravenous drug use. The introduction of diagnostic tests used in blood screening has led to a downward trend in the incidence of HCV after transfusion. However, due to the progress of advanced liver disease, it is slow Existing infections will continue to present serious medical and economic burdens for decades.

The first generation of HCV therapy is based on (pegylated) a combination of interferon-alpha (IFN-alpha) and ribavirin. This combination therapy produces a sustained viral response in more than 40% of patients infected with genotype 1 virus and about 80% of those infected by genotype 2 and 3. In addition to the limited potency of HCV genotype 1, this combination therapy has significant side effects and is poorly tolerated in many patients. The main side effects include flu-like symptoms, hematological abnormalities, and neuropsychiatric symptoms. Second-generation HCV treatment increases the HCV protease inhibitor teleravir or boceprevir, which allows for shorter treatment times but produces a variety of serious side effects. The introduction of the protease inhibitor simeprevir and the HCV polymerase inhibitor sofosbuvir can produce major improvements in treatment. These agents were initially co-administered with interferon and ribavirin, but recent co-administration of cimevir (WO2007/014926) and sulphide (WO2008/121634) has further reduced treatment time and significant reduction Side effects of interferon-free and HCV treatment without ribavirin.

The advantage of nucleoside/nucleotide HCV polymerase inhibitors (e.g., Sububuvir) is that they are often active against several HCV genotypes. For example, Succeed Bubweb has been approved by the FDA and EMA for the treatment of HCV genotypes 1 and 4. However, in the split phase III clinical trial reported in Lawitz et al., N. Eng. J. Med. 2013; 368: 1878-87, it was noted that the response rate in the Subu-Bupiva -Ribavirin group is in the gene. Patients with type 3 infection were lower than those with genotype 2 infection (56% vs 97%) . Therefore, there is a need for a treatment that is more effective, convenient, and better tolerated.

The use of HIV drugs, specifically the use of HIV protease inhibitors, teaches that suboptimal pharmacokinetics and complex dosing regimens quickly cause inadvertent compliance failure. This in turn means that the 24-hour trough concentration (minimum plasma concentration) of the individual drugs in the HIV regimen typically falls below the IC ₉₀ or ED ₉₀ threshold of most of the day. That at least IC ₅₀ IC _90, and more realistically ED ₉₀ or 24 hours of content trough for slowing the development of drug escape mutants of the body based necessary. Achieving essential pharmacokinetics and drug metabolism to allow for these trough levels provides a rigorous challenge for drug design.

NS5B RdRp is absolutely essential for the replication of single-stranded, forward HCV RNA genomes, making it an attractive target for the development of antiviral compounds. There are two main classes of NS5B inhibitors: non-nucleoside inhibitors (NNI) and nucleoside analogs. NNI binds to the ectopic region of the protein, while the nucleoside inhibitor composition is metabolized to the corresponding nucleotide and serves as a surrogate for the polymerase. The formed nucleotides are then incorporated into the nascent RNA polymer chain and the growth of the polymer chains can be terminated. To date, nucleoside and non-nucleoside inhibitors of NS5B have been known.

As indicated above, the inhibition mechanism of nucleoside inhibitors involves the phosphorylation of nucleosides to the corresponding triphosphates. Phosphorylation is generally mediated by host cell kinases and is essential for the nucleoside to be active as a surrogate for NS5B polymerase. Typically, the first phosphorylation step (i.e., conversion of the nucleoside to the 5'-monophosphate nucleoside) is a rate limiting step. Subsequent conversion of the monophosphate to the diphosphate and triphosphate is generally easy and usually does not limit the rate. The strategy for increasing nucleoside triphosphate production is to use a cell permeable monophosphate nucleoside prodrug, a nucleoside with a masked phosphate moiety (the "prodrug moiety"), which is susceptible to intracellular enzyme activation. Thereby producing a nucleoside monophosphate. The monophosphate thus formed is subsequently converted to the active triphosphate by cellular kinase.

Chemical modification of the active compound to obtain a potential prodrug produces a completely new molecular entity that exhibits undesirable physical, chemical, and biological properties, and therefore, the identification of the best prodrug remains an uncertain and challenging task.

There is a need for HCV inhibitors that overcome the shortcomings of current HCV therapies (eg, side effects (eg, toxicity), limited efficacy, no genomic coverage, emergence of resistance, and compliance failure) and also improve sustained viral responses.

The present invention provides novel HCV-inhibiting compounds having properties useful for one or more of the following parameters: antiviral potency; pan-genotype coverage; favorable properties of resistance; non-toxicity and genotoxicity; favorable pharmacokinetics Learning and pharmacodynamics; and easy to deploy and donate. Those skilled in the art will appreciate that the HCV inhibiting compounds of the present invention need not be confirmed. Each of the known compounds is modified in every respect, but instead provides a balance of properties, which means that the HCV inhibiting compound is a valuable alternative to a pharmaceutical agent.

The compounds of the invention may also be attractive due to the fact that they are inactive against other viruses, i.e., specifically for HIV. Patients with HIV infection usually have a co-infection, such as HCV. Treatment of such patients with HCV inhibitors that also inhibit HIV can result in the emergence of resistant HIV strains.

In one aspect, the invention provides a compound represented by Formula I:

Wherein: B is selected from the group consisting of nucleobases of groups (a) to (d): Wherein Y is N or -C(R ¹⁹ )-; R ¹ is H, C(=O)R ³⁰ , C(=O)CHR ³¹ NH ₂ , CR ³² R ^32' OC(=O)CHR ³³ NH ₂ , or R ^{1 is} selected from the group consisting of groups (i) to (vi):

R ² is H, C(=O)R ³⁰ , C(=O)CHR ³¹ NH ₂ , CR ³² R ^32' OC(=O)CHR ³³ NH ₂ or CR ³² R ^32' OC(=O)R ³⁰ Or R ¹ together with R ² form a divalent linker of the formula:

R ³ is OH, C ₁ -C ₆ alkoxy, C ₃ -C ₇ cycloalkoxy, C ₃ -C ₇ cycloalkyl C ₁ -C ₃ alkoxy, benzyloxy, O-(C ₁ -C ₆ alkylene)-TR ²¹ or NHC(R ¹⁵ )(R ^15' )C(=O)R ¹⁶ ; R ⁴ , R ⁵ , R ⁷ and R ⁸ are each independently H, C ₁ - C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ hydroxyalkyl, halo, -OR ¹⁸ , -SR ¹⁸ or -N(R ¹⁸ ) ₂ ; R ⁶ , R ⁹ , R ¹⁰ And R ¹¹ are each independently selected from H, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₇ cycloalkyl, C ₁ -C ₆ halo Alkyl, C ₁ -C ₆ hydroxyalkyl, halo, OR ¹⁸ , SR ¹⁸ , N(R ¹⁸ ) ₂ , -NHC(O)OR ¹⁸ , -NHC(O)N(R ¹⁸ ) ₂ , -CN , -NO ₂ , -C(O)R ¹⁸ , -C(O)OR ¹⁸ , -C(O)N(R ¹⁸ ) ₂ and -NHC(O)R ¹⁸ , wherein the C ₂ -C ₆ alkenyl group And the C ₂ -C ₆ alkynyl group may be optionally substituted by a halo group or a C ₃ -C ₅ cycloalkyl group; R ¹² is H or -(C ₁ -C ₆ alkylene)-TR ²¹ , phenyl, fluorene Or a naphthyl group, the phenyl, indenyl or naphthyl group being optionally substituted by 1, 2 or 3 substituents each independently selected from the group consisting of halo, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₁ -C ₆ haloalkyl, hydroxy C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, C ₁ -C ₆ alkylcarbonyl, C ₃ -C ₆ cycloalkylcarbonyl C ₁ -C ₆ alkoxy, C ₁ -C ₆ haloalkoxy, hydroxy and amine; R ¹³ series H or -(C ₁ -C ₆ alkylene)-TR ²¹ ; or R ¹² and R ¹³ may be bonded between the oxygen atoms to which they are attached to form a C ₂ -C ₄ alkylene group, wherein the C ₂ -C ₄ alkyl is optionally substituted by a C ₆ -C ₁₀ aryl group; R ¹⁴ is H or C ₁ -C ₆ alkyl, phenyl, naphthyl or contains 1, 2 or 3 independently selected from N a 5- to 12-membered monocyclic or bicyclic heteroaryl group of a hetero atom of O and S, the phenyl, naphthyl or heteroaryl being optionally substituted by 1, 2 or 3 R ²² ; R ¹⁵ and R ^15' Each is independently selected from H, C ₁ -C ₆ alkyl, C ₃ -C ₇ cycloalkyl, C ₃ -C ₇ cycloalkyl C ₁ -C ₃ alkyl, phenyl and benzyl, or R ¹⁵ And R ^15' together with the carbon atom to which it is attached form a C ₃ -C ₇ -cycloalkylene group, wherein each C ₁ -C ₆ alkyl group is optionally selected from the group consisting of a halogen group, OR ¹⁸ and SR ¹⁸ Substituted, and each C ₃ -C ₇ cycloalkyl, C ₃ -C ₇ cycloalkyl, phenyl and benzyl are optionally independently selected from C ₁ -C ₃ alkyl, 1 or 2, group of halo and oR ¹⁸ substituents; or R ^{15 'and} R ¹⁵ lines H and R ²⁴ The attached form a 5-membered ring together with atoms; R ¹⁶ lines H, C ₁ -C ₁₀ alkyl, C ₂ -C ₁₀ alkenyl, C ₃ -C ₇ cycloalkyl, C ₃ -C ₇ cycloalkyl C ₁ -C ₃ alkyl, benzyl, phenyl or adamantyl, each of which is optionally selected from the group consisting of halo, OR ¹⁸ and N(R ¹⁸ ) ₂ by 1, 2 or 3, respectively. Substituted; each R ¹⁷ is independently selected from H, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ haloalkyl, C ₃ -C ₇ cycloalkyl, C ₃ -C ₇ cycloalkenyl, phenyl and benzyl; or two R ¹⁷ together with the nitrogen atom to which they are attached form a 3 to 7 membered heterocyclic ring or a 5 to 6 membered heteroaryl ring , such as optionally 1 or 2 independently selected from C ₁ -C ₃ alkyl, halo, C ₁ -C ₃ haloalkyl, amine, C ₁ -C ₃ alkylamino, (C ₁ -C ₃ alkyl) ₂ amine group substituted; each R ^{18 is} independently H, C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl or C ₃ -C ₇ cycloalkyl; R ¹⁹ is H, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₇ cycloalkyl, C ₁ -C ₆ haloalkyl, C ₁ - C ₆ hydroxyalkyl, halo, -OR ¹⁸ or N(R ¹⁸ ) ₂ ; each R ^{20 is} independently H, C ₁ -C ₆ alkyl, C ₁ -C ₆ halo An alkyl group, a C ₃ -C ₇ cycloalkyl group, a C ₁ -C ₆ hydroxyalkyl group or a C ₃ -C ₇ cycloalkyl C ₁ -C ₃ alkyl group; each R ^{21 is} independently H, C ₁ - C ₂₄ alkyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ hydroxyalkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₃ -C ₇ cycloalkyl or C ₃ -C ₇ cycloalkenyl; each R ²² is independently selected from halo, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₁ -C ₆ haloalkyl, phenyl, hydroxy C _1- C ₆ alkyl, C ₃ -C ₆ cycloalkyl, C ₁ -C ₆ alkylcarbonyl, C ₃ -C ₆ cycloalkylcarbonyl, carboxy C ₁ -C ₆ alkyl, pendant oxylate Required), OR ²⁰ , SR ²⁰ , N(R ²⁰ ) ₂ , CN, NO ₂ , C(O)OR ²⁰ , C(O)N(R ²⁰ ) ₂ and NHC(O)R ²⁰ , or attached Any two R ²² groups to an adjacent ring carbon atom may be combined to form -OR ²³ -O-; R ²³ -[C(R ³³ ) ₂ ] _n -; R ²⁴ H, or R ²⁴ and R ¹⁵ together with the atoms to which they are attached form a 5-membered ring; each R ³⁰ is independently selected from C ₁ -C ₆ alkyl and C ₁ -C ₆ alkoxy; each R ³¹ is independently selected from H , C ₁ -C ₆ alkyl, C ₃ -C ₇ cycloalkyl, and benzyl; each R ³² and R ^{32 'are} independently selected from H and lines C ₁ -C ₃ alkyl; each R ³³ are Is independently selected from H and C ₁ -C ₆ alkyl group; the U-based O or S; T are each independently based -S -, - O -, - SC (O) -, - C (O) S-, -SC(S)-, -C(S)S-, -OC(O)-, -C(O)O- and -OC(O)O-; or a pharmaceutically acceptable salt and/or solvent thereof Compound.

The compounds of formula I are optionally provided in the form of pharmaceutically acceptable salts and/or solvates. In one embodiment, the compounds of the invention are provided as a pharmaceutically acceptable salt. In a second embodiment, the compounds of the invention are provided in the form of a pharmaceutically acceptable solvate. In a third embodiment, the compounds of the invention are provided in their free form.

In one aspect, the invention includes prodrugs. In a typical configuration, the prodrug group is located at the 3' position and/or the 5' position of the sugar moiety. Suitable groups for this purpose include esters (i.e., groups of the formula OC(=O)R ³⁰ wherein R ³⁰ is typically a C ₁ -C ₄ alkyl group) and an amino acid ester (i.e., formula OC (=O) A group of CHR ³¹ NH ₂ wherein R ³¹ is typically C ₁ -C ₆ alkyl). Other suitable prodrug groups are phosphate prodrugs, ie, drug groups that are converted in vivo to phosphate esters. Prodrug groups can also be present on nucleobase B.

In one embodiment of the invention, the B group is a group (a). Typically, in this embodiment, group B has the formula (a'):

Wherein R ⁵ is H or F, and R ⁶ is N(R ¹⁸ ) ₂ or NHCOC ₁ -C ₆ alkyl. Typically, R ⁶ is NH ₂ . In still another exemplary embodiment of the invention, B has a group (a"):

Wherein R ⁶ is N(R ¹⁸ ) ₂ or NHCOC ₁ -C ₆ alkyl. Typically, R ⁶ is NH ₂ .

In a second embodiment of the invention, the B group is (b). Typically, in this embodiment, group B has the formula b':

Wherein R ⁸ is H or F. Typically, R ⁸ is H in a third embodiment of the invention, a B group (c').

Wherein R ⁹ is OH or C ₁ -C ₆ alkoxy, and R ¹⁰ is NH ₂ or NHCOC ₁ -C ₆ alkyl.

In a fourth embodiment of the invention, the B group is a group (d).

In one embodiment of the invention, R ² is H.

In an alternative embodiment of the invention, R ² is C(=O)R ³⁰ , C(=O)CHR ³¹ NH ₂ or OCR ³² R ^32' OC(=O)CHR ³³ NH ₂ .

In an embodiment of the invention wherein R ² is C(=O)R ³⁰ , R ³⁰ is typically methyl, isopropyl, isobutyl or second butyl, especially isopropyl. In an embodiment of the invention, wherein R ² is C(=O)CHR ³¹ NH ₂ , R ³¹ suitably corresponds to a side chain of a natural or unnatural amino acid, such as glycine (Gly), alanine ( Ala), a side chain of proline (Val), isoleucine (lIe) or phenylalanine (Phe), ie, R ³¹ is H, methyl, isopropyl, isobutyl or benzyl, respectively. Especially isopropyl. Particular attention is paid to the amino acid ester moiety in which the configuration at the asymmetric carbon atom to which R ³¹ is attached is the configuration of the L-amino acid, specifically L-Ala, L-Val, L-lle and L-Phe Especially L-proline, ie R ³¹ is isopropyl. In an embodiment of the invention, wherein R ² is OCR ³² R ^{32 '} OC(=O)CHR ³³ NH ₂ , R ³² and R ^32' may be the same or different and are typically selected from H and methyl, wherein R ³³ It is usually a C ₁ -C ₃ alkyl group.

In one embodiment of the invention, R ¹ is H.

In an alternate embodiment of the invention, the R ¹ is a prodrug moiety. Suitably according to these embodiments, R ¹ is C(=O)R ³⁰ , C(=O)CHR ³¹ NH ₂ or OCR ³² R ^32' OC(=O)CHR ³³ NH ₂ .

In an embodiment of the invention wherein R ¹ is C(=O)R ³⁰ , R ³⁰ is typically methyl, isopropyl, isobutyl or second butyl, especially isopropyl. In an embodiment of the invention, wherein R ¹ is C(=O)CHR ³¹ NH ₂ , R ³¹ suitably corresponds to a side chain of a natural or unnatural amino acid, such as glycine (Gly), alanine ( Ala), a side chain of proline (Val), isoleucine (lIe) or phenylalanine (Phe), ie, R ³¹ is H, methyl, isopropyl, isobutyl or benzyl, respectively. Especially isopropyl. Particular attention is paid to the amino acid ester moiety in which the configuration at the asymmetric carbon atom to which R ³¹ is attached is the configuration of the L-amino acid, specifically L-Ala, L-Val, L-lle and L-Phe Especially L-proline, ie R ³¹ is isopropyl. R ³¹ may also be a second butyl group. In an embodiment of the invention, wherein R ¹ is OCR ³² R ^{32 '} OC(=O)CHR ³³ NH ₂ , R ³² and R ^32' may be the same or different and are typically selected from H and methyl, wherein R ³³ Usually H or C ₁ -C ₃ alkyl.

In one embodiment of the invention, R ¹ and R ² together form a divalent linker of the formula:

Wherein R ³ is as defined above, thereby providing a compound of the formula:

Typically according to this embodiment, U is O.

Representative configurations for R ³ include C ₁ -C ₆ alkoxy and NHC(R ¹⁵ )(R ^15' )C(=O)R ¹⁶ .

Typically, R ³ is a C ₁ -C ₃ alkoxy group such as isopropoxy or methoxy.

A further typical configuration of R ³ is NHC(R ¹⁵ )(R ^15' )C(=O)R ¹⁶ .

Typically in this configuration, R ¹⁵ and R ^{15 '} are each independently selected from the group consisting of H, C ₁ -C ₆ alkyl, and benzyl. Typically, one of R ¹⁵ and R ^15' is H and the other is a side chain of an amino acid, such as the side chain of alanine, valine, leucine or isoleucine, ie methyl respectively , isopropyl, isobutyl or 1-methylpropan-1-yl. In a preferred configuration, R ¹⁵ and R ^{15 '} are H and the other is methyl.

R ¹⁶ is usually a linear or branched C ₁ -C ₆ alkyl group or a C ₃ -C ₇ cycloalkyl group. Typically, R ¹⁶ is an isopropyl group.

A representative value of R ³ is NHCH(C ₁ -C ₆ alkyl)C(=O)C ₁ -C ₃ alkyl.

An alternative configuration for R ³ is O-(C ₁ -C ₆ alkylene)-TR ²¹ , wherein the C ₁ -C ₆ alkyl moiety is linear or branched.

In one embodiment of the compound of formula (I), R ¹ is a group (i);

Preferably, in the compound of this embodiment, U is O.

In one configuration of the group (i), R ¹³ is H and R ¹² is (C ₁ -C ₆ alkyl)-TR ²¹ , usually in this configuration, R ¹² is an ethyl group, T is a system O and R ²¹ are C ₁₂ -C ₁₉ , thereby forming a structure (ia):

Wherein n is an integer from 11 to 23, such as from 11 to 18. n is preferably an integer from 15 to 16.

Usually in the group (i-a), U is O.

Typically in the compounds of formula (I), wherein R ¹ is a group (ia) and R ² is H.

In an alternative configuration of group (i), R ¹² and R ¹³ are bonded between their attached oxygen atoms to form an optionally substituted C ₂ -C ₄ alkyl group, thereby forming a cyclic phosphate. Typically, alkylene C ₃ alkylene-based, thereby providing a structure (ib):

In general, U is O and Ar is optionally substituted by 1 or 2 substituents independently selected from the group consisting of halo: halo, C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ alkoxy and cyano, usually halo. Representative examples of Ar include a phenyl group and a phenyl group substituted with chlorine at the meta position.

Typically in the compounds of formula (I), wherein R ¹ is a group (ib) and R ² is H.

In still another configuration of group (i), R ¹³ is (C ₁ -C ₆ alkyl)-TR ²¹ , thereby providing a group (ic):

Wherein the C ₁ -C ₆ alkylene moiety is linear or branched. Non-limiting examples of the C ₁ -C ₆ alkylene moiety in the group (ic) include methylene, ethyl, isopropyl and dimethylmethylene.

Usually in the group (i-c), U is O.

In a typical subgroup of the group (ic), U is O, C ₁ -C ₆ alkyl is methylene and T is -C(O)O-, or C ₁ -C ₆ alkyl is alkyl. Ethyl and T-C(O)S- are extended, thereby providing a compound of formula I having a partial structure (i-c1) or (i-c2), respectively:

Wherein R ²¹ is a C ₁ -C ₆ alkyl group, such as a tert-butyl group. R ^{12 in} such structures is typically the same group as R ¹³ or alternatively R ¹² is as defined above.

Typically in the compounds of formula (I), wherein R ¹ is a group (ic) and R ² is H.

In a further embodiment of the compound of formula (I), the R ¹ -based group (iii), ie, R ¹ together with the oxygen atom to which it is attached, forms a triphosphate or tri-thiophosphate, thereby providing the structure Compound:

Or a pharmaceutically acceptable salt thereof, such as a potassium or sodium salt. In a preferred configuration of the embodiments, U is O.

Typically according to this embodiment, R ² is H.

In a further embodiment of the compound of formula (I), the R ¹ group (iv):

In a typical compound of formula (I) wherein R ¹ is a group (iv) and one of R ¹⁵ and R ^{15 '} is H, the stereochemistry is as indicated in the formula:

U is usually O.

R ²⁴ is usually H.

Representative examples of R ¹⁴ include phenyl substituted with 1 or 2 R ²² as appropriate, wherein each R ²² is independently selected from halo, C ₁ -C ₆ alkyl, C ₂ -C ₆ ene And OR ²⁰ and R ²⁰ are C ₁ -C ₆ alkyl; or R ¹⁴ is naphthyl.

Typically according to this embodiment, U is O, R ²⁴ is H and R ¹⁴ is phenyl substituted by 1, 2 or 3 R ²² as appropriate, thereby providing a group (iv-a):

In a typical configuration of the group (iv-a), the phenyl group is substituted with 1 or 2 halo groups such as chlorine or fluorine.

In another group (iv-a) of a representative configuration, a phenyl group substituted with one R ^22, R ²² is selected from the C ₃ -C ₆ cycloalkyl, C ₁ -C ₆ alkylcarbonyl or C _3- C ₆ cycloalkylcarbonyl, the cycloalkyl moiety is optionally substituted by C ₁ -C ₃ alkyl.

In the group (iv-a) of still another representative configuration, phenyl substituted by 2 R ^22, wherein R ²² is selected from a C ₃ -C ₆ cycloalkyl, C ₁ -C ₆ alkylcarbonyl Or a C ₃ -C ₆ cycloalkylcarbonyl group, the cycloalkyl moiety being optionally substituted by C ₁ -C ₃ alkyl, and the other R ^{22 being} methyl, cyclopropyl, fluoro or chloro.

A further representative value R ¹⁴ of the system via two adjacent R located ²² carbon atoms substituted on the phenyl and two of R ²² in combination form a -O-CH ₂ -O-, thereby forming a partial structure:

A further representative configuration of R ¹⁴ is phenyl substituted with R ²² and R ²² is carboxy C ₁ -C ₆ alkyl, and R ²⁴ is H. A representative example of this configuration is illustrated in equation (iv-b)

Usually in the group (iv-b), U is O.

In one configuration of the group (iv), R ¹⁴ is fused to the phenyl group of the 4-membered heterocyclic ring which is substituted by a keto group and a phenyl group. Typical of these structures are shown in the following formula: E.g:

Other representative values for R ¹⁴ include sulfhydryl groups, usually 5-mercapto.

In one embodiment, the R ¹⁴ is heteroaryl containing 5, 12 membered monocyclic or bicyclic aromatic rings independently selected from heteroatoms of N, O and S. And the heteroaryl is optionally substituted with 1, 2 or 3 R ²² as appropriate. Typically in this embodiment, each R ²² is independently selected from C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ alkoxy , hydroxyl and amine groups.

Representative values of R ^{14 in} this example are optionally substituted pyridyl groups.

Typical compounds of this example are those wherein U is O and R ¹⁴ is optionally independently selected from halo, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ alkane by 1 or 2 a pyridyl group substituted with a substituent of a C ₂ -C ₆ alkenyl group, a C ₁ -C ₆ alkoxy group, a hydroxyl group or an amine group.

Typically in the compounds of formula (I), wherein R ¹ is a group (iv) or any of its subgroups, part N(R ²⁴ )C(R ¹⁵ )(R ^{15 '} )-C(=O)OR ¹⁶ Amino acid ester residues are formed, including natural and unnatural amino acid residues. Typically, one of R ¹⁵ and R ^15' is hydrogen and the other is hydrogen or a C ₁ -C ₆ alkyl group such as isopropyl or isobutyl. Particular attention is paid to amino acid residues in which R ^{15 '} is hydrogen, examples being glycine, (Gly) alanine (Ala), valine (Val), isoleucine (Ile) and phenylalanine ( The Phe) residue, i.e., R ^{15 '} is H and R ¹⁵ is methyl, isopropyl, isobutyl or benzyl, respectively. In the compound, wherein R ^{15 '} is hydrogen and R ^{15 is} not hydrogen, the configuration at the asymmetric carbon atom is usually a configuration of an L-amino acid, specifically L-Ala, L-Val, L-Ile And L-Phe.

In a typical configuration of group (iv), R ¹⁵ and R ^{15 '} are H and the other is methyl.

In still another configuration of the group (iv), R ¹⁵ and R ^{15 '} together with the carbon atom to which they are attached form a C ₃ -C ₇ cycloalkyl group, such as a cyclopropyl group or a cyclobutyl group.

In a typical configuration of the group (iv), R ¹⁶ is a C ₁ -C ₁₀ alkyl group.

In one configuration of the group (iv), R ¹⁶ is C ₁ -C ₃ alkyl, such as methyl, ethyl, propyl, isopropyl, preferably isopropyl.

In still another configuration of the group (iv), R ¹⁶ is C ₁ -C ₈ alkyl, such as 2-ethylbutyl, 2-pentyl, 2-butyl, isobutyl, third pentyl Preferred is 2-ethylbutyl.

In still another configuration of the group (iv), R ¹⁶ is a C ₃ -C ₇ cycloalkyl group, such as a cyclohexyl group.

In one embodiment of the compound of formula (I), R ¹ is a group (iv) wherein U is OR ²⁴ H, R ¹⁴ is phenyl, which is C ₃ -C ₆ cycloalkyl, C ₁ -C ₆ alkylcarbonyl or 5 or 6 membered heteroaryl substituted, R ¹⁵ is H, R ¹⁵ is C ₁ -C ₆ alkyl, such as methyl, ethyl or isopropyl, and R ¹⁶ is C ₁ -C ₆ Alkyl or C ₃ -C ₇ cycloalkyl, such as cyclopropyl, cyclobutyl or cyclopentyl.

In one embodiment of the compound of formula (I), R ¹ is a group (iv) wherein R ²⁴ is H, and R ^{14 is} optionally substituted with phenyl or naphthyl; and R ¹⁵ and R ^{15 '} are each independently H. Or C ₁ -C ₆ alkyl, and R ¹⁶ is C ₁ -C ₈ alkyl or C ₃ -C ₇ cycloalkyl.

In a typical configuration of R ¹ of this embodiment, R ²⁴ is H, R ¹⁴ is optionally substituted phenyl; one of R ¹⁵ and R ^15' is H, and the other is C ₁ - C ₃ alkyl, and R ¹⁶ is C ₁ -C ₈ alkyl.

In an alternative configuration of group (iv), R ¹⁵ is H, and R ^{15 '} and R ²⁴ together with the atoms to which they are attached form a pyrrolidine ring, thereby giving a group (iv-c):

Typically in this configuration, the U-form O, R ¹⁴ is optionally substituted phenyl and R ¹⁶ is C ₁ -C ₆ alkyl or C ₃ -C ₆ cycloalkyl.

Typically in the compounds of formula (I), wherein R ¹ is a group (iv) or any of its subgroups, R ² is H.

In a further embodiment of the compound of formula (I), the R ¹ group (v):

Usually in the group (v), U is O.

According to this embodiment, the two N-linked substituents of the P atom are the same, that is, the two R ¹⁵ moieties are identical, the two R ^{15 '} moieties are identical, and the two R ¹⁶ moieties are identical.

In a typical configuration of the group (v), both R ¹⁵ are H or C ₁ -C ₆ alkyl (eg ethyl, n-propyl, isopropyl, n-butyl or isobutyl), two R ^{15 '} are both H, and both R ¹⁶ are C ₁ -C ₆ alkyl (eg methyl, ethyl or isopropyl) or C ₃ -C ₇ cycloalkyl (eg cyclopropyl, ring) Butyl or cyclopentyl).

In one configuration of the group (v), R ¹⁶ is C ₁ -C ₃ alkyl, such as methyl, ethyl, propyl, isopropyl, preferably isopropyl.

In still another configuration of the group (v), R ¹⁶ is C ₁ -C ₈ alkyl, such as 2-ethylbutyl, 2-pentyl, 2-butyl, isobutyl, third pentyl Preferred is 2-ethylbutyl.

In still another configuration of the group (v), R ¹⁶ is a C ₃ -C ₇ cycloalkyl group, for example, a cyclohexyl group. In yet another embodiment of the compound of formula (I), the R ¹ group (vi):

Usually in the group (vi), U is O.

In one configuration of the group (vi), R ¹³ is -(C ₁ -C ₆ alkylene)-TR ²¹ , thereby providing the structure (vi-a):

Wherein the C ₁ -C ₆ alkylene moiety is linear or branched. Non-limiting examples of the C ₁ -C ₆ alkylene moiety in the group (vi-a) include methylene, ethyl, isopropyl and dimethylmethylene.

In one configuration of the subgroup vi-a, R ²¹ is 1-hydroxy-2-methylpropan-2-yl, a group of the formula:

Usually in the group (vi-a), U is O.

In a typical subgroup of the group (vi-a), a C ₁ -C ₆ alkylene group is a methylene group substituted by 1 or 2 C ₁ -C ₃ alkyl groups, and a T-system-OC ( O)O-, thereby providing a compound of formula I having a partial structure (vi-b):

Wherein R ³² and R ^{32' are} independently H or C ₁ -C ₃ alkyl. Typically, one of R ³² and R ^32' is H and the other is H, methyl or isopropyl. Alternatively, both R ³² and R ^32' are methyl.

Usually in the group (vi-b), U is O.

Typical examples of R ²¹ include optionally substituted C ₁ -C ₆ alkyl groups such as methyl, ethyl, propyl and isopropyl.

Typically, one of R ¹⁷ and R ^17' is H and the other is phenyl or benzyl, preferably benzyl.

Typically in the compounds of formula (I), wherein R ¹ is a group (vi) or any of its subgroups, R ² is H.

In a further subgroup of the group (vi-a), the U-form O, C ₁ -C ₆ alkyl-extension-ethyl and T-C(O)S-, thereby providing the following partial structure Compound of formula I:

Typical examples of R ²¹ include optionally substituted C ₁ -C ₆ alkyl groups (especially branched C ₁ -C ₆ alkyl groups) and C ₁ -C ₆ hydroxyalkyl groups.

Typically, one of R ¹⁷ and R ^17' is H and the other is phenyl or benzyl, preferably benzyl.

Typically in the compounds of formula (I), wherein R ¹ is a group (vi) or any of its subgroups, R ² is H.

Accordingly, there is provided a compound of formula I for use as a medicament, in particular for the treatment or prevention of HCV infection, especially for the treatment of HCV infection.

Further provided is the use of a compound of formula I for the manufacture of a medicament, in particular for the treatment or prevention of HCV infection, in particular for the treatment of HCV infection.

Additionally, a method of treating or preventing an HCV infection comprising administering a compound of Formula I, in particular a method of treating an HCV infection comprising administering a compound of Formula I, is provided.

In yet another aspect, the invention relates to the use of a compound of the invention for the inhibition of HCV.

Additionally, there is provided the use of a compound of formula I for the treatment or prevention of HCV infection, for example for the treatment or prevention of HCV infection in a human. In a preferred aspect, the invention provides the use of a compound of formula I for the treatment of HCV infection, for example, treatment of HCV infection in a human.

Furthermore, the invention relates to a process for the manufacture of a compound of formula I, a novel intermediate for the manufacture of a compound of formula I and the manufacture of such intermediates.

In still another aspect, the invention provides a pharmaceutical composition comprising a compound of formula I and a pharmaceutically acceptable adjuvant, diluent, excipient or carrier. The pharmaceutical compositions will generally contain a compound of formula I in an antiviral effective amount (e.g., for humans), but the subtherapeutic amount of the compound of formula I may still be of value when intended to be combined with other agents or in multiple doses.

Those skilled in the art will recognize that any of the subgroups of the compounds of formula I described herein will be included when referring to a compound of formula I.

The HCV genotype in the context of treatment or prevention according to the present invention includes the major HCV genotypes, i.e., genotypes 1a, 1b, 2a, 3a, 4a, 5a and 6a. The invention also provides methods of treating or preventing HCV infection. In general, the invention provides methods of treating HCV infection.

Representative HCV genotypes in the context of treatment or prevention according to the present invention include genotype 1b (popular in Europe) and 1a (popular in North America). The invention also provides methods of treating or preventing HCV infection, in particular HCV infection of genotype 1a or 1b. In general, the invention provides methods of treating HCV infection, in particular HCV infection of genotype 1a or 1b.

Other representative genotypes in the context of treatment or prevention according to the present invention include genotype 3a (eg, wild-type genotype 3a) and mutant strains of genotype 3a (eg, S282T and L159/320F mutant). In general, the invention provides methods of treating HCV infection, in particular HCV infection of genotype 3a (eg, wild-type genotype 3a) and mutant strains of genotype 3a (eg, S282T and L159/320F mutants).

The invention further relates to the treatment or prevention of HCV infection caused by genotypes 2a, 4a, 5a, 6a. The invention also provides a method for treating or preventing HCV infection of genotypes 2a, 4a, 5a, 6a

The good activity of the compounds of the invention against genotype 3 is also noteworthy due to the poor formation of previous generations of nucleotides. Preferably, the compositions of the invention have the genotype extensive coverage for each of the six kinds of genotypes, i.e., the compounds of the present invention, EC ₅₀ between genotypes not significantly different, thereby simplifying treatment.

The compounds of the invention have several pairs of palmitic centers and may be present and isolated in optically active and racemic forms. Some compounds can exhibit polymorphism. It will be understood that any of the racemic, optically active, non-image, heteromeric or stereoisomeric forms of the compounds provided herein, or mixtures thereof, are within the scope of the invention. The absolute configuration of such compounds can be determined using methods known in the art, for example, X-ray diffraction or NMR and/or starting materials from known stereochemistry and/or stereoselective synthesis methods. The pharmaceutical compositions of the present invention will preferably comprise substantially stereoisomerically pure preparations indicating stereoisomers.

Most of the amino acids are palmitic and can exist as individual mirror image isomers. It is a named L- or D-amino acid, wherein the L-image isomer is a natural mirror image isomer. Thus, the pure mirror image isomer of the amino acid is readily available and wherein the amino acid is used to synthesize the compound of the invention, the use of a palmitic amino acid will provide a palmitic product.

Compounds and intermediates in pure stereoisomeric forms as referred to herein are defined as substantially free isomers of the mirror-isomeric or non-image-isomeric forms of the same basic molecular structure of such compounds or intermediates. In particular, the term "stereomerically pure" relates to stereoisomeric having a stereoisomeric excess of at least 80% (ie, a minimum of 90% for one isomer and 10% for other possible isomers) up to 100%. Excess (ie one isomer is 100% and no other isomers a compound or intermediate, more specifically having a stereoisomeric excess of 90% up to 100%, even more specifically having a stereoisomeric excess of 94% up to 100% and most specifically having 97% up to 100% A stereoisomeric excess of a compound or intermediate. The terms "image isomerically pure" and "non-image isomerically pure" should also be understood in a similar manner, but on the other hand, the image isomerized excess and the non-imagewise heterogeneous excess of the mixture, respectively.

Pure stereoisomeric forms of the compounds and intermediates of the invention can be obtained by procedures well known in the art. For example, the mirror image isomer can be resolved by the racemic mixture (ie, by the formation of a non-image-isomerized salt by reaction with an optically active acid or base, followed by the selectivity of the non-imagewise salt formed. Crystallization) is separated from each other. Examples of such acids are tartaric acid, benzhydryl tartaric acid, xylylmercapto tartaric acid and camphorsulfonic acid. Alternatively, the mirror image isomer can be separated by a chromatographic technique using a palmitic stationary phase. Pure stereochemically isomeric forms can also be obtained by synthesis from stereochemically pure forms of the appropriate starting materials, provided that the reaction occurs stereospecifically by palmar synthesis or by the use of a palmitic adjuvant. If a specific stereoisomer is desired, the preparation of the compound is preferably carried out using a stereospecific method. These methods will advantageously employ mirror image isomeric pure starting materials.

The non-image isomerized racemates of the compounds of the invention can be isolated by conventional methods. Suitable physical separation methods which may advantageously be employed are, for example, selective crystallization and chromatography (e.g., column chromatography).

When a phosphorus atom is present in a compound of the invention comprising the same, the phosphorus atom may represent the center of the palm of the hand. According to the Cahn-Ingold-Prelog priority rule, the center of the center is named "R" or "S". When no palmarity is indicated, it is intended to include both R- and S-isomers, as well as mixtures of the two (i.e., non-imagewise isomeric mixtures).

In a preferred embodiment of the invention, a stereoisomer having an S-configuration at the phosphorus atom is included. These stereoisomers are named S _P .

In other embodiments of the invention, stereoisomers having an R-configuration at the phosphorus atom are included. These stereoisomers are named R _P .

In other embodiments of the invention, non-imagewise isomeric mixtures, i.e., mixtures of compounds having an R- or S-configuration at the phosphorus atom, are included.

The invention also includes isotopically-labeled compounds of formula I or any of the subgroups of formula I, wherein one or more atoms are isotopically of the atom (ie, having the same number of atoms but different atomic mass than the atoms normally found in nature) Atomic) permutation. Examples of isotopes that may be included in a compound of any of Formula I or Formula I include, but are not limited to, hydrogen isotopes (e.g., ² H and ³ H (also expressed as D for 氘 and T for 氚, respectively) Carbon isotope (eg ¹¹ C, ¹³ C and ¹⁴ C), nitrogen isotopes (eg ¹³ N and ¹⁵ N), oxygen isotopes (eg ¹⁵ O, ¹⁷ O and ¹⁸ O), phosphorus isotopes (eg ³¹ P) And ³² P), sulfur isotopes (eg ³⁵ S), fluorine isotopes (eg ¹⁸ F), chlorine isotopes (eg ³⁶ Cl), bromine isotopes (eg ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br and ⁸² Br) and Isotopes of iodine (eg, ¹²³ I, ¹²⁴ I, ¹²⁵ I, and ¹³¹ I). The choice of isotopes included in an isotope-labeled compound will depend on the specific application of the compound. For example, analysis of drug or substrate distribution Compounds incorporating radioisotopes (eg, ³ H or ¹⁴ C) will generally be most useful. For radioactive imaging applications (eg, positron emission tomography (PET)), positron-emitting isotopes (eg, ¹¹ C, ¹⁸ F, ¹³ N) Or ¹⁵ O) will be useful. The inclusion of heavier isotopes (eg, 氘, ie ² H) can be any of the subgroups of Formula I or Formula I. The compounds of the group provide greater metabolic stability, which can result, for example, in increasing the in vivo half-life or reduced dosage requirements of the compound.

Isotopically labeled compounds of any of the formula I or formula I may be replaced by reagents or starting materials which are suitably labeled with isotopes by methods analogous to those described in the Reaction Schemes and/or Examples below. The corresponding non-isotopically labeled reagents or starting materials are prepared by conventional techniques known to those skilled in the art.

Pharmaceutically acceptable addition salts comprise the therapeutically active non-toxic acid and base addition salt forms of the compounds of formula I. Concerned about the free, i.e., non-salt form of the compound of formula I.

Pharmaceutically acceptable acid addition salts are conveniently obtained by treating the base form with the appropriate acid. Suitable acids include, for example, inorganic acids such as hydrohalic acids (eg, hydrochloric acid or hydrobromide) Acid, sulfuric acid, nitric acid, phosphoric acid and similar acids; or organic acids such as acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid (ie oxalic acid), malonic acid, succinic acid (ie succinic acid) , maleic acid, fumaric acid, malic acid (ie hydroxysuccinic acid), tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclamic acid, salicylic acid, Amino acid, pamomic and similar acids. Instead, the salt forms can be converted to the free base form by treatment with a suitable base.

Compounds of formula I containing an acidic proton may also be converted to their non-toxic metal or amine addition salt form by treatment with a suitable organic and inorganic base. Suitable base salt forms include, for example, ammonium salts, alkali metal and alkaline earth metal salts (e.g., lithium, sodium, potassium, magnesium, calcium salts, and the like), salts with organic bases (e.g., benzathine, N-A) Base-D-reducing glucosamine, hydrabamine salts, and salts with amino acids (eg, arginine, lysine, and the like).

Some of the compounds of formula I may also exist in their tautomeric form. For example, the tautomeric form of the indole group (-C(=O)-NH-) is an imino alcohol (-C(OH)=N-), which may be in a ring having aromatic character stable. Although not explicitly indicated in the structural formulae shown herein, such forms are intended to be included within the scope of the invention.

Terms and expressions used throughout the Abstract, the specification and the scope of the patent application are to be interpreted as defined below, unless otherwise indicated. The meaning of each term is independent at each occurrence. Unless otherwise indicated, the terms apply regardless of whether the terms are used alone or in combination with other terms. Terms or expressions that are not explicitly defined herein are to be interpreted as having their ordinary meaning as used in the art. Chemical names, common names, and chemical structures are used interchangeably to illustrate the same structure. If a chemical compound is referred to by both chemical structure and chemical name and there is an ambiguity between the structure and the name, the structure will prevail.

"C _m -C _n alkyl" itself or in complex expression (eg, C _m -C _n haloalkyl, C _m -C _n alkylcarbonyl, C _m -C _n alkylamine, etc.) represents a specified carbon atom The number of linear or branched aliphatic hydrocarbon groups, for example, C ₁ -C ₄ alkyl having 1 to 4 carbon atoms means an alkyl group. The C ₁ -C ₆ alkyl group has the corresponding meaning and also includes all straight chain and branched isomers of the pentyl group and the hexyl group. Preferred alkyl C ₁ -C ₆ alkyl groups for use in the present invention include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, tert-butyl Base, n-pentyl and n-hexyl, especially C ₁ -C ₄ alkyl, such as methyl, ethyl, n-propyl, isopropyl, tert-butyl, n-butyl and isobutyl. Methyl and isopropyl are generally preferred. The alkyl group may be unsubstituted or substituted by one or more substituents which may be the same or different, each substituent being independently selected from the group consisting of halo, alkenyl, alkynyl, aryl, cycloalkyl, Cyano, hydroxy, -O-alkyl, -O-aryl, -alkyl-O-alkyl, alkylthio, -NH ₂ , -NH(alkyl), -N(alkyl) ₂ , -NH(cycloalkyl), -OC(=O)-alkyl, -OC(=O)-aryl, -OC(=O)-cycloalkyl, -C(=O)OH and -C( =O) O-alkyl. Generally, the alkyl group is unsubstituted unless otherwise indicated.

"C ₂ -C _n alkenyl" represents a straight-chain or branched aliphatic hydrocarbon group having at least one carbon-carbon double bond and having the specified number of carbon atoms, for example, C ₂ -C ₄ alkenyl means having 2 to 4 Alkenyl of one carbon atom; C ₂ -C ₆ alkenyl means an alkenyl group having 2 to 6 carbon atoms. Non-limiting alkenyl groups include ethenyl, propenyl, n-butenyl, 3-methylbut-2-enyl, n-pentenyl and hexenyl. The alkenyl group may be unsubstituted or substituted by one or more substituents which may be the same or different, each substituent being independently selected from the group consisting of halo, alkenyl, alkynyl, aryl, cycloalkyl, Cyano, hydroxy, -O-alkyl, -O-aryl, -alkyl-O-alkyl, alkylthio, -NH ₂ , -NH(alkyl), -N(alkyl) ₂ , -NH(cycloalkyl), -OC(=O)-alkyl, -OC(=O)-aryl, -OC(=O)-cycloalkyl, -C(=O)OH and -C( =O) O-alkyl. Generally, the alkenyl group is unsubstituted unless otherwise indicated.

"C ₂ -C _n alkynyl" represents a straight-chain or branched aliphatic hydrocarbon group having at least one carbon-carbon triple bond and having the specified number of carbon atoms, for example, C ₂ -C ₄ alkynyl means having 2 to 4 An alkynyl group of one carbon atom; a C ₂ -C ₆ alkynyl group means an alkynyl group having 2 to 6 carbon atoms. Non-limiting alkenyl groups include ethynyl, propynyl, 2-butynyl and 3-methylbutynylpentynyl and hexynyl. An alkynyl group may be unsubstituted or substituted by one or more substituents which may be the same or different, each substituent being independently selected from the group consisting of halo, alkenyl, alkynyl, aryl, cycloalkyl, Cyano, hydroxy, -O-alkyl, -O-aryl, -alkyl-O-alkyl, alkylthio, -NH ₂ , -NH(alkyl), -N(alkyl) ₂ , -NH(cycloalkyl), -OC(O)-alkyl, -OC(O)-aryl, -OC(O)-cycloalkyl, -C(O)OH and -C(O)O- alkyl. Generally, the alkynyl group is unsubstituted unless otherwise indicated.

The term "C _m -C _n haloalkyl" as used herein denotes a C _m -C _n alkyl group in which at least one C atom is substituted by a halogen (for example, a C _m -C _n haloalkyl group may have 1 to 3 halogen atoms), Preferred is chlorine or fluorine. A typical haloalkyl group is a C ₁ -C ₂ haloalkyl group, wherein the halo group suitably represents fluorine. Exemplary haloalkyl groups include fluoromethyl, difluoromethyl, and trifluoromethyl.

The term "C _m -C _n hydroxyalkyl" as used herein denotes a C _m -C _n alkyl group in which at least one C atom is substituted with one hydroxy group. A typical C _m -C _n hydroxyalkyl group is a C _m -C _n alkyl group in which one C atom is substituted with one hydroxy group. Exemplary hydroxyalkyl groups include hydroxymethyl and hydroxyethyl.

The term "C _m -C _n aminoalkyl" as used herein denotes a C _m -C _n alkyl group in which at least one C atom is substituted with an amine group. A typical C _m -C _n aminoalkyl group is a C _m -C _n alkyl group in which one C atom is substituted with an amine group. Exemplary aminoalkyl groups include aminomethyl and aminoethyl.

As used herein, the term "C _m -C _n alkylene" represents an indication of the number of carbon atoms in a straight or branched divalent alkyl group. Preferred C _m -C _n alkylene C ₁ -C ₃ alkylene groups for use in the present invention. Non-limiting examples of alkylene groups include -CH ₂ -, -CH ₂ CH ₂ -, -CH ₂ CH ₂ CH ₂ -, -CH(CH ₃ )CH ₂ CH ₂ -, -CH(CH ₃ )-, and -CH ( CH(CH ₃ ) ₂ )-.

The term "Me" means methyl and "MeO" means methoxy.

The term "C _m -C _n alkylcarbonyl" means C _m -C _n alkyl of formula -C (= O) - of a group, wherein C _m -C _n alkyl portion based as defined above. Usually, "C _m -C _n alkylcarbonyl" is C ₁ -C ₆ alkyl-C(=O)-.

"C _m -C _n alkoxy" means the group C _m -C _n alkyl group -O-, where C _m -C _n alkyl-based as defined above. Particularly concerned C ₁ -C ₄ alkoxy, including methoxy, ethoxy, propoxy, isopropoxy, tert-butoxy, n-butoxy and isobutoxy. Methoxy and isopropoxy groups are generally preferred. The C ₁ -C ₆ alkoxy group has the corresponding meaning and extends to all straight-chain and branched isomers including pentyloxy and hexyloxy.

The term "C _m -C _n alkoxycarbonyl" represents a group of the formula C _m -C _n alkoxy-C(=O)- wherein the C _m -C _n alkoxy moiety is as defined above. Typically, "C _m -C _n alkoxycarbonyl" is C ₁ -C ₆ alkoxy-C(=O)-.

The term "amino" refers to the group -NH ₂ .

The term "halo" refers to a halogen group such as fluorine, chlorine, bromine or iodine. Usually, the halogen group is fluorine or chlorine.

The term "aryl" means phenyl, biphenyl or naphthyl.

The term "heterocycloalkyl" denotes a stable saturated monocyclic 3 to 7 membered ring containing from 1 to 3 heteroatoms independently selected from O, S and N. In one embodiment, the stable saturated monocyclic 3 to 7 membered ring contains one hetero atom selected from the group consisting of O, S, and N. In a second embodiment, the stable saturated monocyclic 3 to 7 membered ring contains 2 heteroatoms independently selected from O, S and N. In a third embodiment, the stable saturated monocyclic 3 to 7 membered ring contains 3 heteroatoms independently selected from O, S and N. A stable saturated monocyclic 3 to 7 membered ring containing from 1 to 3 heteroatoms independently selected from O, S and N can generally be a 5 to 7 membered ring, such as a 5 or 6 membered ring. The heterocycloalkyl group may be unsubstituted or substituted by one or more substituents which may be the same or different, each substituent being independently selected from the group consisting of halo, alkenyl, alkynyl, aryl, naphthenic , cyano, hydroxy, -O-alkyl, -O-aryl, -alkyl-O-alkyl, alkylthio, -NH ₂ , -NH(alkyl), -N(alkyl) ₂ , -NH(cycloalkyl), -OC(O)-alkyl, -OC(O)-aryl, -OC(O)-cycloalkyl, -C(O)OH and -C(O) O-alkyl. Generally, unless otherwise indicated, a heterocycloalkyl group is unsubstituted.

The term "heteroaryl" denotes a stable monocyclic or bicyclic aromatic ring system containing from 1 to 4 heteroatoms independently selected from O, S and N, each ring having 5 or 6 ring atoms. In one embodiment of the invention, the stabilized monocyclic or bicyclic aromatic ring system contains a heteroatom selected from the group consisting of O, S and N, each ring having 5 or 6 ring atoms. In the second embodiment of the present invention In the examples, the stable monocyclic or bicyclic aromatic ring system contains two heteroatoms independently selected from O, S and N, each having 5 or 6 ring atoms. In a third embodiment, the stabilized monocyclic or bicyclic aromatic ring system contains three heteroatoms independently selected from O, S and N, each ring having 5 or 6 ring atoms. In a fourth embodiment, the stabilized monocyclic or bicyclic aromatic ring system contains four heteroatoms independently selected from O, S and N, each having 5 or 6 ring atoms. One embodiment of a heteroaryl group comprises a flavonoid.

The term "C ₃ -C _n cycloalkyl" denotes a cyclic monovalent alkyl group having the number of carbon atoms, for example, C ₃ -C ₇ cycloalkyl means a cyclic monovalent alkyl group having 3 to 7 carbon atoms. Preferred cycloalkyl C ₃ -C ₄ alkyl groups for use in the present invention are cyclopropyl and cyclobutyl. The cycloalkyl group may be unsubstituted or substituted by one or more substituents which may be the same or different, each substituent being independently selected from the group consisting of halo, alkenyl, alkynyl, aryl, cycloalkyl. , cyano, hydroxy, -O-alkyl, -O-aryl, -alkyl-O-alkyl, alkylthio, -NH ₂ , -NH(alkyl), -N(alkyl) ₂ , -NH(cycloalkyl), -OC(O)-alkyl, -OC(O)-aryl, -OC(O)-cycloalkyl, -C(O)OH, and -C(O)O -alkyl. Generally, the cycloalkyl group is unsubstituted unless otherwise indicated.

The term "C _m -C _n alkyl group" means the above defined herein as C _m -C _n alkyl which is substituted with an amine, i.e. a hydrogen atom from the alkyl portion of the NH ₂ - a displaceable group. Usually, the "amino group C _m -C _n alkyl group" is an amine group C ₁ -C ₆ alkyl group.

The term "C _m -C _n alkyl amino carbonyl group" means the above C _m -C _n alkylcarbonyl group, as defined herein of which a hydrogen atom of the alkyl moiety by a NH ₂ - a displaceable group. Generally, the "amino C _m -C _n alkylcarbonyl group" is an amine C ₁ -C ₆ alkylcarbonyl group. Examples of amine C _m -C _n alkylcarbonyl groups include, but are not limited to, glycidinyl groups: C(=O)CH ₂ NH ₂ , propylamine groups: C(=O)CH(NH ₂ )CH ₃ , Proline group: C=OCH(NH ₂ )CH(CH ₃ ) ₂ , leucine group: C(=O)CH(NH ₂ )(CH ₂ ) ₃ CH ₃ ,isoleucine group:C( =O)CH(NH ₂ )CH(CH ₃ )(CH ₂ CH ₃ ) and orthraenic acid group: C(=O)CH(NH ₂ )(CH ₂ ) ₃ CH ₃ and the like. This definition is not limited to natural amino acids.

Terms are interpreted in accordance with the definitions provided above and the common usages in the technical field.

The term "(=O)" as used herein, forms a carbonyl moiety when attached to a carbon atom. It should be noted that the atom may have only a pendant oxy group when the valence of the atom permits.

The term "monophosphate, diphosphate and triphosphate" refers to a group:

The terms "thio-monophosphate, thio-diphosphate and thio-triphosphate" refer to the group:

As used herein, the position of a group on any of the molecular moieties used in the definition can be anywhere on the moiety as long as it is chemically stable. Each definition is independent when any of the variables appear more than once in any part.

As used herein, "a compound of formula I", or a "compound of the invention" or like terms is intended to include a subgroup of a compound of formula I and a compound of formula I, including possible stereochemically isomeric forms and pharmaceutically acceptable salts thereof. And solvates.

The term "solvate" encompasses a compound of formula I and any pharmaceutically acceptable solvate thereof which can be formed. Such solvates are, for example, hydrates, alkoxides (e.g., ethoxides, propoxides), and the like, especially hydrates.

In general, the names of the compounds used in this application were generated using ChemDraw Ultra 12.0. In addition, if a stereochemistry of a structure or a portion of a structure is not indicated by, for example, bold or dashed lines, the structure or a portion of the structure should be interpreted as encompassing all stereoisomers thereof.

General synthetic method

Compounds of the invention may be synthesized by, for example, the illustrative synthetic reactions shown and described below Various methods are illustrated in the drawings. The starting materials and reagents used may be obtained from commercial suppliers or may be prepared according to the literature procedures set forth in the references using methods well known to those skilled in the art.

FIG 1 illustrates the reaction of a compound of formula route I, wherein R ¹ and R ² system H, and a base line B of uracil or uracil derivatives, i.e., B-based formula (b) of the group.

It can be used in the presence of a base such as imidazole or the like or any other suitable protecting group such as a mercapto group such as an ethyl fluorenyl group, a benzamidine group or a p-chlorobenzylidene group or a trityl group. (4S,5R)-4-hydroxy-5-(hydroxymethyl)dihydrofuran-2(3H)-one using a TIPS-chloride treatment, for example, a triisopropylsulfonyl (TIPS) group Protection of the hydroxyl group. Alternatively, an orthogonal protecting group strategy can be employed to enable later selection of one hydroxyl group to deprotect without contacting other hydroxyl groups. Typically, the 5'-hydroxy group is subsequently protected with a trityl, methoxytrityl or decyl group, followed by protection of the 3'-hydroxy group using, for example, a thiol group. The thus protected derivative is then subjected to electrophilic alpha-fluorination by treatment with N-fluorobenzenesulfonimide (NFSI) in the presence of a base such as bis(trimethyldecyl)guanamine to provide Fluoride (1b). The α-chloro substituent is then conveniently introduced by reaction with N-chlorosuccinimide in the presence of a base such as lithium bis(trimethyldecyl)guanamine or the like. Subsequent reduction of the ketone functional group using any suitable reducing agent (e.g., DIBAL or the like) followed by conversion of the resulting hydroxyl group to a leaving group (e.g., a sulfonic acid, halide, or phosphate derivative) provides a glycosyl donor ( 1e). The sulfonic acid derivative (e.g. methyl sulfone) with usually by a base (e.g. Et ₃ N) with the presence of methanesulfonamide acyl chloride or an equivalent process made; sugar bromide is generally via lines by using acetic anhydride Or an analog of the acetoacetate of the hydroxy group, followed by treatment with hydrogen bromide in acetic acid. Subsequent use of standard conditions well known in the art of nucleoside chemistry, for example, in hexamethyldioxane (HDMS) and Lewis acid (such as TMS triflate or tin tetrachloride or the like) Condensation with a desired base or a protected derivative thereof in the presence of a nucleoside (1f). In the case where a glycosyl bromide is used as the glycosyl donor, an accelerator of a glycosylation reaction such as tin tetrachloride or a silver salt such as silver trifluoromethanesulfonate or the like is suitably used. The hydroxy protecting group and, if present, the protecting group on the base are then removed by appropriate methods according to standard methods well known in the art to provide the nucleoside (1 g). If desired, the obtained nucleoside (1 g) can then be converted to 5'-monophosphate, diphosphate or triphosphate, 5'-thio-monophosphate using any of the methods described below or according to literature procedures. Thio-diphosphate or thio-triphosphate or conversion to a prodrug.

Compounds of the invention having a cyclic phosphate prodrug moiety that links the 3' and 5' positions together (ie, R ¹ and R ² together with the oxygen atom to which they are attached form a cyclic phosphate) may, for example, be The method described in WO2010/075554 is made. Obtaining such compounds (wherein R ³ is OR ^{3 '} and R ^{3 '} is H, C ₁ -C ₆ alkyl, C ₃ -C ₇ cycloalkyl, C ₃ -C ₇ cycloalkyl C ₁ -C ₇ alkane The route of the benzyl or benzyl group and the phosphorus (III)-reagent used to introduce the phosphorus moiety is shown in Figure 2A of the reaction.

Glycol (2a) and phosphorus (III)-reagent prepared as described above (for example, alkyl-N,N,N',N'-tetraisopropylphosphonamide having the desired group R ^{3 '} The ester) is reacted in the presence of an activator such as tetrazole or dicyanoimidazole or the like to provide a cyclic phosphite (2b). The phosphite to phosphoric acid is then carried out using any convenient oxidation method known in the art, such as oxidation with a peroxide reagent such as m-chloroperoxybenzoic acid, tert-butyl hydroperoxide, hydrogen peroxide or the like. Subsequent oxidation of the ester (2c). Alternatively, TEMPO-oxidation or oxidation based on iodine-THF-pyridine-water or any other suitable oxidation method can be used.

Similarly, a corresponding cyclic phosphorothioate prodrug (i.e., U-based S) having a 3',5'-cyclic prodrug moiety (2d) in the compound of the present invention can be obtained by a sulfurized phosphite derivative (2b). )obtain. Suitable sulfurizing agents include, but are not limited to, elemental sulfur, Lawesson's reagent, cyclooctasulfide, bis(triethoxymethyl)propyl-tetrasulfide (TEST).

Alternatively, the cyclic phosphate (2c) can be prepared directly in one step by reacting a diol with a P(V)-reagent (e.g., an alkyl dichlorophosphate), thereby avoiding a separate oxidation step.

The phosphorus (III) and phosphorus (V) reagents to be used to form cyclic phosphites and phosphates, respectively, can be prepared as described in WO2010/075554. Briefly, commercially available chloro-N,N,N',N'-tetraisopropylphosphonium amide reacts with the desired alcohol R ^{3 '} -OH in the presence of a tertiary amine (eg, Et ₃ N) to provide phosphorus ( III) a reagent, and phosphorus oxychloride (POCl ₃ ) is reacted with a desired alcohol R ^{3 '} -OH in the presence of Et ₃ N or the like to provide a phosphorus (V) reagent.

The cyclic phosphate prodrug of the present invention (wherein U is O, R ³ is NHC(R ¹⁵ )(R ^15' )C(=O)R ¹⁶ ) can be prepared as shown in the reaction scheme of Figure 2B.

The cyclic phosphate (2Ab) is formed by, for example, using a solvent such as MeCN or the like to diol (2a) with a desired amino acid ester and two leaving groups (2Aa) (for example, two The phosphorylating agent of the p-nitrophenol group) is achieved by reaction in the presence of a base such as DBU or equivalent.

In a similar manner, a corresponding cyclic phosphorothioate prodrug (i.e., U-S) having a 3',5'-cyclic prodrug moiety in a compound of the invention can be used as the phosphoric acid by using the corresponding thiophosphoramide Chemical reagents are obtained.

For the preparation of the compound of the invention wherein R ² is H and R ¹ is phosphonium ester, ie, the prodrug moiety of formula (iv), a higher reactivity 5' can be utilized compared to the secondary 3'-hydroxyl group. -Hydroxyl, and the phosphonium ester can be introduced directly onto the 3',5-diol without any special protecting group strategy. This method is illustrated in Reaction Scheme 3.

Nucleosides prepared as described above in the presence of a base such as N-methylimidazole (NMI) or the like in an inert solvent such as an ether (for example diethyl ether or THF) or a halogenated hydrocarbon (for example dichloromethane) The derivative (3a) is condensed with a desired chloroguanamine phosphate to provide a phosphonium amide derivative (3b).

Similarly, a compound of the invention (wherein R ² is H and R ¹ is thiophosphonamide, ie, a prodrug moiety of formula (iv), wherein U is S) is obtained by making sugar (3a) and thioguanidine Amino phosphate reaction is obtained.

The chloroguanamine phosphate used in the above reaction scheme can be obtained by a two-step reaction starting from phosphonium chloride (POCl ₃ ). Reaction Scheme 4 illustrates a chloroguanamine phosphate which can be used to prepare a compound of formula I wherein R ¹ is a group of formula (iv) wherein U is O and R ²⁴ is H and can be used to prepare a compound of formula I (wherein R ^{A group} of formula (iv-c) wherein U is O and R ²⁴ and R ^{15 '} together with the atoms to which they are attached form a pyridoxine ring of a pyrrolidine ring.

Condensation of POCl ₃ with the desired alcohol R ¹⁴ OH in an inert solvent such as Et ₂ O provides the alkoxy phosphate dichlorophosphate or the aryloxy dichlorophosphate (4a). Subsequent reaction with the amino acid derivative (4b) or (4b') respectively provides chlorophosphonamide (4c) or (4c'). If desired, the resulting chlorophosphonamides (4c) and (4c') can be converted to the corresponding phosphorylating reagents having activated phenols as leaving groups (eg, pentafluorophenol or p-NO ₂ -phenol), as shown in the figure 4d and 4e are roughly illustrated. This conversion is conveniently carried out by reacting the chlorine derivative (4c) or (4c') with a desired activated phenol in the presence of a base such as triethylamine or the like.

Thiochloroguanamine phosphate (i.e., a phosphorylating reagent which can be used to prepare a compound of formula (I), wherein R ¹ is a group of formula (iv) and U is S) can be prepared using a strategy similar to that outlined above. , as illustrated in the reaction diagram 5.

The thiophosphonium chloride is reacted with the desired alcohol R ¹⁴ OH in the presence of a base such as Et ₃ N or the like to provide an alkoxy thiodichlorophosphate or an aryloxy thiodichlorophosphate (5a). Subsequent reaction with the amino acid derivative (4b) or (4b') respectively provides the thiochloroguanamine phosphate (5b) or (5b').

A route to obtain a phosphorylating reagent which can be used to prepare a compound of formula (I) wherein R ¹ is a group (v) and U is O is shown in Figure 6.

Phosphorylating reagents (such as 4-nitrophenyl dichlorophosphate, phosphonium trichloride or the like) in a solvent such as DCM in the presence of Et ₃ N or the like are reacted with a suitable amine to provide the desired chlorophosphonium dichloride. Amine ester.

a compound of formula (I) wherein the R ¹ group (i) is a prodrug moiety, and both R ¹² and R ¹³ are R ²¹ (=O)S-(C ₁ -C ₆ alkyl)- and U O) can be made according to the literature procedure. For example, the method described in Bioorg. & Med. Chem. Let., Vol. 3, No. 12, 1993, pp. 2521-2526, as illustrated generally in Figure 7A.

By treatment with phosphonic acid in the presence of an activator such as neopentyl chloride, followed by reaction with S-(2-hydroxyalkyl)alkane sulfate and neopentyl chloride in pyridine and subsequent oxidation ( For example, conversion of the 5'-hydroxy compound (7a), such as iodine in pyridine/water, to the corresponding hydrophosphate (7b) provides a phosphate triester. The protecting group is ultimately removed using standard methods to provide a nucleotide prodrug (7c).

Alternatively, the nucleotide prodrug (7c) can be prepared by phosphorylating a nucleoside (7a) with a phosphorylating reagent which already has an appropriate substituent. This method is described in WO 2013/096679 and illustrated in Reaction Scheme 7B.

The nucleoside (7a) is reacted with a phosphorylating reagent in the presence of 5-ethylthiotetrazole (ETT), followed by oxidation with, for example, m CPBA to provide the desired prodrug (7c). The phosphorylation reagent is suitably prepared according to the literature procedure as generally depicted in Figure 8.

In the presence of a tertiary amine such as triethylamine or equivalent, it is desirable to react the hydrazine chloride R ²¹ C(=O)Cl with the decyl aliphatic alcohol of the desired configuration, followed by 1,1-dichloro-N,N The obtained mercaptothioalkanol derivative (8a) is treated with diisopropylphosphine to provide a phosphorylating reagent (8b).

a compound of formula I (wherein the R ¹ group (i) is a prodrug moiety and R ¹² and R ¹³ have the formula R ²¹ C(=O)OC ₁ -C ₆ alkyl- or R ²¹ OC(=O)OC ₁ -C ₆ alkylene-) can be prepared according to the methods described in, for example, WO 2013/096679 and the references cited therein. This method is briefly illustrated in Reaction Scheme 9A.

The optionally protected nucleoside 9a is preferably present in a solvent such as THF using suitable coupling conditions (e.g., BOP-Cl and 3-nitro-1,2,4-triazole) in the presence of DIEA or the like. Suitable diphosphates 9b or 9b' are coupled in the form of an ammonium salt (e.g., triethylammonium salt or the like) to provide prodrugs 9c and 9c', respectively.

a compound of formula I (wherein the R ¹ group (i) is a prodrug moiety and R ¹² and R ¹³ have the formula R ²¹ C(=O)OC ₁ -C ₆ alkyl- or R ²¹ OC(=O)OC _In an alternative method of _{1- C6} alkylene-), nucleoside 9a is reacted with phosphonium chloride in a first step and then further reacted with the phosphorylating reagent that has been obtained, as illustrated in reaction Figure 9B.

Phosphate esters 9c and 9c' are reacted with phosphinium chloride by using a solvent such as triethyl phosphate, followed by the desired chloroalkyl carbonate (9b") or ester in the presence of DIEA at elevated temperature ( 9b''') reaction was obtained.

a compound of formula I (wherein the R ¹ group (i) is a prodrug moiety, wherein U is O, R ¹² is H and R ¹³ has the formula R ²¹ -OC ₁ -C ₆ alkyl - and R ²¹ is C ₁ - _C24 alkyl) can be prepared in accordance with, for example, the methods described in J. Med. Chem., 2006, 49, 6, pages 2010-2013 and WO 2009/085267, and references cited therein. The general method is illustrated in Reaction Scheme 10A.

The appropriate alkoxy alcohol (10a) is reacted with the phosphorus chloride by using, for example, diethyl ether or the like as a solvent in the presence of triethylamine, followed by phosphorylation of the optionally protected nucleoside and finally deprotection of the implemented phosphoric acid The formation of the reagent (10b) provides the protein (10c).

a compound of formula I (wherein the R ¹ group (i) is a prodrug moiety, wherein U is O, R ¹² is H and R ¹³ has the formula R ²¹ -OC ₁ -C ₆ alkyl - and R ²¹ is C ₁ -C ₂₄ alkyl) the alternative method, using a phosphorus (III) - as the reagent phosphorylating reagent, such as a reaction as illustrated in FIG. 10B.

The phosphorus (III) reagent is prepared by reacting an alkoxy alcohol (10a) with a phosphonamine (10d) in the presence of a tertiary amine such as DIEA or the like. The nucleoside is then phosphorylated with the obtained phosphoniumamine derivative (10e), followed by oxidation using, for example, a peroxide such as a third butoxy peroxide or the like to provide a nucleotide (10f). The cyanoethyl moiety is hydrolyzed and the protecting group, if present, is removed to provide the desired nucleotide (10c).

a compound of formula I (wherein the R ¹ group (vi) prodrug moiety and the R ¹³ system R ²¹ C(=O)O-CH ₂ - or R ²¹ OC(=O)O-CH ₂ -) may be The method described in WO 2013/039920 and the references cited therein are made. This method is briefly illustrated in Reaction Figure 11A.

Phospholipid esters 11c and 11c' are reacted with phosphatidylcholine chloride in triethyl phosphate, followed by reaction with the desired amine NHR ¹⁷ R ^17' in the presence of DIEA and finally in the presence of DIEA at elevated temperatures Obtained by reaction with chloroalkyl carbonate (11b) or ester (11b').

Compounds of the formula I (wherein the R ¹ group (vi) prodrug moiety and the R ¹³ system R ²¹ C(=O)S-CH ₂ CH ₂ -) can be as described in WO 2008/082601 and the references cited therein Method made. This method is briefly illustrated in Reaction Scheme 12A.

Providing a hydrogenation of the 5'-hydroxy compound (12a) with a suitable tetraalkylammonium salt of the desired hydrogen hydride (e.g., tetraethylammonium salt) by activation with neopentyl chloride in pyridine to provide the hydrogen phosphinate (12b). The amine NR ¹⁷ R ^{17 ' is} then introduced by reacting with the desired amine in carbon tetrachloride under anhydrous conditions, followed by removal of the protecting group, thereby producing the phosphonium amide (12c).

Alternatively, the phosphoxylamine ester (12c) can be reacted with the hydrogen phosphinate (7b) of Figure 7A by the presence of a coupling agent (e.g., PyBOP or the like) with the desired S-alkane sulfuric acid (2-hydroxyethyl). The ester R ²¹ C(C=O)SCH ₂ CH ₂ OH is reacted, then aminated and deprotected as described above. This pathway is illustrated in Reaction Scheme 12B.

As will be apparent to those skilled in the art, the procedures illustrated in the reactions of Figures 12A and 12B will be suitable not only for the preparation of S-mercaptothioethanol derivatives, but also for the preparation of other alkyl groups between sulfur and oxygen atoms. a derivative of the configuration.

At the 5' position and optionally also at the 3' position, there is a thiol prodrug moiety (ie, R ¹ and optionally R ^{2 is} also C(O=)R ³⁰ or C(=O)R ³¹ NH ₂ ) The compounds of the invention can be obtained by subjecting a suitable 3' protected compound to suitable deuteration conditions, as illustrated in the reaction scheme of Figure 13.

Nucleoside (13b) (wherein the prodrug group ester at the 5' position, ie a group of the formula OC(=O)R ¹⁰ ) is by pyridine or alkyl hydrazine chloride R ³⁰ C(=O)Cl or In the presence of such a standard method (for example using an alkyl anhydride R ³⁰ C(=O)OC(=O)R ³⁰ )), the 5'-hydroxy compound (9a) is reacted with a suitable hydrating agent, and at the 5' position The nucleoside (13d) bearing an amino acid ester can be obtained by reacting a 5'-hydroxy compound (13a) with an N-protected aliphatic amino acid in the presence of a suitable peptide coupling agent such as EDAC or the like. Removing the 3'-hydroxyl protecting group then produces a compound of the present invention, wherein R ² lines H. On the other hand, the 3'-hydroxyl compounds (13b) and (13c) are subjected to the purification conditions just described above, thereby producing dimercapto derivatives (13d) and (13e), respectively.

Compounds of the invention having an ester or amino acid ester prodrug moiety at the 5'- and/or 3' position can be prepared as illustrated in Reaction Scheme 14.

Since the reactivity of the 5' position of the diol (14a) is higher, the position can be selectively reacted with a suitable oxime to obtain the 5'-mercapto derivative (14b) and (14c), or it can be suitably The protecting group is protected to allow subsequent deuteration of the 3' position. Nucleoside (14b) (wherein the prodrug group ester in the 5' position, ie a group of the formula OC(=O)R ³⁰ ) is a deuterating agent (for example an alkane) in the presence of pyridine or hydrazine or the like The basal anhydride) reaction is conveniently obtained, while the nucleoside (14c) bearing the amino acid ester at the 5' position will protect the diol (14a) and N by the presence of a suitable peptide coupling agent (eg EDAC or the like). The aliphatic amino acid is obtained by a reaction. If the thiol prodrug group is desired to be at the 3' position, the protection-deuteration-deprotection sequence will be suitable for obtaining the cleaning reactant in decreasing yield. Typically, a protecting group such as decyl, trityl or monomethoxytrityl (MMT) will be suitable for protecting the 5'-hydroxyl group. The use of such groups is extensively described in the literature, and is generally used for its introduction, such as reaction with a corresponding chloride (e.g., chloride) in a solvent such as pyridine. Subsequent deuteration as described above, followed by removal of the 5'-O-protecting group, and in the case where the amino acid ester is introduced as an N-protected amino acid, ie an N-protecting group, depending on the protecting group used The group is treated with an appropriate condition (for example, acidic treatment in the case of a trityl or methoxytrityl protecting group), followed by a 3'-deuterated derivative (14d) and (14e). If desired, the phosphonium ester can be introduced, for example, at the 5' position of the 5'-hydroxy derivatives (14d) and (14e) obtained using the procedures described above, or can be introduced using standard literature phosphorylation procedures. Phosphate, diphosphate or triphosphate, or the 5' position can be deuterated using the method described above for the 3' position.

a compound of the invention having an acetal prodrug moiety at the 5' position or at both the 5' and 3' positions (ie, a compound of formula I (wherein R ¹ or both R ¹ and R ² are CR ³² R ^32' OC(=O)CHR ³³ NH ₂ )) can be prepared from the 5'-hydroxy compound using, for example, the method described in Bioorg. Med. Chem. 11 (2003) 2453-2461.

a compound of the invention having a "HepDirect" prodrug moiety at the 5' position (i.e., a compound of formula I wherein the R ¹ group (i), and R ¹² and R ¹³ are bonded between the oxygen atoms to which they are attached The propyl group can be obtained according to the method described in J. Am. Chem. Soc, Vol. 126, No. 16, 2004, pp. 5154-5163.

A route for obtaining a compound of formula I wherein the B group is a (a) or (b), R ² is H and the R ¹ is a triphosphate, ie a group of formula (iii), wherein U is O) is illustrated Figure 15.

A suitable phosphorylating reagent for the preparation of a triphosphate of a compound of formula (I) wherein the B group (a) or (b) is a 5-nitrocyclosalgenyl chlorophosphite (I-6), It is prepared by reacting phosphorus trichloride with 2-hydroxy-5-nitrobenzyl alcohol as detailed in the experimental section below.

A suitable 3'-O protected derivative of the nucleoside (15a) of the present invention and a nitrocycloalkenyl chlorophosphite (I-1) in the presence of Et ₃ N in an inert solvent such as DCM or MeCN ) The reaction, after use (e.g.) Oxone ^® oxide, thereby providing a cyclic phosphate triester (15b). The triphosphate (15c) is then obtained by reaction with a pyrophosphate such as tributylamine pyrophosphate followed by treatment with ammonia. To give the desired salt form, so triphosphate subjected to a suitable ion exchange procedures, e.g. potassium salt form if desired, then the residue was purified by column Dowex ^® -K ^+.

The pathway for obtaining a compound of formula I wherein B is uracil, R ² is H and R ¹ is thio-triphosphate, ie a group of formula (iii), wherein U is S, is illustrated in Scheme 16.

A suitable reagent for the introduction of a first phosphate group in the preparation of a thio-triphosphate of a U-nucleoside of a compound of formula (I) is 2-chloro-4H-1,3,2-benzodioxine- 4-ketone, which was prepared according to literature procedures. Thus, a suitable 3'-O protected nucleoside is reacted with 2-chloro-4H-1,3,2-benzodioxan-4-one in a solvent such as pyridine/THF or equivalent. It is then treated with tributylammonium pyrophosphate in the presence of tributylamine in a solvent such as DMF. The resulting intermediate is then converted to the thiotriphosphate by treatment with sulfur in DMF. To obtain the desired salt form, the triphosphate is subjected to a suitable ion exchange procedure, for example, if a lithium salt form is desired, the residue is passed through a column Dowex®-Li ⁺ .

An alternative route to obtaining a thio-triphosphate is illustrated in Figure 17 of the reaction.

In this method, a phosphorothioate reagent is used in the phosphorylation step. The reagent is prepared by reacting PSCl ₃ with a triazole in a solvent such as MeCN or the like. The reagent thus formed is then coupled to the 3'-O protected nucleoside 13a and thereafter reacted with a pyrophosphate such as tris(tetrabutylammonium pyrophosphate) to provide a thio-triphosphate. (17b).

The use of the various protecting groups (PG) used in the above reaction schemes is known to those skilled in the art, and their use and other alternatives are extensively described in the literature, for example, see Greene TW, Wuts PGMProtective Groups in organic synthesis, 2nd edition, New York: Wiley; 1995.

The term "N-protecting group" or "N-protecting" as used herein refers to a group which is intended to protect the N-terminus of the amino acid or peptide or to protect the amine group against undesired reactions during the synthetic procedure. The commonly used N-protecting groups are disclosed in Greene. The term "N-protecting group" as used herein includes fluorenyl, for example, methyl, ethyl, propyl, propyl, butyl butyl, 2-chloroethyl, 2-bromo. Ethyl, trifluoroethyl, trichloroethane, phthalic acid, o-nitrophenoxyethyl fluorenyl, α-chlorobutyl fluorenyl, benzamidine, 4-chlorobenzhydryl , 4-bromobenzylidene, 4-nitrobenzhydryl and the like; sulfonyl, for example, benzenesulfonyl, p-toluenesulfonyl and the like; urethane forming groups such as benzyl Oxycarbonyl, p-chlorobenzyloxy-carbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethyl Oxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxy Carbonyl, 1-(p-biphenyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzylhydroxycarbonyl, Tributoxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2-trichloroethoxy Carbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl, decyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl And the like; alkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl and the like; and mercapto groups such as trimethylsulfonyl and the like. Advantageous N-protecting groups include methyl fluorenyl, ethyl fluorenyl, benzhydryl, propyl fluorenyl, tert-butyl ethenyl, phenyl sulfonyl, benzyl (Bz), tert-butoxy Carbonyl (BOC) and benzyloxycarbonyl (Cbz).

Hydroxy and/or carboxyl protecting groups are also extensively reviewed in Greene as above and include ethers such as methyl, substituted methyl ethers (eg, methoxymethyl, methylthiomethyl, benzyloxymethyl, Third butoxymethyl, 2-methoxyethoxymethyl and the like), mercapto ether (eg trimethylsulfonyl (TMS), tert-butyldimethylhydrazino (TBDMS) tribenzyl Substituted ethyl ether (eg 1-ethoxymethyl, 1-methyl), fluorenyl, triphenylphosphonium, tert-butyldiphenylfluorenyl, triisopropyldecyl and the like 1-methoxyethyl, tert-butyl, allyl, benzyl, p-methoxybenzyl, diphenylmethyl, triphenylmethyl, and the like, aralkyl (eg, triphenyl) Methyl and 9-phenyl-xanthene (pixyl, 9-hydroxy-9-phenyl) Derivatives, especially chlorides). Ester hydroxy protecting groups include, for example, formate, benzylformate, chloroacetate, methoxyacetate, phenoxyacetate, pivalate, crotonate, diester ( Mesitoate), benzoate and the like. Carbonate hydroxy protecting groups include methylvinyl, allyl, cinnamyl, benzyl, and the like.

In one aspect, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I and a pharmaceutically acceptable carrier. A therapeutically effective amount in this context is sufficient to stabilize or reduce viral infection and, in particular, HCV infection, in an infected individual (eg, a human) The amount. The "therapeutically effective amount" will vary depending on the individual needs of each particular situation. The influencing dose is characterized, for example, by the severity of the condition to be treated, the age, weight, general health, etc. of the subject to be treated, the route of administration and the form.

In one aspect, the invention relates to the use of a compound of formula I for the treatment of "untreated" patients, i.e., patients infected with HCV who have not previously been treated for infection.

In another aspect, the invention relates to the use of a compound of formula I for the treatment of a "experienced" patient, ie a patient infected with HCV who has previously been treated for infection and subsequently relapsed.

In another aspect, the invention relates to the use of a compound of formula I for the treatment of a "non-responder", ie a patient infected with HCV who has previously been treated but is not responsive to treatment.

In still another aspect, the invention is directed to a pharmaceutical composition comprising a prophylactically effective amount of a compound of formula I as specified herein and a pharmaceutically acceptable carrier. A prophylactically effective amount in this context is an amount sufficient to act in a prophylactic manner against HCV infection in an individual at risk of infection.

In still another aspect, the invention relates to a method of preparing a pharmaceutical composition as specified herein, comprising intimately admixing a pharmaceutically acceptable carrier with a therapeutically or prophylactically effective amount of a compound of formula I as specified herein.

Thus, the compounds of the invention may be formulated into a variety of pharmaceutical forms for administration purposes. As a suitable composition, there may be all compositions commonly used for the reference of systemic administration of a drug. For the preparation of the pharmaceutical composition of the present invention, an effective amount of a specific compound (as an addition salt form or a solvate) as an active ingredient is mixed with a pharmaceutically acceptable carrier, and the desired preparation is administered as an end effect. Depending on the form, the carrier can take a wide variety of forms. It is expected that such pharmaceutical compositions will be in a single dosage form which is especially suitable for oral, rectal, transdermal or parenteral administration. For example, in the preparation of a composition in an oral dosage form, any of the usual pharmaceutical media may be employed, for example, in oral liquid preparations (eg, suspensions, syrups, elixirs, In the case of emulsions, solutions) are water, glycols, oils, alcohols and the like; or in the case of powders, pills, capsules and lozenges, solid carriers such as starch, sugar, kaolin, lubricants, binders, disintegration Agents and the like. Tablets and capsules represent the most advantageous oral dosage unit form due to their ease of administration, in which case solid pharmaceutical carriers are obviously employed. For parenteral compositions, the carrier will typically comprise at least a substantial portion of sterile water, but may include other ingredients to, for example, aid in dissolution. For example, an injectable solution can be prepared which comprises a saline solution, a dextrose solution or a mixture of saline and dextrose solution. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspensions, and the like may be employed. Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations. In compositions suitable for transdermal administration, the carrier optionally comprises a combination of a permeation enhancer and/or a suitable wetting agent, as appropriate, and a minor proportion of any suitable combination of additives which do not introduce significant deleterious effects on the skin. . The compounds of the invention may also be orally inhaled or insufflated as solutions, suspensions or dry powders using any of the delivery systems known in the art.

It is especially advantageous to formulate the pharmaceutical compositions mentioned above in unit dosage form for ease of administration and to achieve dose consistency. Unit dosage form as used herein refers to a physically discrete unit suitable as a single dosage unit, each unit containing a predetermined amount of active material calculated to produce the desired therapeutic effect, and the desired pharmaceutical carrier. Examples of such unit dosage forms are lozenges (including scored or coated lozenges), capsules, pills, suppositories, powder packs, flakes, injectable solutions or suspensions, and the like, and the like.

The compounds of formula I show activity against HCV and are useful in the treatment and/or prevention of HCV infection or diseases associated with HCV. In general, the compounds of formula I are useful in the treatment of HCV infection or diseases associated with HCV. Diseases associated with HCV include progressive liver fibrosis, inflammation and necrosis leading to cirrhosis, advanced liver disease, and HCC. A variety of compounds of the invention are active against mutant strains of HCV. In addition, many of the compounds of the invention may exhibit advantageous pharmacokinetic properties and are attractive in terms of bioavailability, including acceptable half-life, AUC (area under the curve) and peaks, and no adverse phenomena (eg, insufficient fastness) Effect and organization stay).

The in vitro antiviral activity of a compound of formula I against HCV can be tested by the cellular HCV replication subsystem based on Lohmann et al. (1999) Science 285: 110-113, with the further modifications described by Krieger et al. (2001) Journal of Virology 75: 4614-4624 (incorporated herein by reference), which is further exemplified in the Examples section. This model, although not a complete infection model for HCV, is widely accepted as the most powerful and effective model for autonomous HCV RNA replication currently available. It will be appreciated that it is important to distinguish between compounds that specifically interfere with HCV function and those who exert cytotoxic or cytostatic effects in the HCV replicon model, and thus cause a decrease in HCV RNA or linked reporter gene concentrations. Fluorescent redox dyes (e.g., resazurin) are known in the art for the analysis of cytotoxicity based on, for example, mitochondrial enzymes. In addition, cell rescreening is used to assess non-selective inhibition of linked reporter gene activity (eg, firefly luciferase). Appropriate cell types can be equipped with a luciferase reporter gene by stable transfection, the performance of which depends on the active gene promoter, and the cells can be used as a rescreen to eliminate non-selective inhibitors.

Due to the antiviral properties of the compounds of formula I, including any possible stereoisomers, pharmaceutically acceptable addition salts or solvates thereof, in particular their anti-HCV properties, they can be used to treat warm blood cells infected with HCV Animals, specifically humans. The compounds of formula I are further useful for the prevention of HCV infection. The invention further relates to a method of treating a warm-blooded animal, in particular a human, infected with HCV or at risk of contracting HCV, the method comprising administering an anti-HCV effective amount of a compound of formula I.

Thus, the compounds of the invention are useful as pharmaceuticals, in particular as anti-HCV medicines. Such use or treatment as a medicament comprises systemically administering to an HCV infected individual or an individual susceptible to HCV infection an amount effective to combat a condition associated with HCV infection.

The invention also relates to the use of a compound of the invention for the manufacture of a medicament for the treatment or prevention of HCV infection.

In a preferred embodiment, the invention relates to the use of a compound of formula I for the manufacture of a medicament for the treatment of HCV infection.

In general, an antiviral effective daily amount is expected to be from about 0.01 mg/kg to about 700 mg/kg, or from about 0.5 mg/kg to about 400 mg/kg, or from about 1 mg/kg to about 250 mg/kg, or about 2 mg/kg to about 200 mg/kg or from about 10 mg/kg to about 150 mg/kg body weight. It may be suitable to administer the desired dose in two, three, four or more sub-doses at appropriate intervals throughout the day. The sub-doses can be formulated to contain, for example, from about 1 mg to about 5000 mg, or from about 50 mg to about 3000 mg, or from about 100 mg to about 1000 mg, or from about 200 mg to about 600 mg, or from about 100 mg to about 400 mg of active ingredient per unit dosage form. Unit dosage form.

The invention also relates to a combination of a compound of formula I, a pharmaceutically acceptable salt or solvate thereof, and another antiviral compound, in particular another anti-HCV compound. The term "combination" may be in reference to a product comprising (a) a compound of formula I and (b) optionally another anti-HCV compound, for use as a combined preparation for the simultaneous, separate or sequential use in the treatment of HCV infection.

The anti-HCV compounds that can be used in combination include HCV polymerase inhibitors, HCV protease inhibitors, inhibitors of other targets in the HCV life cycle, and immunomodulators, and combinations thereof. HCV polymerase inhibitors include NM283 (vallopabine), R803, JTK-109, JTK-003, HCV-371, HCV-086, HCV-796 and R-1479, R-7128, MK-0608, VCH- 759, PF-868554, GS9190, XTL-2125, NM-107, GSK625433, R-1626, BILB-1941, ANA-598, IDX-184, IDX-375, INX-189, MK-3281, MK-1220, ABT-333, PSI-7851, PSI-6130, GS-7977 (speed complex Buwe), VCH-916. Inhibitors of HCV protease (NS2-NS3 inhibitors and NS3-NS4A inhibitors) include BILN-2061, VX-950 (telaprevir), GS-9132 (ACH-806), SCH-503034 (Bo赛瑞韦), TMC435350 (西美瑞韦), TMC493706, ITMN-191, MK-7009, BI-12202, BILN-2065, BI-201335, BMS-605339, R-7227, VX-500, BMS650032, VBY -376, VX-813, SCH-6, PHX-1766, ACH-1625, IDX-136, IDX-316. Examples of HCV NS5A inhibitors are BMS790052, A-831, A-689, NIM-811 and DEBIO-025 are examples of NS5B cyclophilin inhibitors.

Inhibitors of other targets in the HCV life cycle, including NS3 helicase; metalloproteinase inhibitors; antisense oligonucleotide inhibitors such as ISIS-14803 and AVI-4065; siRNAs, such as SIRPLEX-140-N; Vector short hairpin RNA (shRNA); DNase; HCV-specific ribozymes such as heptazyme), RPI.13919; entry inhibitors such as HepeX-C, HuMax-HepC; alpha glucosidase inhibitors, eg sig Celgosivir, UT-231B and the like; KPE-02003002; and BIVN 401.

Immunomodulators include natural and recombinant interferon isoforms, including alpha-interferon, beta-interferon, gamma-interferon, and ω-interferon, such as Intron A®, Roferon-A®, Canferon-A300®, Advaferon ®, Infergen®, Humoferon®, Sumiferon MP®, Alfaferone®, IFN-beta® and Feron®; polyethylene glycol-derived (PEGylated) interferon compounds such as PEG interferon-α-2a (Pegasys®) , PEG interferon-α-2b (PEG-Intron®) and pegylated IFN-α-con1; long-acting formulations and derivatives of interferon compounds, such as albumin-fused interferon abufen (albuferon) a; a compound that stimulates interferon synthesis in a cell, such as resiquimod; interleukin; a compound that enhances a type 1 helper T cell response, such as SCV-07; a purine-like receptor agonist, such as CpG -10101 (actilon) and isatoribine; thymosin α-1; ANA-245; ANA-246; histamine dihydrochloride; propapatium (propagermanium); Tetrachloride; polymyocyte (ampligen); IMP-321; KRN-7000; antibodies, such as civacir and XTL-6865; And therapeutic vaccines such as InnoVac C and HCV E1E2 / MF59.

Other antiviral agents include ribavirin, amantadine, viramidine, nitazoxanide, telbivudine, NOV-205, and talivirin ( Taribavirin); internal ribosome entry inhibitor; broad spectrum viral inhibitors such as IMPDH inhibitors and mycophenolic acid and its derivatives, including but not limited to VX- 497 (merimepodib), VX-148 and/or VX-944); or a combination of any of the above.

Specific agents for use in such combinations include interferon-[alpha] (IFN-[alpha]), pegylated interferon-[alpha] or ribavirin, and therapeutic agents based on antibodies directed against HCV epitopes, small Interfering RNA (Si RNA), ribozymes, DNase, antisense RNA, small molecule antagonists such as NS3 protease, NS3 helicase, and NS5B polymerase.

In another aspect, a combination of a compound of formula I as specified herein and an anti-HIV compound is provided. The latter are preferably HIV inhibitors that have a positive effect on the drug metabolism and/or pharmacokinetics of improved bioavailability. An example of such an HIV inhibitor is ritonavir. Accordingly, the present invention further provides a combination comprising: (a) a compound of formula I or a pharmaceutically acceptable salt or solvate thereof; and (b) ritonavir or a pharmaceutically acceptable salt thereof. The compound ritonavir, its pharmaceutically acceptable salts and processes for its preparation are described in WO 94/14436. US 6,037,157 and the references cited therein: US 5,484,801, US 08/402,690, WO 95/07696 and WO 95/09614 disclose preferred dosage forms of ritonavir.

The invention also relates to a method of preparing a combination as described herein, comprising the steps of combining a compound of formula I with another agent, such as an antiviral agent, including an anti-HCV or anti-HIV agent, specifically as mentioned above By.

These combinations are found to be useful in the manufacture of a medicament for the treatment of HCV infection in a mammal infected with HCV, in particular comprising a compound of formula I as indicated above and interferon-alpha (IFN-alpha), PEGylation interference Su-α or ribavirin. Or the invention provides a method of treating a mammal, in particular a human, infected with HCV comprising administering to the mammal an effective amount of a combination as specified herein. In particular, the treatment comprises systemic administration of the combination, and an effective amount is that amount effective to treat a clinical condition associated with HCV infection.

In one embodiment, the combinations mentioned above are formulated in the form of a pharmaceutical composition comprising the above active ingredient and a carrier as described above. Each of the active ingredients can be individually Formulations and formulations may be co-administered, or a formulation containing both and, if desired, other active ingredients may be provided. In the former case, the combination may also be formulated as a combined preparation for simultaneous, separate or sequential use in HCV therapy. The composition may take any of the above forms. In one embodiment, the two components are formulated in a dosage form (eg, a fixed dose combination). In a particular embodiment, the invention provides a pharmaceutical composition comprising: (a) a therapeutically effective amount of a compound of formula I, including a possible stereoisomeric form thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof a solvate, and (b) a therapeutically effective amount of ritonavir or a pharmaceutically acceptable salt thereof and (c) a carrier.

The individual components of the combination of the invention may be administered separately at different times during the course of the therapy or simultaneously in a divided or single combination. The present invention is intended to cover all such simultaneous or alternate treatment regimes and the term "administering" should be interpreted accordingly. In a preferred embodiment, separate dosage forms are administered simultaneously.

In one embodiment, the combination of the present invention contains ritonavir or a pharmaceutically acceptable salt thereof in an amount sufficient to clinically improve the compound of formula I relative to the bioavailability of the compound of formula I alone. bioavailability. Alternatively, the combination of the invention comprises ritonavir or a pharmaceutically acceptable salt thereof in an amount relative to at least one pharmacokinetic variable when administered to a compound of formula I (selected from t _1/2 , C _min , C _Max , C _ss , AUC at 12 hours or AUC at 24 hours) is sufficient to increase the at least one pharmacokinetic variable of the compound of formula I.

Combinations of the invention may be administered to a human in a dosage range specific for each component (e.g., a compound of formula I as specified above) contained in such combinations, and ritonavir or a pharmaceutically acceptable salt may be There is a dose amount ranging from 0.02 g/day to 5.0 g/day.

The weight ratio of the compound of formula I to ritonavir may range from about 30:1 to about 1:15, or from about 15:1 to about 1:10, or from about 15:1 to about 1:1, or about 10: 1 to about 1:1, or about 8:1 to about 1:1, or about 5:1 to about 1:1, or about 3:1 to about 1:1, or about 2:1 to 1:1 Within the scope. The compound of formula I and ritonavir may be administered orally once or twice a day, wherein the amount of the compound of formula I per dose is as described above; and the amount of ritonavir per dose is 1 mg to About 2500 Mg, or from about 50 mg to about 1500 mg, or from about 100 mg to about 800 mg, or from about 100 mg to about 400 mg, or from 40 mg to about 100 mg of ritonavir.

Thus, the various examples and intermediates of the present invention will now be illustrated by the following examples. The examples are intended to further illustrate the invention and in no way limit the scope of the invention. Compound names were generated by ChemDraw Ultra software, Cambridgesoft, version 12.0.2.

In addition to the above definitions, the following abbreviations are used in the examples below and in the synthetic reaction schemes. If the abbreviation used herein is undefined, it has its recognized meaning Bn benzyl.

Bz benzamidine

BOP-Cl bis-(2-o-oxy-3-oxazolidinyl)phosphinium chloride

Bz benzamidine

DCC dicyclohexylcarbodiimide

DCM dichloromethane

DIEA diisopropylethylamine

DMAP 4-dimethylaminopyridine

DMF N,N-dimethylformamide;

DMPU 1,3-dimethyl-3,4,5,6-tetrahydro-2-pyrimidinone

EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride

ES electrospray

Et ₃ N triethylamine

EtOAc ethyl acetate

EtOH ethanol

Et ₂ O diethyl ether

LC liquid chromatography

HOAc acetic acid

HPLC high performance liquid chromatography

MeCN acetonitrile

MeOH methanol

MS mass spectrometry

NT 3-nitro-1,2,4-triazole

NTP nucleoside triphosphate

Pg protecting group

Ph phenyl

Standard error of SEM mean

TEST bis(triethoxymethyl)propyl-tetrasulfide

THF tetrahydrofuran

TFA trifluoroacetic acid

TFAA trifluoroacetic anhydride

TIPS triisopropyl fluorenyl

The following phenols were prepared and used to prepare intermediates of the compounds of the invention:

Phenol 1

Step a) 1-(3-((t-butyldimethylmethyl)oxy)phenyl)ethanone (Ph1-a)

To a solution of 3-hydroxyacetophenone (4.46 g, 32.8 mmol) in DMF (6 mL). After 5 min, a solution of TBDMS-CI (4.69 g, 31.1 mmol) in DMF (4 mL). The reaction mixture was stirred at rt EtOAc (EtOAc) EtOAc (EtOAc (EtOAc) Wash with brine (60 mL). The organic layer was dried over Na ₂ SO _4, filtered and concentrated and flash chromatographed on silica by eluting with hexane / EtOAc eluted the residue obtained was purified, the title compound (5.7g, 69%).

Step b) Tert-butyldimethyl(3-(prop-1-en-2-yl)phenoxy)decane (Ph1-b)

Methyl(triphenylphosphonium) bromide (10.2 g, 28.4 mmol) was suspended in anhydrous THF (30 mL) and the suspension was cooled to 0. n-Butyllithium (17.8 mL, 28.4 mmol) was added dropwise to the mixture and the resulting solution was stirred at room temperature for 30 min. Ph1-a (5.7 g, 22.8 mmol) was added to the mixture and the reaction was allowed to stand at room temperature for 60 min. The reaction was quenched with aqueous sodium bicarbonate and extracted with diethyl ether (50 mL). The organic layer was washed with sodium bicarbonate solution, dried (Na ₂ SO _4), filtered and concentrated. The residue obtained was purified by EtOAc EtOAc elut elut elut elut

Step c) Tert-butyldimethyl(3-(1-methylcyclopropyl)phenoxy)decane (Ph1-c)

To a cooled (0 ° C) solution of the olefin Ph1-b (3.9 g, 15.7 mmol) in 1,2-dichloroethane (60 mL) was added dropwise hexane (439.2 mmol) under nitrogen over 10 min. Diethylzinc. Diiodomethane (6.32 mL, 78.5 mmol) was added dropwise and the mixture was stirred at 0 ° C for 30 min and then allowed to reach room temperature overnight. The mixture was poured into an ice cold solution of ammonium chloride and extracted with diethyl ether. The organic layer was washed with saturated sodium bicarbonate, dried (Na ₂ SO _4), filtered and concentrated. The crude material was taken up in hexane and the remaining diiodomethane was discarded. The hexane layer was concentrated to a crude material which was taken up in the next step without further purification.

Step d) 3-(1-methylcyclopropyl)phenol (phenol 1)

The Ph1-c (3.45 g, 13.1 mmol) was taken up in a 1 M solution of THF (20 mL, 20 mmol). The reaction was quenched with 1M EtOAc (EtOAc)EtOAc. The organic layer was washed with brine (2 × 50mL), dried (Na ₂ SO _4), filtered and concentrated. The title compound (0.56 g, 29%) eluted elut elut elut elut elut MS 147.1 [MH] ^- .

Phenol 2

The title compound was obtained from 4-hydroxyacetophenone (6.0 g, 44.1 mmol) using the method described for the preparation of phenol. The yield was 53%.

Phenol 3

Step a) 1-(3-(Benzyloxy)phenyl)cyclopentanol (Ph3-a)

To the suspension of magnesium bar (1.29 g, 52.8 mmol) in anhydrous THF (50 mL) was added iodine warmed with magnesium. The mixture was refluxed and an approximately 5% solution of 3-bromophenol (13.9 g, 52.8 mmol) was then. At the start of the reaction, the bromide solution was added dropwise and the mixture was further refluxed for 1 hour. The mixture was cooled to about 5 ° C and a solution of cyclopentanone (4.44 g, 52.8 mmol) in THF (50 mL). The mixture was stirred at rt for 72 h then EtOAc (EtOAc)EtOAc. The organic phase was washed with brine, dried (Na ₂ SO _4), filtered and concentrated. The product was purified by EtOAc EtOAc (EtOAc)

Step b) 1-(Benzyloxy)-3-(cyclopent-1-en-1-yl)benzene (Ph3-b)

To a solution of Ph3-a (8.4 g, 28.2 mmol) in benzene (100 mL) was added p-toluenesulfonic acid. The mixture was refluxed for 3 hours using a DMF separator, then cooled to rt, diluted with diethyl ether and washed with saturated sodium hydrogen carbonate and brine. The organic phase was dried (Na ₂ SO _4), filtered and concentrated. The product was purified by EtOAc EtOAc (EtOAc) MS 249.4 [MH] ^- .

Step c) 3-cyclopentylphenol (phenol 3)

A solution of Ph3-b (6.4 g, 26 mmol) in EtOAc (EtOAc) (EtOAc) (EtOAc) The catalyst was filtered off and washed with EtOAc and EtOAc. The solvent was evaporated under reduced EtOAcqqqqqqqqqq MS 161.2 [MH] ^- .

Phenol 4

Step a) Ternyl (3-cyclopropylphenoxy) dimethyl decane (Ph4-a)

(3-Bromophenoxy) (t-butyl) dimethyl decane (5.46 g, 19 mmol), cyclopropyl Acid (2.12 g, 24.7 mmol), tripotassium phosphate (14.1 g, 66.5 mmol), tricyclohexylphosphine (0.53 g, 1.9 mmol) and Pd(OAc) ₂ (0.21 g, 0.95 mmol) in toluene (80 mL) The suspension in water (4 mL) was stirred at 110 ° C overnight. The slurry was diluted with diethyl ether and washed with water and brine. The organic phase was dried (MgSO _4), filtered and concentrated. The crude was purified by EtOAc EtOAc EtOAc EtOAc

Step b) 3-cyclopropylphenol (phenol 4)

To a solution of Ph4-a (1.94 g, 7.81 mmol) in THF (25 mL), 1M EtOAc. The solution was stirred for 2 hours, then the solvent was evaporated and the residue was dissolved in EtOAc and treated with conc. NH ₄ Cl was washed twice (aq) and once with brine. The organic phase was dried (MgSO _4), filtered and concentrated. The crude was purified by EtOAc EtOAc EtOAc (EtOAc:EtOAc

Phenol 5

Step a) 2-(4-Bromophenoxy)tetrahydro-2H-pyran (Ph5-a)

4-bromophenol (3.75 g, 21.7 mmol) was dissolved in 3,4-dihydro-2H-pyran (16 ml, 175 mmol), and a catalytic amount of p-toluenesulfonic acid (15 mg, 0.09 mmol) was added and the mixture was Stir at 22 ° C for 45 min. The mixture was diluted with diethyl ether and washed with 1M NaOH (aq) twice, washed with water, dried (Na ₂ SO ₄₎ and concentrated to give the title of this compound (5.57g, 99%).

Step b) 2-(4-Cyclopropylphenoxy)tetrahydro-2H-pyran (Ph5-b)

To Ph5-a (552.5 mg, 2.15 mmol), ZnBr (144 mg, 0.64 mmol), tri-tert-butylphosphine tetrafluoroborate (35.6 mg, 0.12 mmol) and Pd(OAc) ₂ (29.5) during 15 min. A solution of 0.5 M cyclopropylmagnesium bromide in THF (6.5 mL, 3.25 mmol). The mixture was stirred at 22 <0>C for 90 min then cooled on ice-bath and ice water (10 mL). The mixture was extracted 3 times with EtOAc and the extract was washed with brine and then dried (Na ₂ SO _4), filtered and concentrated. The residue was purified by EtOAc EtOAcjjjjjj

Step c) 4-cyclopropylphenol (phenol 5)

p-Toluenesulfonic acid monohydrate (18.9 mg, 0.1 mmol) was added to a solution of Ph5-b (2.28 g, 10.45 mmol) in MeOH (15 mL). The mixture was heated in a microwave reactor at 120 °C for 5 min, then concentrated and purified by EtOAc (EtOAc)EtOAc. The solid obtained was crystallized from petroleum ether to give the title compound (1.08 g, 77%).

Phenol 6

Step a) 1-(3-Methoxyphenyl)cyclobutanol (Ph6-a)

3-methoxyphenylmagnesium bromide in THF (2.11 g, 99.8 mmol) was added dropwise to a stirred solution of cyclobutanone (6.66 g, 95 mmol) in diethyl ether (65 mL). 1M solution in ). The mixture was stirred at 0 °C to 10 °C for 3 hours, then the mixture was added to a saturated solution of saturated NH ₄ Cl (300 mL) and water (300 mL). The mixture was stirred for 10 min and then extracted three times with diethyl ether. The organic phase was dried (Na ₂ SO _4), filtered, and concentrated. The crude product was purified by EtOAc EtOAc (EtOAc)

Step b) 1-cyclobutyl-3-methoxybenzene (Ph6-b)

To a solution of Ph6-a (15.4 g, 86.1 mmol) in ethanol (200 mL) was added 10% Pd (2.5 g) on carbon and the mixture was hydrogenated at 60 psi in Parr. After 18 h, an additional 10% carbon on Pd (1.5 g) was added and the mixture was hydrogenated at 60 psi for an additional 18 hours. The catalyst was filtered off and washed with EtOAc and EtOAc. The solution was concentrated under reduced EtOAcqqqqqqqli

Step c) 3-cyclobutylphenol (phenol 6)

A solution of 1 M boron tribromide (18.1 g, 72.2 mmol) in DCM was added dropwise to a solution of Ph.sub.6-b (10.6 g, 65.6 mmol) in anhydrous DCM (65 mL). The mixture was stirred at -5 ℃ 2.5 hours and then the reaction was quenched with NH ₄ Cl saturated solution of cooled and extracted three times with DCM. The organic phase was dried (Na ₂ SO _4), filtered and concentrated. The crude product was purified by EtOAc EtOAc (EtOAc)

Phenol 7

Step a) 1-(4-(Benzyloxy)phenyl)cyclobutanol (Ph7-a)

in To a suspension of magnesium strips (2.43 g) and traces of iodine in diethyl ether (50 mL), 1 -(benzyloxy)-4-bromobenzene (2.63 g, 100 mmol) Diethyl ether: a solution of THF in 1:1 (100 mL). After the addition was complete, the mixture was refluxed for 4 hours and then cooled to 0 ° C. Anhydrous THF (50 mL) was added, then a solution of <RTI ID=0.0>> After stirring for 2h, add cold saturated NH ₄ Cl solution (500ml) and the mixture was stirred for 15 minutes, then extracted with EtOAc. The organic phase was washed with brine, dried over sodium sulfate and evaporated The product was purified by EtOAc EtOAc elut elut elut elut elut

Step b) 4-cyclobutylphenol (phenol 7)

To a solution of Ph7-a (12.4 g, 41.4 mmol) <RTI ID=0.0></RTI> <RTI ID=0.0></RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> </ RTI> <RTIgt; The catalyst was filtered off, washed with ethanol and concentrated. The product was purified by silica gel chromatography (isohexane-EtOAc). The appropriate fractions were combined and concentrated and crystallised eluted eluted elute

Phenol 8

4-(1-methylcyclopentyl)phenol (Ph-8)

Was added dropwise 1-methyl cyclopentanol (2.00g, 20.0mmol) and phenol (2.07g, 22.0mmol) fresh AlCl (100mL) suspension of the by-pentane ₃ (1.33g, 10mmol) during the 30min A solution in pentane (50 mL). The resulting mixture under N ₂ at rt for 72h, then the reaction mixture was poured into water / ice and HCl (12M, 20mmol, 1.66mL) in. The organic phase was washed with water (50mL) and brine (50mL), dried (Na ₂ SO _4), filtered and concentrated. The crude was purified by EtOAc EtOAc (EtOAc)

Phenol 9

Step a) 2-(4-Bromo-3-methylphenoxy)tetrahydro-2H-pyran (Ph9-a)

To a solution of 4-bromo-3-methylphenol (4.0 g, 21.4 mmol) in 3,4-dihydro-2-H-pyran (16 mL, 175 mmol). The reaction mixture was stirred at room temperature for 1 h then diluted with diethyl ether and washed with 1M EtOAc The organic phase was dried (Na ₂ SO _4), filtered and concentrated. The crude was purified by EtOAc EtOAc EtOAc (EtOAc)

Step b) 2-(4-Cyclopropyl-3-methylphenoxy)tetrahydro-2H-pyran (Ph9-b)

Ph9-a (3.12 g, 11.5 mmol), ZnBr ₂ (2.59 g, 11.5 mmol), tri-tert-butylphosphonium tetrafluoroborate (0.2 g, 0.69 mmol) and Pd(OAc) ₂ (258 mg, 1.15) mmol) placed in a flask and the flask was flushed with N ₂ several times. THF (10 mL) was added while stirring, then 0.5 M cyclopropylmagnesium bromide in THF (35 mL, 17.4 mmol) was added dropwise over 5 min. The mixture was stirred at rt then filtered through a pad of Celite and eluted with MeOH. The solution was concentrated and purified EtOAc EtOAcjjjjjjj

Step c) 4-cyclopropyl-3-methylphenol (phenol 9)

The Ph9-b (1.70g, 7.30mmol) was dissolved in MeOH (20ml) and add _{p TsxH 2 O (318mg, 1.67mmol} ). The mixture was stirred at 22 ° C for 30 minutes and then concentrated. The crude was purified by EtOAc EtOAc EtOAc EtOAc

Phenol 10

Step a) 4-cyclopropyl-1-methoxy-2-toluene (Ph10-a)

Reaction of 4-bromo-1-methoxy-2-toluene (4.39 g, 21.9 mmol) with cyclopropylmagnesium bromide according .

Step b) 4-cyclopropyl-2-methylphenol (phenol 10)

Was added to the solution of Ph10-a (1.54g, 9.49mmol) in DCM (7.5mL) under N ₂ at 0 ℃ under BBr ₃ (5mL, 5mmol). The reaction was stirred for 2 h then quenched with EtOAc (EtOAc) The crude was dissolved in EtOAc and washed with brine. The organic phase was dried (Na ₂ SO _4), filtered and concentrated. The crude product was purified by EtOAc EtOAc EtOAc EtOAc MS 147.11 [MH] ^- .

Phenol 11

4-cyclopropyl-3-methoxyphenol (phenol 11)

The title compound was obtained from 4-bromo-3-methoxyphenol (1.11 g, 5.49 mmol) according to the procedure described for the preparation of phenol. The yield was 40%.

Phenol 12

Step a) 3-(Dimethylamino)-1-(3-hydroxyphenyl)propan-1-one (Ph12-a)

Add a few drops of HCl to a solution of 3-hydroxyacetophenone (4.08 g, 30 mmol), formaldehyde (4.05 g, 45 mmol) and dimethylamine hydrochloride (2.69 g, 33 mmol) in absolute EtOH (100 mL) The reaction mixture was refluxed for 18 h. Additional dimethylamine hydrochloride (0.55 eq., 1.22 g), p-carbaldehyde (0.5 eq., 1.35 g), and HCl (0.5 mL) were added and the reaction mixture was further refluxed for 4 h then cooled to rt. The precipitated white solid was collected and washed with EtOAc EtOAc (EtOAc) g, 38%) which was used in the next step without further purification.

Step b) cyclopropyl(3-hydroxyphenyl)methanone (phenol 12)

NaH (60% mineral oil dispersion) (1.13 g, 28.2 mmol) was added portionwise to a stirred suspension of trimethyl iodide yttrium oxide (6.20 g, 28.2 mmol) in DMSO (100 mL). After 1 h, solid Ph12-a (2.59 g, 11.3 mmol) was added portionwise during stirring and cooling. The reaction mixture was stirred at rt EtOAc (EtOAc)EtOAc. The organic phase was washed with a saturated aqueous solution of NH ₄ Cl (2 × 100mL), dried (Na ₂ SO _4), filtered and concentrated. The crude material was purified by EtOAcjjjjjjjjj

Phenol 13

Step a) cyclopropyl(4-hydroxyphenyl)methanone (Ph13)

To the NaOH solution (8 mL, aqueous solution, 50% w/w), p-hydroxy-γ-chlorobutanone (4.95 g) was added portionwise over about 30 min, followed by the addition of NaOH (35 mL, aqueous solution, 25% w/w) Then, p-hydroxy γ-chlorobutanone (4.95 g) was added in one portion. The temperature was lowered to 140 ° C and NaOH (8 g) was added. After 90 min, H ₂ O (10 ml) was added, and after an additional 60 min, the reaction mixture was cooled and diluted with H ₂ O 27-30ml) neutralized to pH 7 . The precipitate formed was filtered, washed with H ₂ O and dried in vacuo. The solid was triturated at 40 ℃, then overnight at RT in CHCl ₃ (200ml) during 10min. The slurry was heated to 40 ° C during 30 min and then filtered. The filtrate was dried (MgSO _4), filtered and concentrated to 70ml. Hexane was added and an oil formed which eventually turned into a crystal. The slurry was filtered with CHCl ₃ / solid was washed with hexane and dried to give the title of this compound (4.15g, 51%).

Phenol 14

Step a) 3-(1-Hydroxy-2,2-dimethylpropyl)phenol (Ph14-a)

To a cold (-10 °C) mixture of 3-hydroxybenzaldehyde (2.00 g, 16.4 mmol) in diethyl ether (20 mL) was added dropwise t.Bu-MgBr (1.5 eq.). THF (20 mL) was added during the addition. The mixture was brought to 23 ° C and stirred for 6 hours. Add t .Bu-MgBr (0.7 equivalents) and the mixture was stirred overnight, then cooled and quenched with aqueous saturated NH ₄ Cl to produce. EtOAc was added to the mixture followed by 1 M aqueous HCl solution until a homogeneous mixture was obtained. The organic phase was separated and washed with brine and the phases were dried (Na ₂ SO _4), filtered and concentrated. The resulting crude was purified by EtOAc EtOAc EtOAc EtOAc

Step b) 1-(3-Hydroxyphenyl)-2,2-dimethylpropan-1-one (Ph14)

3 Å MS and pyridinium chlorochromate (PCC) (1.97 g, 9.15 mmol) were added to an oven-dried round bottom flask, followed by anhydrous DCM (5 mL). The mixture was stirred at 20 &lt;0&gt;C for 5 min then a mixture of EtOAc EtOAc (EtOAc &lt

After complete oxidation, the mixture was filtered through a pad of celite and the pad was washed with diethyl ether. The filtrate was concentrated. The crude was purified by EtOAc EtOAc EtOAc: MS 179.25 [M+H]+.

Phenol 15

1-(4-hydroxyphenyl)-2,2-dimethylpropan-1-one (Ph15)

4-Hydroxybenzaldehyde (3 g, 24.6 mmol) was obtained according to EtOAcqqqqqq

Amino acid 1

Step a) (S)-(S)-2-((Tertidinoxycarbonyl)amino)propionic acid dibutyl ester (AA1-a)

The L-Boc- alanine (2.18g, 11.5mmol) was dissolved in anhydrous DCM (40mL) was added and the alcohol (R) - butan-2-ol (938mg, 12.6mmol). The mixture was cooled to about 5 ° C and EDC (3.31 g, 17.2 mmol) was added in one portion, then DMAP (140 mg, 1. The mixture was allowed to reach room temperature and stirred overnight, then diluted with ethyl acetate (~300 mL) and the organic phase was washed three times with saturated sodium The organic phase was dried over sodium sulfate and concentrated under reduced pressure. The product was isolated by EtOAc EtOAc (EtOAc)

Step b) (S)-(S)-2-Aminopropionic acid second butyl ester (AA1-b)

A mixture of AA1-a (2.77 g, 11.3 mmol) and p-toluenesulfonic acid monohydrate (2.15 g, 11.3 mmol) in EtOAc (45 mL). The residue obtained was crystallized from EtOAc (EtOAc)

Amino acid 2

(S)-(R)-2-aminopropionic acid pentan-2-yl ester (AA2)

The procedure described for the preparation of AA1 was followed but (R) -pentan-2-ol was used instead of (R) -butan-2-ol, which gave the title compound (4.6 g).

Amino acid 3

(S)-(S)-2-aminopropionic acid pentan-2-yl ester (AA3)

Following the procedure described for the preparation of AA1 but using (S)-pentan-2-ol instead of (R) -butan-2-ol, the title compound (8.3 g) was obtained.

The following intermediates were prepared and which can be used to prepare the compounds of the invention:

Intermediate 1

Step a) (R)-2-((Tertibutoxycarbonyl)amino)propionic acid 4-fluorobenzyl ester (I-1a)

The Boc-L-AlaOH (19.92mmol) , DMAP (1.99mmol) and (4-fluorophenyl) methanol (23.9 mmol) was dissolved in CH ₂ Cl ₂ (100mL). Triethylamine (23.9 mmol) To this solution, after addition of EDC (23.9mmol) and the resulting reaction mixture was stirred overnight under N ₂ at room temperature. The reaction mixture was diluted with CH ₂ Cl ₂ (100mL), washed with saturated aqueous NaHCO ₃ (2 × 50mL), saturated aqueous NaCl (2 × 50mL), dried (Na ₂ S0 ₄₎ and concentrated. The residue was purified by EtOAc EtOAc EtOAc (EtOAc:EtOAc MS: 296 [MH] ^- .

Step b) (R)-2-Aminopropionic acid 4-fluorobenzyl ester (I-1b)

Compound I-1a (14.93 mmol) was dissolved in EtOAc EtOAc m. MS: 198 [M+H] ⁺ .

Step c) (2R)-2-((Chloro(phenoxy)phosphonyl)amino)propionic acid 4-fluorobenzyl ester (I-1)

PhOPOCl ₂ (4.28 mmol) was added dropwise to a solution of Compound I-5b (4.28 mmol) in CH ₂ Cl ₂ , then triethylamine (8.56 mmol). The resulting reaction mixture was stirred at -78 °C under Ar and allowed to reach room temperature overnight. The reaction mixture was evaporated with EtOAc EtOAcjjjjjjj ³¹ P-NMR (CDCl ₃ ) δ: 7.85 (s) and 7.54 (s) (R _P and S _P non-image isomers).

Intermediate 2

Step a) (S)-(R)-2-((Tert-butoxycarbonyl)amino)propionic acid dibutyl ester (I-2a)

L-Boc-alanine (2.18 g, 11.5 mmol) was dissolved in anhydrous DCM (40 mL) and EtOAc (EtOAc) The mixture was cooled to about 5 ° C and EDC (3.31 g, 17.2 mmol) was added in one portion, then DMAP (140 mg, 1. The mixture was allowed to reach room temperature and stirred overnight, then diluted with ethyl acetate (~300 mL) and the organic phase was washed three times with saturated sodium The organic phase was dried over sodium sulfate and concentrated under reduced pressure. The product was isolated by EtOAc EtOAc (EtOAc)

Step b) (S)-(R)-2-Aminopropionic acid second butyl ester (I-2b)

A mixture of I-10a (2.77 g, 11.3 mmol) and p-toluenesulfonic acid monohydrate (2.15 g, 11.3 mmol) in EtOAc (45 mL) The residue obtained was crystallized from EtOAc (EtOAc)

Step c) (2S)-(R)-2-(((4-Nitrophenoxy)(phenoxy)phosphonium)amino)propionic acid tert-butyl ester (I-2)

To a solution of the compound I-10b (3.15 g, 9.92 mmol) in DCM (75 mL), EtOAc (1 eq.). equivalent).

The mixture was allowed to reach room temperature and stirred overnight, then cooled to about 5 ° C and solid 4-nitrophenol (1 eq, 15 mmol) was added, then triethylamine (1 eq, 15 mmol) was added dropwise and mixture After stirring for 4 hours, it was concentrated under reduced pressure, diluted with ethyl acetate (40 ml) and ether (40 ml) and stood at room temperature overnight. The triethylamine-HCl salt was filtered and the filtrate was concentrated under reduced pressure. The residue was purified by EtOAc EtOAc EtOAc elutcd

The following compounds were prepared according to the procedure described for the preparation of I-2 using the appropriate alcohol:

Intermediate 7

Step a) (S)-2-Aminopropionic acid cyclooctyl ester (I-7a)

To a slurry of L-alanine (1.7 g, 19.1 mmol) and cyclooctanol (25 ml, 191 mmol) in toluene (100 ml) was added p-toluenesulfonic acid monohydrate (3.6 g, 19.1 mmol). will The reaction mixture was heated at reflux temperature for 25 h and water was removed from the reaction using a Dean-Stark. The mixture was concentrated under reduced pressure and the residue was taken in vacuo overnight. To the residue (27 g) was added diethyl ether (100 ml). The title compound (4.84 g, 68%).

Step b) (2S)-2-((4-Nitrophenoxy)(phenoxy)phosphonium)amino)propionic acid cyclooctyl ester (I-7)

Compound I-7a was reacted according to the procedure described for the preparation of step 1-2, which gave the title compound (4.7 g, 76%).

Intermediate 8

(2S)-2-(((4-Nitrophenoxy)(phenoxy)phosphonium)amino)propionic acid cycloheptyl ester (I-22)

Following the procedure described for the preparation of compound I-7 but using cycloheptanol (27 ml, 224 mmol) instead of cyclooctanol afforded the title compound (5.72 g. 55%).

Intermediate 9

(2S)-2-((4-nitrophenoxy)(phenoxy)phosphonium)amino)propionic acid cyclohexyl ester (I-23)

Follow the procedure described for the preparation of step I-2, but using (S)-2-aminopropionic acid cyclohexyl ester instead of (S)-2-aminopropionic acid 3,3-dimethylbutyl ester This gave the title compound (10.6 g, 82%).

Intermediate 10

(S)-2-((bis(4-nitrophenoxy)phosphonium)amino)propionic acid 2-ethylbutyl ester (I-10)

To a solution of bis(4-nitrophenyl) chlorophosphate (6.14 g, 17.1 mmol) in DCM (50 ml), (2) 2-ethyl butyl 2-aminopropionate (5 g, 14.49mmol), the mixture was cooled and added dropwise in an ice bath was added _{Et 3 N (4.77mL, 34.2mmol)} . After 15 min, the cooling was removed and the reaction mixture was stirred at 23 ° C until the reaction was completed according to TLC. After the addition of diethyl ether, the mixture was filtered and evaporated.

Intermediate 11

Step a) (S)-2-Aminopropyl isopropylate (I-11a)

To the HCl salt L- alanine (17.8g, 200mmol) in isopropanol (700 mL of) at 0 ℃ in the suspension was added dropwise SOCl ₂ (29mL, 400mmol). The suspension was stirred at rt EtOAc (EtOAc)

Step b) (2S)-2-((((S)-1-isopropoxy-1-yloxypropan-2-yl)amino)(4-nitrophenoxy)phosphonium )-Amino)isopropyl isopropylate (I-11)

4-Nitrophenyl dichlorophosphate (1.8 g 7 mmol) was added dropwise to a solution of the amine I-11a (2.35 g, 14 mmol) and triethylamine (7.7 mL, 56 mmol) in DCM. Solution in DCM. The reaction mixture was allowed to reach room temperature, stirred overnight, concentrated and then diluted with ethyl acetate and ether. The title compound (1.6 g, 50%) was obtained eluted eluted elute

Intermediate 12

Step a) (S)-2-((Tertidinoxycarbonyl)amino)propionic acid neopentyl ester (I-12a)

EDAC and DMAP were added portionwise to a solution of Boc-alanine (18.9 g, 100 mmol) and neopentyl alcohol (13.0 mL, 120 mmol) in DCM (200 mL). The reaction mixture was allowed to reach room temperature and stirred for 72 h. Add EtOAc (700mL) and the solution was washed three times with saturated NaHCO ₃ and the organic phase was washed once with brine, then concentrated. The residue was purified by EtOAc EtOAc EtOAcjEtOAc

Step b) (S)-2-Aminopropionic acid neopentyl ester (I-12b)

To a solution of the Boc-protected amine I-12a (21.1 g, 82.0 mmol) in EtOAc (330 mL). The reaction mixture was stirred at -65 &lt;0&gt;C for 8 h then allowed to reach room temperature overnight. The mixture was filtered and concentrated to give the title compound (lj,

(2S)-2-((((S)-1-(Pentyloxy))-l-oxypropyl-2-yl)amino)(4-nitrophenoxy)-phosphonium Amino) neopentyl propionate (I-12)

4-Nitrophenol dichlorophosphate was added dropwise to a solution of the amine I-12b (3.90 g, 24.5 mmol) in DCM (100 mL). The reaction mixture was allowed to reach room temperature, stirred overnight, concentrated and then diluted with diethyl ether. The mixture was filtered, and the~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Intermediate 13

(2S)-2-((chloro(phenoxy)phosphino)amino)propionic acid ethyl ester (I-13)

Add thiophosphoryl acyl chloride (0.27mL, 2.62mmol) under N ₂ in a mixture of phenol (247mg, 2.62mmol) in anhydrous DCM (8.8mL) and anhydrous THF (4.4mL) of the solution at the -35 ℃ . After 5 min, triethylamine (365 [mu]L, 2.62 mmol). Ethyl propylamine x HCl (403 mg, 2.62 mmol) was added and the reaction mixture was stirred at -35 ° C for 5 min then triethylamine (731 μL, 5.24 mmol). The temperature was allowed to slowly reach rt overnight (17 h). The reaction mixture was diluted with Et ₂ O, filtered and concentrated under reduced pressure. The crude product was purified by flash chromatography eluting elut elut elut elut elut MS 306.18 [MH] ^- .

Intermediate 14

(2S)-2-((chloro(4-chlorophenoxy)phosphino)amino)propionic acid neopentyl ester (I-14)

4-Chlorophenol (381 μL, 3.87 mmol) was added in one portion to a solution of thiophosphonium chloride (400 μL, 3.87 mmol) in DCM at -30 ° C, then triethylamine (1.62 mL, 11.6) Mm). The reaction was stirred for 2 h while bringing the temperature to +5 °C. The pTs salt of (S)-2-aminopropionic acid neopentyl ester (1.28 g, 3.87 mmol) was added and the mixture was cooled to -30 °C. Triethylamine (1.62 L, 11.6 mmol) was added dropwise and the mixture was taken to room temperature and stirred over night. The mixture was concentrated to EtOAc (EtOAc m. MS 368.34 [M+H] ⁺ .

Intermediate 15

(2S)-2-((chloro(naphthalen-1-yloxy)phosphino)amino)propionic acid methyl ester (I-15)

The mixture was stirred at -35 ℃ naphthol (1 eq) in dry DCM (10mL) and anhydrous THF (5mL) under N ₂ in the solution of the acyl chloride was added thiophosphoryl (1 equiv). After 5 min, triethylamine (1 eq.) was added dropwise and the reaction mixture was stirred at -35 ° C for 3 h. (S)-2-Aminopropionic acid methyl ester (1 eq.) was added and the reaction mixture was stirred at -35 °C for 5 min then triethylamine (2 eq.) was added dropwise. The temperature was allowed to slowly reach rt overnight. The reaction mixture was diluted with Et ₂ O, filtered and concentrated under reduced pressure. The obtained crude product was subjected to flash chromatography (hexane: EtOAc 8: 1), to give the title compound, 8.0%, MS 564.24 [M + H] +.

The following intermediate system was prepared according to the procedure described for Intermediate 13 using the appropriate phenol and amino acid ester.

Intermediate 32

(2S)-(R)-2-(((Perfluorophenoxy)(phenoxy)phosphonium)amino)propionic acid dibutyl ester (I-32)

Stirring of pTs salt (12.0 g, 37.7 mmol) of (S)-(R)-2-aminopropionic acid as the second butyl ester in DCM (50 mL) over 15 min. Et ₃ N (10.9 mL, 78.1 mmol) was added dropwise to the solution. To this mixture was added a solution of phenyl dichlorophosphate (5.61 mL, 37.7 mmol) in DCM (50 mL). The reaction mixture was stirred for an additional 30 minutes at -70 °C, then allowed to warm to 0 °C during 2 h and stirred for 1 h. The added pentafluorophenol (6.94g, 37.7mmol) and Et ₃ N (5.73mL, 41.1mmol) in DCM (30mL) was added to the mixture over 20 minutes. The crude mixture was stirred at 0 ° C for 18 h and then concentrated. The residue was taken up in THF (100 mL). The solvent was evaporated and the residue was triturated with tributylmethyl ether. The insoluble material was filtered off and washed with a third butyl methyl ether. The combined filtrate was concentrated and the crude solid was taken &lt;RTI ID=0.0&gt;&gt; The solid was filtered and washed with EtOAc EtOAc (EtOAc:EtOAc

Intermediate 33

(2S)-2-(((Perfluorophenoxy)(phenoxy)phosphonium)amino)propionic acid ethyl ester (I-33)

The title compound was prepared according to the procedure described for I-32 but from EtOAc (EtOAc: EtOAc) The yield was 8.56 g, 27%.

Intermediate 34

(2S)-2-(((Perfluorophenoxy)(phenoxy)phosphonium)amino)propionic acid 2-ethylbutyl ester (I-34)

The title compound was prepared according to the procedure described for I-32 but from p-s salt (18.8 g, 54.4 mmol) of 2-ethyl butyl (S)-2-aminopropionate. Yield 27.0 g, 99%. LC-MS 495.44 [M+H] ⁺ .

Intermediate 35

(2S)-2-(((Perfluorophenoxy)(phenoxy)phosphonium)amino)propionic acid butyl ester (I-35)

To a cooled (-20 ° C) slurry of (S)-2-aminopropionic acid butyl ester (26.4 g, 83.1 mmol) in DCM (200 mL) EtOAc (12.4 mL, . The mixture was stirred for 10min, followed by the dropwise addition of Et ₃ N (25.5mL, 183mmol) was 15min. The mixture was stirred at -20 ° C for 1 h and then at 0 ° C for 30 min. The mixture was kept cooled in an ice bath and added pentafluorophenol (15.3g, 0.08mol), then added dropwise _{Et 3 N (11.6mL, 0.08mol)} . The mixture was stirred overnight and slowly reached 20 °C. Diethyl ether was added and the mixture was filtered through EtOAc (EtOAc)EtOAc.EtOAc. Appropriate fractions were taken, EtOAc (EtOAc:EtOAc)

Intermediate 36

(2S)-2-(((Perfluorophenoxy)(phenoxy)phosphonium)amino)propionic acid cyclohexyl ester (I-36)

To a solution of L-alanine cyclohexyl ester (25.5 g, 74.4 mmol) in DCM (250 mL), EtOAc (11.1 mL, 74.4 mmol). The resulting mixture was stirred for 10 min, then triethylamine (2.2 eq.) was added over a period of 10 min and the reaction was cooled at -15 ° C for 30 min and then at room temperature for 72 h. The reaction was cooled on ice and pentafluorophenol (13.7 g, 74.4 mmol) was then added and then triethylamine (1 eq.) was added over 10 min. The reaction was allowed to reach rt and stirred for 30 min. The insoluble material was filtered through a pad of celite and washed with DCM (100 mL). The solvent was evaporated and the residue was evaporated mjjjjjjjjj The insoluble material was filtered through a pad of celite and washed with EtOAc (75 mL) and the solution was allowed to stand overnight at 5 °C. The formed crystals were dissolved in EtOAc and the solution was washed with 2M NaOH (× 1), 2M HCl (× 1) , dried (Na ₂ SO ₄₎ and concentrated to produce this (2.37g, 6%) of the title compound of almost Pure diastereomer (decomposition rate = about 90%).

Intermediate 37

(2S)-2-(((Benzo[d][1,3]dioxol-5-yloxy)(perfluorophenoxy)phosphonium)amino)propionic acid isopropyl Base ester (I-37)

Under N ₂ at -78 deg.] C was added POCl ₃ (1.79ml, 19.2mmol) to the sum of sesamol (2.65g, 19.2mmol) in DCM (60mL) solution, followed by dropwise added Et ₃ N (2.67ml, 19.2 mmol). The mixture was stirred at -20 ° C to -30 ° C for 4 h. The mixture was cooled to -78 deg.] C and a solution of (S) -2- propionic acid isopropyl ester (3.22g, 19.2mmol) in DCM (10mL) in the solution after 15min was added Et ₃ N (5.62 Ml, 40.3 mmol). The reaction mixture was allowed to reach rt and stirred overnight. Subsequently, the temperature of the reaction mixture was lowered to 0 ° C and pentafluorophenol (3.53 g, 19.2 mmol) was added in one portion, followed by dropwise addition of Et ₃ N (2.67 ml, 19.2 mmol). The resulting slurry was stirred at 0 °C. Upon completion of the reaction as judged by LC-MS, the mixture was filtered and washed with cold DCM. The filtrate was concentrated and redissolved in tributyl ether, filtered again and then concentrated. EtOAc:hexane 20:80 was added and the resulting slurry was gently heated until a clear solution was obtained. The solution was brought to rt and then placed at -20 °C. After 1 hour, crystals formed, which were filtered, washed several times with hexane and then dried under vacuum, yield: 1.8 g. The mother liquor was concentrated and the crystals formed were filtered and dried under vacuum, yield: 5.5 g. Total yield: 7.3 g, 69%. MS ES+ 498.06 [M+H] ⁺ .

The following intermediate system was prepared according to the procedure described for Intermediate 37 using the appropriate phenol and amino acid ester.

¹ pentafluorophenol was added at -78 ° C instead of at 0 ° C as in I-37.

Intermediate 41

(2S)-(S)-2-(((Perfluorophenoxy)(phenoxy)phosphonium)amino)propionic acid dibutyl ester (I-41)

The title compound was replaced by (S)-(R)- according to the method described for I-32 but from (S)-(S)-2-aminopropionic acid dibutyl ester (12.0 g, 37.8 mmol). 2-Aminopropionic acid dibutyl ester is obtained. Yield: 3.33 g, 19%.

Intermediate 42

(2S)-2-(((Perfluorophenoxy)(phenoxy)phosphonium)amino)propionic acid propyl ester (I-42)

The title compound was replaced by (S)-(R)-2- according to the procedure described for the procedure of 1-35, but starting from the HCl salt of (S)-2-aminopropionic acid propyl ester (5.62 g, 33.53 mmol). The pTs salt of the second butyl alanide is prepared. The product was recrystallized from isopropyl ether. Yield: 5.8 g (38%). LC-MS ES+ 454.1 [M+H] ⁺ .

Intermediate 46

Step a) (S)-(R)-2-((Tert-butoxycarbonyl)amino)propanoic acid 1-methoxypropan-2-yl ester (I-46a)

EDC (6.08) was added to a solution of Boc-L-alanine (5 g, 0.03 mol) and (R)-(-)-1-methoxy-2-propanol (2.59 ml, 0.03 mol) at 0 °C. g, 0.03 mol) and 4-(dimethylamino)pyridine (0.48 g, 0.004 mol). The reaction mixture was stirred on a chilled ice water bath and then stirred at room temperature for 72 h.

The reaction mixture was concentrated to about 1/2 volume, diluted with ethyl acetate (400 mL) and an aqueous solution (200ml) and washed with saturated _aqueous NH ₄ Cl (200mL), 10% aqueous citric acid (50mL) and saturated NaHCO. The organic layer was dried (Na ₂ SO _4), filtered and concentrated.

The crude product was purified by EtOAc EtOAc EtOAc EtOAc EtOAc

Step b) (S)-(R)-2-Aminopropionic acid 1-methoxyprop-2-yl ester (I-46b)

A solution of I-46a (5.88 g) in EtOAc (EtOAc)EtOAc. (5.19g, 99%).

Step c) (2S)(R)-2-(((Perfluorophenoxy)(phenoxy)phosphonium)amino)-propionic acid 1-methoxyprop-2-yl ester (I- 46)

To a cooled (0 ° C) solution of (S)-(R)-2-aminopropionic acid 1-methoxypropan-2-yl ester hydrochloride (5.18 g, 22.1 mmol) in DCM (35 mL) Triethylamine (9.25 mL, 66.4 mmol) was added dropwise. The mixture was cooled to -78.degree. C. and a solution of &lt;RTI ID=0.0&gt;&gt; The mixture was stirred for 10min, followed by the dropwise addition of Et ₃ N (25.5mL, 183mmol) was 15min. The mixture was stirred at -78 °C for 5 min and then at 0 °C for 2 h. Was added dropwise DCM (20mL) in the pentafluorophenol (4.07g, 22.1mmol) and _{Et 3 N (3.39mL, 23.3mmol)} , the reaction mixture was then allowed to slowly reach room temperature and stirred overnight. The mixture was concentrated and THF (50 mL) was added. The solid was filtered and washed with EtOAc (3×25 mL). The filtrate was concentrated and the residue was dissolved in EtOAc EtOAc (EtOAc) Heptane (50 ml) was added and the product was precipitated from the solution after standing at room temperature for 1 h. Additional heptane (50 ml) was added and the solid was removed by filtration. The precipitate was washed with tert-butyl methyl ether / heptane 1:2 (50 ml) and heptane (50 ml). The precipitate was dried under vacuum, which gave the title compound as a pure s. (4.32g, 40%). LC-MS ES+ 484.34 [M+H] ⁺ .

Intermediate 47

Step a) (S)-2-((Tertidinoxycarbonyl)amino)propionic acid 1,3-dimethoxypropan-2-yl ester (I-47-a)

Add EDC (2.79 g, 14.5 mmol), crystal 4 to a solution of Boc-L-alanine (2.42 g, 12.8 mmol) and 1,3-dimethoxypropan-2-ol (1.52 g, 12.6 mmol) - (dimethylamino) pyridine (229mg, 1.88mmol) and _{Et 3 N (5.27ml, 37.8mmol)} . The reaction mixture was stirred at room temperature for 72h, then diluted with EtOAc and washed with NaHCO ₃ (aq, × 2), 0.1M HCl (aq, × 2), dried (Na ₂ SO ₄₎ and concentrated. The crude product obtained was used directly in the next step.

Step b) (2S)-2-(((Perfluorophenoxy)(phenoxy)phosphonyl-amino)propionic acid 1,3-dimethoxypropan-2-yl ester (I-47) )

I-47a (3 g, 10.8 mmol) <RTI ID=0.0></RTI><RTIID=0.0> An oil obtained by dissolving in DCM (40 ml) and phenyl dichlorophosphate (1.62 mL, 10.8 mmol) was added. The mixture was cooled on an ice bath and after 15 min, Et3N ( ₃ . The mixture was stirred at 4 ° C for 18 h, then slowly reached 22 ° C. The mixture was cooled again to 0 ℃ and added pentafluorophenol (2.01g, 10.9mmol), then added dropwise _{Et 3 N (1.51mL, 10.8mmol)} . The mixture was stirred at 0 ° C for 1 h then at 22 ° C for 5 h. The mixture was filtered and the solid was washed with EtOAc <RTI ID=0.0> The combined organic phases were washed with NaHCO ₃ (aq, × 2) and brine, then dried (Na ₂ SO _4). The solution was passed through a short column of cerium oxide which was eluted with petroleum ether / EtOAc (8:2). The appropriate fractions were collected and concentrated and the obtained oil was dissolved in diisopropyl ether and treated with heptane to give a slightly cloudy solution that solidified upon standing. The mixture was allowed to stand at 4 ° C for 72 h then EtOAc (EtOAc) LC ES+ 514.0 [M+H] ⁺ .

Intermediate 48

(2S)-2-(((Perfluorophenoxy)(phenoxy)phosphonium)amino)propanic acid pent-3-yl ester (I-48)

The title compound was substituted for (S)-(R) according to the method described for I-32 but from the HCl salt of (S)-2-aminopropionate pent-3-yl ester (3.25 g, 16.6 mmol). Prepared by pTs salt of 2-butylaminopropionate. Yield: 8.0 g (18%). LC-MS ES+ 482.4 [M+H] ⁺ .

Intermediate 49

The title compound was prepared according to the procedure described in WO 2014078427.

Intermediate 50

(2S)-2-(((Perfluorophenoxy)(quinolin-6-yloxy)phosphonium)amino)propionic acid isopropyl ester (I-50)

Phosphorus chloride (1.5 mL, 16.4 mmol) was added to DCM (40 mL) and the mixture was cooled in a dry ice/EtOH bath. Add 6-hydroxy quinoline (2.38g, 16.4mmol), then added dropwise DCM Et (5mL) in the ₃ N _(2.28mL, 16.4mmol). The mixture was stirred under cooling 3h, followed by addition of isopropyl-alanine (2.75g, 16.4mmol), then added dropwise _{Et 3 N (4.57ml, 32.8mmol)} . The mixture was stirred for 5 h under cooling. Was added pentafluorophenol (3.02g, 16.4mmol), after addition of Et ₃ N (2.28ml, 16.4mmol) and the mixture was stirred for 72h. The mixture was diluted with EtOAc (200mL) and washed with 0.1M HCl (aq) × 2, dried (Na ₂ SO ₄₎ and concentrated. The residue was purified with EtOAc (EtOAc) (EtOAc) The solid was collected by filtration to give the title compound (787mg, 9.5%).

Intermediate 51

(2S)-(S)-2-(((Perfluorophenoxy)(phenoxy)phosphonium)amino)propionic acid 1-methoxyprop-2-yl ester (I-51)

The title compound was substituted for (R)-(-)-1 starting from (S)-(+)-1-methoxy-2-propanol (0.87 mL, 8.89 mmol) according to the procedure described for I-46. -Methoxy-2-propanol. Yield: 604 mg, 14%. LC-MS ES-481.5 [MH] ^- .

Example 1

Step a) (4S,5R)-4-((Triisopropyldecyl)oxy)-5-(((triisopropyldecyl)oxy)) Dihydrofuran-2(3H)-one (1a)

To (4S,5R)-4-hydroxy-5-(hydroxymethyl)dihydrofuran-2(3H)-one (3.30 g, 25.0 mmol) and imidazole (10.2 g, 150 mmol) in DMF (35 mL) TIPS-chloride (16.4 g, 85 mmol) was added dropwise to the ice-cooled stirred solution. The mixture was stirred at 0 ° C for 1 h then stirred at rt for 40 h. The reaction was quenched with water and EtOAc EtOAc EtOAc. The organic phase was dried (Na ₂ SO _4), filtered, and concentrated by silica gel column chromatography and eluted with a gradient of isohexane and from 0% to 10% EtOAc elution of product was isolated. The mixed fraction was purified again by EtOAc (EtOAc) elute

Step b) (3S, 4R, 5R)-3-fluoro-4-((triisopropyldecyl)oxy)-5-(((triisopropylcapto)oxy)methyl)-di Hydrofuran-2(3H)-one (1b)

To a solution of 1a (4.45 g, 10.0 mmol) and NFSI (4.73 g, 15.0 mmol) in dry THF (50 mL), EtOAc &lt;RTI ID=0.0&gt; 1 M solution (2.18 g, 13.0 mmol). The mixture was stirred at -70 ° C for 90 min and then added to a saturated aqueous solution of ammonium chloride and crushed ice. The mixture was extracted three times with EtOAc, the organic phase was dried (Na ₂ SO _4), filtered, and concentrated and chromatographed by silica gel with a gradient of isohexane and from 0% to 5% EtOAc elution of product was isolated. Yield: 4.63 g, 67%.

Step c) (3S,4R,5R)-3-chloro-3-fluoro-4-((triisopropyldecyl)oxy)-5-(((triisopropyldecyl)oxy)) Base)-dihydrofuran-2(3H)-one (1c)

To a solution of 1b (3.08 g, 6.65 mmol) and N-chlorosuccinimide (1.07 g, 7.99 mmol) in dry THF (25 mL) A 1M solution of hydrazinium lithium amide. The mixture was stirred at -70 ° C for 90 min and then added to a saturated aqueous solution of ammonium chloride and crushed ice. The mixture was extracted three times with EtOAc, the organic phase was dried (Na ₂ SO _4), filtered, and concentrated and chromatographed by silica gel with a gradient of isohexane and from 0% to 5% EtOAc elution of product was isolated. Yield 2.40 g, 73%.

Step d) (3S, 4R, 5R)-3-chloro-3-fluoro-4-((triisopropyldecyl)oxy)-5-(((triisopropylcapto)oxy)) Base)-tetrahydrofuran-2-ol (1d)

A solution of DIBAL (2.23 g, 15.7 mmol) in DCM in 1M was added dropwise to a solution of 1c (5.20 g, 10.5 mmol) in anhydrous toluene (50 mL). The mixture was stirred at -70 &lt;0&gt;C for 2 h then the temperature was taken to -30 &lt;0&gt;C and the reaction was quenched with 2 mL MeOH and then added to a mixture of Rochelle salt and crushed ice. The mixture was stirred for 30 minutes and then extracted three times with EtOAc. The organic phase was dried (Na ₂ SO _4), filtered and concentrated under reduced pressure. The product was isolated by chromatography on a pad of hexanes eluting with EtOAc EtOAc. The yield was 5.22 g, 85%.

Step e) (2S, 3S, 4R, 5R)-Methanesulfonic acid 3-chloro-3-fluoro-4-((triisopropyldecyl)oxy)-5-(((triisopropylhydrazino) )oxy)methyl)tetrahydrofuran-2-yl ester (1e)

To a cooled solution of 1d (2.00 g, 4.01 mmol The mixture was stirred at RT 3 h, then diluted with EtOAc (80mL), washed with saturated NaHCO ₃ (aq), HCI, water and brine. The organic phase was dried (Na ₂ SO _4), filtered and concentrated. The crude product was dried in vacuo and then used in the next step without further purification.

Step f) 1-((2R,3S,4R,5R)-3-chloro-3-fluoro-4-((triisopropyldecyl)oxy)-5-(((triisopropylhydrazino) Oxy)methyl)tetrahydrofuran-2-ylpyrimidine-2.4(1H,3H)-dione (1f)

A suspension of uracil (699 mg, 6.24 mmol) and ammonium sulfate (25.8 mg, 0.195 mmol) in hexamethyldioxane (HD) (40 mL) was refluxed overnight. The solvent was removed in vacuo and the residue was taken crystalljjjjjjj 1e (2.25 g, 3.90 mmol) was added under argon and then TMS triflate was slowly added. The mixture was stirred at RT for 10 minutes and then refluxed for 4 hours. The mixture was added to a cooled sodium bicarbonate solution and extracted three times with EtOAc. The organic phase was washed with brine and dried over sodium sulfate. The solution was evaporated under reduced pressure and the mixture was purified with EtOAc EtOAc EtOAc EtOAc EtOAc (EtOAc) .

Step g) 1-((2R,3S,4R,5R)-3-Chloro-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H )-dione (1g)

A solution of 1f (1.27 g, 2.14 mmol) in 80%EtOAc was stirred at <RTI ID=0.0></RTI><RTIgt; The residue was dissolved in dry THF (10 mL), EtOAc (EtOAc (EtOAc) % MeOH in DCM was purified by chromatography. The mixed fraction was purified by HPLC eluting with EtOAc EtOAc (EtOAc) MS 281.2 [M+H] ⁺ .

¹ H NMR (500 MHz, DMSO) δ 10.39 (s, 1H), 7.78 (d, J = 8.1 Hz, 1H), 6.74 (s, 1H), 6.22 (d, J = 16.1 Hz, 1H, 7), 5.73 (d, J = 8.1 Hz, 1H), 5.52 (s, 1H), 4.21 (dd, J = 19.6, 9.2 Hz, 1H), 3.87-3.77 (m, 2H), 3.64 (dd, J =12.7, 2.8 Hz, 1H).

¹³ C NMR (126 MHz, DMSO) δ 162.76, 150.26, 139.06, 115.71, 113.71, 102.28, 86.98, 86.69, 81.01, 73.28, 73.14, 58.19.

Example 2

(2S)-2-((((2R,3R,4S,5R)-4-chloro-5-(2,4-di-oxy-3.4-dihydropyrimidin-1(2H)-yl)-) 4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphonium)amino)propionic acid isopropyl ester (2)

To a solution of 1 g of sugar (28 mg, 0.1 mmol) in THF (1.5 mL), EtOAc (EtOAc) The suspension was stirred at 0 ° C for 1 h, then DMPU (0.5 mL) was added, then (2S)-2-(((perfluorophenoxy)(phenoxy)phosphorate was added at 0 °C during about 5 min. A solution of decyl)amino)propionic acid isopropyl ester (57 mg, 0.12 mmol) (prepared as described in WO2011/123672) in THF (0.5 mL). The mixture was stirred at 0 &lt;0&gt;C for 5 h then RT was obtained and quenched with saturated aqueous ammonium chloride. The mixture was extracted three times with EtOAc. The organic phase was dried (Na ₂ SO _4), concentrated and the product was isolated by HPLC under reduced pressure. (Gemini NX 30 mm 20% to 60% acetonitrile, 10 mmol ammonium acetate gradient for 17 minutes and flow rate 40 ml/min.) Yield 22 mg, 40%.

Example 3

Step a) (2R, 3R, 4S, 5R)-acetic acid 4-chloro-5-(2,4-di-oxo-3,4-dihydropyrimidin-1(2H)-yl)-4-fluoro- 2-(hydroxymethyl)tetrahydrofuran-3-yl ester (3a)

To a solution of compound 1f (81 mg, 0.29 mmol) in EtOAc (EtOAc) The resulting mixture was stirred at room temperature for 40h, diluted with DCM and washed with NaHCO _3. The organic phase was concentrated and purified EtOAcjjjjjjjj

The obtained compound was dissolved in anhydrous pyridine (1.4 mL), Ac ₂ O (29 μL, 0.31 mmol) was added and the solution was stirred at rt. After 2h, MeOH was added, and the mixture was concentrated and the combined organic layers were extracted with DCM (× 3) with saturated aqueous NaHCO _3, washed with ₄ Na ₂ SO, concentrated and co-evaporated once with THF. The residue was taken up in 80% HOAc (35 mL) and stirred at 45 &lt;0&gt;C for 3 h then concentrated. The residue was purified by EtOAc EtOAc EtOAc EtOAc

Step b) Lithium ((2R,3R,4S,5R)-trichloro-4-chloro-5-(2,4-di-oxy-3,4-dihydropyrimidin-1(2H)-yl)-4 -fluoro-3-hydroxytetrahydrofuran-2-yl)methyl ester (3b)

To a stirred solution of compound 3a (78 mg, 0.24 mmol) in a mixture of anhydrous pyridine (560 μL) and dry THF (560 μL) was added 2-chloro-1,3,2-benzodioxole-4 a freshly prepared solution of the ketone (64 mg, 0.31 mmol) in dry THF (280 uL). The mixture was stirred at room temperature under nitrogen for 15 minutes then added under nitrogen tributylammonium _{P 2 O 7 (146mg, 0.27mmol} ) and tributyl amine (127μL, 0.53mmol) in dry DMF (560μL) A previously prepared solution. The obtained solution was stirred at room temperature under nitrogen for an additional 15 minutes, followed by adding I ₂ (123mg, 0.48mmol) in pyridine / water (98/2, v / v ) (1.1mL) and the solution form of The reaction mixture was stirred for 15 minutes. Excess iodine was eliminated by adding about 19 drops of a 5% aqueous Na ₂ SO ₃ solution and the reaction solution was concentrated. The residue was taken up in water / acetonitrile (95:5) (5 mL). Concentrated ammonia (10 mL) was added and the~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

Approximately 430 mg of crude material was dissolved in 10% MeCN / water (3 mL) and filtered and purified by HPLC on a Gilson apparatus using a Phenomenex Luna 5μ NH ₂ (150 x 21.2 mm) column, solvent A: 95% water: 5 % acetonitrile: 0.05 M ammonium bicarbonate, solvent B: 95% water: 5% acetonitrile: 0.8 M ammonium bicarbonate gradient: 0% B to 50% B in 30 min.

The NTP fractions were pooled and concentrated, and the residue was dissolved in 10% MeCN / water and lyophilized. The obtained solid was taken up in 10% MeCN/water, the insoluble matter was filtered through a 0.45 μm glaze filter and the clear filtrate was evaporated to dryness, dissolved in water/acetonitrile (95:5), passed through Dowex-Li ⁺ and frozen. Drying gave the title compound (39.3 mg, 28%).

¹ H NMR (500 MHz, D ₂ O) δ 7.87 (d, J = 8.2 Hz, 1H), 6.41 (d, J = 15.9 Hz, 1H, 1), 5.98 (d, J = 8.2 Hz, 1H), 4.56 (dd, J = 19.1, 9.4 Hz, 1H, 5), 4.35 (dddd, J = 42.1, 12.3, 5.1, 2.2 Hz, 3H), 4.19 (d, J = 9.4 Hz, 1H, 8).

¹³ C NMR (126 MHz, D ₂ O) δ 165.94, 151.67, 140.78, 114.54, 112.55, 103.12, 87.95, 87.62, 79.45, 79.38, 73.16, 73.02, 62.60, 62.56.

Example 4

(2S)-2-((((2R,3R,4S,5R)-4-chloro-5-(2,4-di-oxy-3,4-dihydropyrimidin-1(2H)-yne) 4-fluoro-3-hydroxytetrahydrofuran-2-ynemethoxy)(phenoxy)phosphonium)amino)propionic acid cyclohexyl ester (4)

(, 0.053mmol 15mg) in dry THF was added under N ₂ at 0 ℃ downward nucleoside 1g (2mL) in a solution of t.BuMgCl (13.7mg, 0.12mmol). The resulting suspension was stirred at 0 ° C for 1 h, then DMPU (0.5 ml) was added, followed by dropwise addition of (2S)-2-(((perfluorophenoxy)(phenoxy)phosphonium)amino) A solution of cyclohexyl propionate (33 mg, 0.067 mmol) in THF (0.5 mL) was maintained at EtOAc. After 4h, add NH ₄ Cl (saturated aqueous solution) was added and the mixture was extracted three times with EtOAc. The extract was washed with water and the brine, then dried (Na ₂ SO ₄₎ and concentrated under reduced pressure. The residue obtained was purified by gradient elution with DCM / MeOH using EtOAc (EtOAc EtOAc). Appropriate fractions were combined, concentrated and evaporated from EtOAc EtOAc. LC-MS 590.09 [M+H] ⁺ .

The following compounds were synthesized by phosphorylating nucleoside 1 g with the indicated phosphorylation reagent using the procedure of Example 4:

Example 18

Step a) 1-((2R,3S,4R,5R)-3-chloro-3-fluoro-4-hydroxy-5-(((6-nitro-2-oxo-yl)- 4H-benzo[d][1,3,2]dioxaphosphon-2-yl)oxy)methyl)tetrahydrofuran-2-ylpyrimidine-2,4(1H,3H)-dione (18a)

The nucleoside 3a (69 mg, 0.21 mmol) was dissolved in a mixture of acetonitrile / dichloromethane (2.7 / 1.3, ca. 4 mL) and the solution was cooled to -20 ° C under nitrogen. Et ₃ N (77 μL, 0.56 mmol) was added to the solution, followed by the addition of a solution prepared in DCM (1.34 mL; 2 mmol to 5 mL to give a stock solution) 2-chloro-6-nitro-4H-benzo[ d] [1,3,2] dioxaphosphorane (125 mg, 0.54 mmol). The cooling bath was removed and the reaction was stirred at room temperature. After 11⁄2 h, the reaction was cooled to -5 ° C and a solution of &lt;RTI ID=0.0&gt;&gt; Followed by extraction with EtOAc in a mixture, the phases were separated and the organic phase was washed with cold water (2 ×), dried (Na ₂ SO _4). Concentrate and co-evaporate from heptane / DCM, LCMS 536 [M+H]. This crude material was used in the next step.

Step b) ((2R,3R,4S,5R)-tetrahydrodiphosphate 4-chloro-5-(2,4-di-oxy-3,4-dihydropyrimidin-1(2H)-yl)- 4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl ester (18b)

Compound 18a was co-evaporated with anhydrous DMF, then dissolved in anhydrous DMF (2. <RTI ID=0.0></RTI></RTI><RTIgt; The solution was stirred at room temperature for about 17 h, then concentrated in vacuo and a few portions of water was added, then concentrated ammonia (25-30 mL) and THF (1-2 mL) were added and the mixture was stirred at room temperature. After 2h, most of the NH ₃ is removed by evaporation and the residue was extracted with DCM (4 × 40mL). The aqueous layer was concentrated and the residue was dissolved in 10%MeCN/Milli Q water. The insoluble material was filtered off and the filtrate was concentrated to dryness.

The obtained residue was dissolved in 10% MeCN/water (1.5 mL), loaded onto a column of activated carbon (0.85×3.00 cm) and eluted with 10%MeCN/Milli Q water. Appropriate fractions were pooled, concentrated, co-evaporated with MeCN (x2) and finally dried on a freeze dryer. The crude residue (76 mg) was dissolved in 10% MeCN / EtOAc (1 mL) and applied on a Luna NH ₂ column on a Luna NH ₂ column using a gradient of 0% B to 30% B (30 mL) /min) Purified over 20 min (solvent A: 0.05 M ammonium bicarbonate, 5% acetonitrile; solvent B: 0.8 M ammonium bicarbonate, 5% acetonitrile). Appropriate fractions were pooled and concentrated to dryness and the residue was dissolved in &lt The loose residue was taken up in 10% MeCN in EtOAc (EtOAc). LCMS ES ^- 438.8[MH] ^-

Example 19, an alternative route to Compound 1

Step a) (3S, 4R, 5R)-3-chloro-3-fluoro-4-((triisopropyldecyl)oxy)-5-(((triisopropyldecyl)oxy)acetate) Methyl)-tetrahydrofuran-2-yl ester (19a)

Under argon at -35 ℃ solution of compound 1c (16.3g, 32.8mmol) in THF (120mL) are added dropwise a solution of Li (Ot-Bu) ₃ AlH in THF (39mL, 39mmol) in a solution of 1M. The mixture was stirred at -35 °C for 1 h then stirred at rt for 1 h. The mixture was cooled to -25 ° C, DMAP (4.00 g, 32.8 mmol) was added and the mixture was stirred for 15 min, then acetic anhydride (33.5 g, 328 mmol) was added dropwise and the mixture was stirred for 2 h. The mixture was brought to 0.degree. C. and EtOAc (200 mL) and water. The phases were separated and the aqueous extracted with EtOAc (x 2). The combined organic phases were washed with water (x 2) and brine (×1). The organic phase was dried (Na ₂ SO _4), filtered and concentrated under reduced pressure. The residue was co-evaporated twice with EtOAc (EtOAc)EtOAc.

Step b) (3S, 4R, 5R)-3-chloro-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl acetate (19b)

Triethylamine trihydrofluoride (20.5 g, 126 mmol) was added to a stirred solution of compound 19a (17.0 g, 31.4 mmol) in EtOAc (EtOAc) The mixture was stirred at rt for 72 h, at 50 ° C for 20 h and then stirred at rt overnight. The solution was concentrated on EtOAc (EtOAc)EtOAc.

Step c) (2R, 3R, 4S)-benzoic acid 5-ethoxycarbonyl-2-((benzylideneoxy)methyl)-4-chloro-4-fluorotetrahydrofuran-3-yl ester (19c )

To a stirred solution of the compound 19b (6.80 g, 26.8 mmol), triethylamine (10.8 g, 107 mmol) was added under ice cooling, followed by the addition of benzamidine chloride (9.41 g, 66.9 mmol). The mixture was allowed to reach rt and stirred overnight. EtOH (5 mL) was added and the mixture was stirred 30 min then concentrated in vacuo. Water was added and the mixture was extracted with EtOAc (×3). The organic layer was washed with water and brine, dried (Na ₂ SO _4), filtered and concentrated under reduced pressure. The product was purified by EtOAc EtOAc (EtOAc)

Step d) ((2R,3R,4S)-benzoic acid 3-(benzylideneoxy)-4-chloro-4-fluoro-5-hydroxytetrahydrofuran-2-yl)methyl ester (19d)

To a stirred solution of compound 19c (10.1 g, 23.0 mmol) EtOAc (EtOAc) The mixture was stirred at rt for EtOAc (EtOAc) (EtOAc) The combined aqueous phases were extracted with EtOAc then EtOAc EtOAc. The combined organic phases were dried (Na ₂ SO _4), filtered and concentrated under reduced pressure. The product was purified by EtOAc (EtOAc) elut elut

Step e) ((2R,3R,4S)-benzoic acid 3-(benzylideneoxy)-4-chloro-4-fluoro-5-((methylsulfonate) Ethyloxy)tetrahydrofuran-2-yl)methyl ester (19e)

At -15 ℃ (100mL) was added in the under N ₂ to compound 19d (8.36g, 21.2mmol) in dry _{DCM Et 3 N (3.54mL, 25.4mmol} ), after addition of MsCl (1.97mL, 25.4mmol ). The reaction mixture was stirred at -15 &lt;0&gt;C for 2 h then EtOAc (EtOAc &lt The phases were separated and the aqueous layer was extracted with DCM. The combined organic extracts were washed (sat aq) NH ₄ Cl, dried (MgSO ₄₎ and concentrated under reduced pressure to give a clear oil of the title compound (9.86g, 98%).

Step f) ((2R,3R,4S,5R)-benzoic acid 3-(benzylideneoxy)-4-chloro-5-(2,4-di-oxy-3,4-dihydropyrimidine -1(2H)-yl)-4-fluorotetrahydrofuran-2-yl)methyl ester (19f)

The uracil (3.09g, 27.5mmol) and ammonium sulphate (48.5mg, 0.367mmol) was heated in HMDS (49.3mL, 236mmol) under N ₂ at reflux for 16h. The reaction mixture was cooled to rt, concentrated under reduced pressure and dried in vacuo. In the case of N ₂ to the compound 19e (8.68g, 18.4mmol) in dry DCE (75mL) was added anhydrous DCE (50mL) in the residue. TMSOTf was added slowly to the solution under N ₂ (6.12g, 27.5mmol). After the addition, the reaction mixture was heated to 80 ° C for 5 h and then heated at 65 ° C for 16 h. The reaction mixture was cooled to rt, with NaHCO ₃ (saturated aqueous solution) was quenched, filtered, and extracted twice with DCM. The combined organic extracts were dried (MgSO ₄₎ and concentrated under reduced pressure. EtOAc and DCM were added and the formed precipitate was collected by filtration, which afforded the purified <RTIgt; The filtrate was evaporated to ruthenium dioxide and purified by flash chromatography (EtOAc:EtOAc:EtOAc:EtOAc: 95 (942 mg, 11%).

Step e) 1-((2R,3S,4R,5R)-3-chloro-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H )-dione (19e)

Compound 19f (670mg, 1.37mmol) was suspended in NH (7N, in MeOH) _3. After 30 min, EtOH (5 mL) was added and the suspension was stirred at rt. After an additional 1 hour, the suspension became a solution and the reaction mixture was stirred at rt for 15 h. The solvent was evaporated under reduced br~~~~~~ LC-MS ES-279.31 [MH] ^- .

The following compounds were synthesized by phosphorylating nucleoside 1 g with the indicated phosphorylation reagent using the procedure of Example 4:

¹ DMPU is not present in the reaction mixture

² 18h after adding an additional 0.8 equivalents of a phosphorylating agent (I-49)

Example 26

Step a) (2R,3R,4S,5R)-4-methylbenzoic acid 4-chloro-5-(2,4-di-oxy-3,4-dihydropyrimidin-1(2H)-yl) 4-fluoro-2-(((4-methylbenzylidyl)oxy)methyl)tetrahydrofuran-3-yl ester (26a)

1 g (253 mg, 0,9 mmol) of nucleoside was dissolved in pyridine (5 ml) and DCM (5 ml). Triethylamine (630 μl, 4.52 mmol) was added and the mixture was cooled on ice. After 15 min, 4-methylbenzhydrin chloride (300 μl, 2.27 mmol) was added and the mixture was stirred for 10 min under cooling and then stirred at 22 ° C for 90 min. Was added NaHCO ₃ (aq) and the mixture was diluted with DCM and washed with 1M HCl (aq) × 3, dried (Na ₂ SO ₄₎ and concentrated. The residue was purified by EtOAc EtOAcjjjjjjj LC-MS

Step b) 4-Amino-1-((2R,3S,4R,5R)-3-chloro-3-fluoro-4-hydroxy-5-(hydroxymethyl)-tetrahydrofuran-2-yl)pyrimidine-2 (1H)-ketone (26b)

Compound 26a (279 mg, 0.54 mmol) was dissolved in pyridine (5 mL), molecular sieves (4 Å, half a spoon) was added and the mixture was stirred on ice bath for 15 min. Phosphorus chloride (200 μl, 2.18 mmol) was added and after 5 min, 1,2,4-1H-triazole (373 mg, 5.4 mmol) was added. The mixture was stirred under cooling for 15 min and then at 22 ° C for 5 h. Ammonia (32%, 10 mL, 82.2 mmol) was added and the mixture was stirred at 22 ° C overnight. The mixture was concentrated, dissolved in water and washed with EtOAc EtOAc. The combined organic layers were extracted with EtOAc EtOAc (EtOAc m. %). MS ES+ 279.9 [M+H] ⁺ .

Step c) (2S)-2-(((((2(, 2R,4R,4S,5R)-5-(4-amino-2-yloxypyrimidin-1(2H)-yl)-4-) Chloro-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methoxy)(phenoxy)phosphonium)amino)propionic acid isopropyl ester (26c)

Compound 26b (27.4 mg, 0.1 mmol) was dissolved in anhydrous THF (6 mL) containing molecular sieves, and the mixture was stirred at 22 ° C for 30 min, then 2 M butyl butyl chloride in THF (0.11 ml) was added and mixture Stir for another 30 minutes. Add (2S)-2-(((Perfluorophenoxy)(phenoxy)phosphonium)amino)propanoate (51.4 mg, 0.11 mmol) and stir the mixture for 15 h then EtOAc and washed (aq) NaHCO _3, dried (Na ₂ SO _4), filtered and concentrated. The residue was purified by EtOAc (EtOAc:EtOAc) Bring the appropriate parts together and concentrate. By prep HPLC using Gemini C18 column using acetonitrile / water _{(pH 7,0.01M NH 4 OAc, 20-40} %) gradient elution of the residue was purified. The product was concentrated, followed by MeOH / water _{(pH 7,0.01M NH 4 OAc, 33-50} %) gradient elution of the column purified fluorophenyl. The product was collected, dissolved in acetonitrile / water (1: 4) and lyophilized to give the title of this compound (13mg, 24%) LC- MS 548.9 [M + H] +.

Example 27

Step a) N-(1-((2R,3S,4R,5R)-3-chloro-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl-2-yloxy- 1,2-dihydropyrimidin-4-yl)isobutylamine (27a)

To a solution of nucleoside 26b (139 mg, 0.497 mmol) in dioxane (1.7 mL) and water (0.19 mL). The solution was stirred at 58 ° C for 3 h then concentrated. The residue was dissolved in DCM and the 20% EtOH saturated aqueous NaHCO ₃ / brine 30:70 v / v washed (× 4), dried (Na ₂ SO _4), filtered and concentrated. The residue was purified by EtOAc EtOAc elut elut elut elut elut elut

Step b) (2R, 3R, 4S, 5R)-Isobutyric acid 4-chloro-4-fluoro-5-(4-isobutylammonium-2-yloxypyrimidine-1(2H)-yl)- 2-(((4-Methoxyphenyl)diphenylmethoxy)methyl)tetrahydrofuran-3-yl ester (27b)

To a solution of compound 27a (62 mg, 0.177 mmol) in EtOAc (1.sub.1 mL) Additional 4-methoxytritylhydrazine chloride (16 mg, 0.3 eq.) was added and the mixture was shaken again for 18 h. Isobutyric anhydride (33.6 mg, 0.212 mmol) was added and the solution was shaken at rt for 4 h. The reaction was quenched with MeOH, then concentrated and saturated aqueous NaHCO ₃ and extracted with DCM (× 3) /. The organic phase was dried (Na ₂ SO _4), filtered and concentrated and the residue was co-evaporated twice with toluene and co-evaporated twice with THF.

The solid residue obtained was used directly in the next step.

Step c) (2R, 3R, 4S, 5R)-Isobutyric acid 4-chloro-4-fluoro-2-(hydroxymethyl)-5-(4-isobutylammonium-2-oxo-pyrimidine- 1(2H)-yl)tetrahydrofuran-3-yl ester (27c)

Compound 27b (123 mg, 0.177 mmol) was dissolved in EtOAc (EtOAc) (EtOAc) (EtOAc) . The residue was purified by EtOAc EtOAc EtOAc. LC-MS 420.0 [M+H] ⁺ .

Step d) (((2R,3R,4S,5R)-5-(4-Amino-2-oxo-pyrimidine-1(2H)-yl)-4-chloro-4-fluoro-3-hydroxytetrahydrofuran -2-yl)methyl)triphosphate (27d)

Compound 27c (36.0 mg, 0.086 mmol) was dissolved in a mixture of MeCN / DCM: 1.06 / 0.54 (~ 1.6 mL) and the solution was cooled to -20 ° C under nitrogen. To the solution was added Et ₃ N (31.1 μL, 0.223 mmol), followed by 2-chloro-6-nitro-4H-benzo[d][1,3,2]dioxaphosphorane (50.1 mg, 0.214 mmol) in DCM (0.71 mL). The cooling bath was removed and the reaction was stirred at room temperature for 11⁄2 h. The reaction was cooled to -5 ℃ and added Oxone ^® (0.343mmol) in aqueous solution (1.73 mL) and the two-phase system in the vigorously stirred for 15min. The mixture was extracted with ethyl acetate, and the organic phase was washed with cold water (2 ×), dried (Na ₂ SO ₄₎ and concentrated. The residue was co-evaporated with toluene once and co-evaporated once with anhydrous DMF then dissolved in anhydrous DMF (1 mL). Tributylamine pyrophosphate (0.1 mmol, 54.6 mg) was added under nitrogen and the solution was shaken at room temperature for about 18 h then concentrated. 30% MeCN/H ₂ O (about 20 mL) was added to the residue and the solution was shaken at rt for 20-25 min. The volatiles were evaporated and the residual oil-solid mixture was dissolved in concentrated ammonia (10-15 mL) and shaken at room temperature for about 5 h.

By an evaporator to remove most of the NH _3, and then the residue was extracted with DCM (4 × 40mL). The organic extract was discarded and the aqueous layer was concentrated. The residue was dissolved in 5% MeCN (1.5-2.0 mL) in water and loaded onto an activated carbon column (0.85 x 2.5). The column was washed with 5% MeCN in water and 6-7 mL of the eluent was collected and concentrated and lyophilized. The residue was dissolved in 5% MeCN / water (1.6 mL of) and by semipreparative HPLC using a Phenomenex Luna 5μ NH ₂ column with 0% B to 40% B gradient of (30mL / min) through the machine on a Gilson It was purified by elution with 30 min (solvent A: 0.05 M ammonium hydrogencarbonate, 5% acetonitrile; solvent B: 0.8 M ammonium hydrogencarbonate, 5% acetonitrile). Appropriate NTP fractions were pooled and concentrated to dryness and the residue was dissolved in MeOH & EtOAc &&lt The residue was taken up in 5% MeCN (4-5 mL) in water and filtered and filtered. The residue was dissolved in 5% MeCN (0.5 mL) in water and applied to a short Li+ Dowex column (6 x 1 cm) and washed with 5% MeCN in water. The first batch, approximately 10 mL, was pooled, concentrated and lyophilized to give the title compound (11.7 mg, 30%). MS ES+ 519.9 [M+H] ⁺ .

NMR data for the selection of the exemplified compounds:

Compound 9

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 7.55 (d, J = 7.8 Hz, 1H), 6.87 (d, J = 8.4 Hz, 1H), 6.84 (d, J = 1.7 Hz, 1H), 6.76- 6.60 (m, 2H), 6.32-6.19 (m, 1H), 6.10-6.01 (m, 1H), 6.02 (s, 2H), 5.62 (d, J = 8.1 Hz, 1H), 4.86 (p, J = 6.3 Hz, 1H), 4.37-4.15 (m, 4H), 4.07-3.97 (m, 1H), 3.79 (tq, J = 10.1, 7.1 Hz, 2H), 1.23 (d, J = 7.1 Hz, 3H), 1.16 (d, J = 6.3 Hz, 5H).

¹³ C NMR (126 MHz, DMSO-d ₆ ) δ 172.50, 147.46, 144.86, 144.81, 143.91, 115.06, 115.05, 113.05, 113.05, 112.41, 112.40, 112.37, 112.37, 107.88, 102.36, 102.34, 101.52, 78.74, 74.44, 74.30, 67.90, 64.28, 49.65, 40.63, 40.40, 40.34, 40.27, 39.99, 39.90, 39.83, 39.73, 39.66, 39.57, 39.40, 39.23, 39.07, 38.90, 21.28, 21.26, 19.72, 19.67, -0.00.

Compound 10

¹ H NMR (500 MHz, DMSO-d ₆ ) δ 7.58 (d, J = 8.1 Hz, 1H), 7.28 (d, J = 8.0 Hz, 1H), 7.20-7.05 (m, 2H), 6.90 (td, J = 7.9, 1.6 Hz, 1H), 5.60 (d, J = 8.1 Hz, 1H), 4.87 (dq, J = 12.5, 6.2 Hz, 1H), 4.41-4.20 (m, 5H), 4.09-3.99 (m, 1H), 4.00-3.77 (m, 2H), 3.79 (s, 3H), 1.79 (s, 1H), 1.22 (d, J = 7.1 Hz, 3H), 1.16 (d, J = 6.3 Hz, 5H).

¹³ C NMR (126 MHz, DMSO-d ₆ ) δ 172.59, 172.55, 162.64, 150.28, 150.24, 150.09, 139.37, 139.32, 125.29, 120.90, 120.88, 120.25, 115.03, 113.02, 112.85, 102.25, 78.82, 74.38, 74.24, 67.85, 64.26, 55.59, 49.57, 40.26, 40.20, 40.17, 39.99, 39.90, 39.82, 39.73, 39.66, 39.57, 39.40, 39.23, 39.07, 38.90, 21.30, 21.26, 19.63, 19.58.

Compound 11

^{1 H NMR (500MHz, DMSO-} d 6) δ 7.57 (d, J = 8.1Hz, 1H), 7.13 (d, J = 8.3Hz, 2H), 6.98-6.86 (m, 3H), 6.25 (t, J = 16.6 Hz, 1H), 5.62 (d, J = 8.1 Hz, 1H), 4.86 (hept, J = 6.2 Hz, 1H), 4.37-4.15 (m, 4H), 4.07-3.97 (m, 1H), 3.78 (tq, J = 10.2, 7.1 Hz, 1H), 3.72 (s, 2H), 1.23 (d, J = 7.1 Hz, 3H), 1.22-1.11 (m, 8H).

¹³ C NMR (126 MHz, DMSO-d ₆ ) δ 172.52, 172.48, 162.65, 155.91, 150.08, 143.96, 143.91, 120.95, 120.92, 114.99, 114.42, 112.98, 102.27, 78.77, 74.44, 74.30, 67.86, 64.21, 55.29, 49.65, 40.25, 40.15, 39.99, 39.90, 39.83, 39.74, 39.66, 39.57, 39.40, 39.24, 39.07, 38.90, 21.30, 21.27, 19.69, 19.64.

Compound 13

^{1 H NMR (500MHz, DMSO-} d 6) δ 0.82 (t, 3H), 1.12 (d, 3H), 1.25 (d, 3H), 1.49 (m, 2H), 3.83 (dtd, 1H), 4.02 (m , 1H), 4.26 (dt, 2H), 4.34 (m, 1H), 4.72 (h, 1H), 5.58 (d, 1H), 6.12 (dd, 1H), 6.26 (m, 1H), 7.20 (m, 3H), 7.37 (t, 2H), 7.53 (d, 1H).

¹³ C NMR (126 MHz, DMSO-d ₆ ) δ 9.35, 19.05, 19.77 (d), 28.00, 40.08, 49.72, 64.32, 72.27, 74.35 (d), 78.72 (m), 102.36, 114.08 (d), 119.95 ( d), 124.49, 129.54, 150.53 (d), 163.41 (m), 172.62 (d).

Compound 15

^{1 H NMR (500MHz, DMSO-} d 6) δ 1.16 (d, 6H), 1.24 (d, 3H), 1.80 (m, 1H), 1.96 (m, 1H), 2.05 (pdd, 2H), 2.26 (m , 2H), 3.49 (p, 1H), 3.81 (tq, 1H), 4.04 (m, 1H), 4.30 (m, 3H), 4.86 (hept, 1H), 5.60 (d, 1H), 6.07 (dd, 1H), 6.24 (d, 1H), _6.68 (d, 1H), 7.03 (m, 3H), 7.28 (t, 1H), 7.58 (d, 1H). ¹³ C NMR (126 MHz, DMSO-d ₆ ) 6 17.58, 19.65 (d), 21.26, 21.29, 29.13, 49.65, 64.33, 67.87, 74.37 (d), 78.78, 102.28, 113.98 (d), 117.35 (d), 117.76(d), 122.45, 129.25, 139.49, 147.50, 150.05, 150.56, 162.56, 172.48(d).

Compound 16

^{1 H NMR (500MHz, DMSO-} d 6) δ 0.78 (m, 8H), 1.15 (d, 12H), 1.23 (d, 7H), 1.35 (s, 6H), 3.80 (tq, 2H), 4.03 (m , 2H), 4.25 (m, 4H), 4.34 (m, 2H), 4.86 (p, 2H), 5.59 (d, 2H), 6.07 (dd, 2H), 6.24 (d, 2H), 6.72 (s, 1H), 7.01 (m, 6H), 7.26 (t, 2H), 7.57 (d, 2H).

¹³ C NMR (126 MHz, DMSO-d ₆ ) δ 16.03, 18.82, 19.65 (d), 21.26, 21.30, 24.42, 49.63, 64.29, 67.87, 74.35 (d), 78.78, 87.51, 102.30, 114.00 (d), 116.92 (d), 117.60(d), 122.06, 129.18, 139.60(m), 148.45, 150.13, 150.50(d), 162.69, 172.47(d).

Compound 17

¹³ C NMR (126MHz, DMSO-d ₆ ) δ 15.52, 18.66, 19.65 (d), 21.25, 21.30, 24.98, 49.67, 64.23, 67.87, 74.35 (d), 78.76, 87.49, 102.25, 113.97 (d), 119.69 (d), 127.19, 139.65(d), 142.69, 148.17(d), 150.02, 162.52, 172.46(d).

Compound 21

^{1 H NMR (500MHz, DMSO)} δ 1.25 (d, 3H), 3.23 (m, 6H), 3.41 (m, 4H), 3.87 (ddt, 1H), 4.04 (m, 1H), 4.31 (m, 3H) , 5.02 (p, 1H), 5.61 (d, 1H), 6.20 (m, 2H), 7.21. (m, 3H), 7.38 (t, 2H), 7.57 (d, 1H).

¹³ C NMR (126 MHz, DMSO) δ 19.72 (d), 49.60, 58.36, 64.28, 70.33, 70.46, 71.53, 74.38 (d), 78.81 (d), 102.26, 114.00 (d), 119.99 (d), 124.53, 129.55, 150.03, 150.51 (d), 162.54, 172.59 (d).

Compound 24

^{1 H NMR (500MHz, DMSO)} δ 1.15 (dd, 6H), 1.22 (d, 3H), 3.52 (m, 1H), 3.78 (tq, 1H), 4.05 (m, 1H), 4.16 (m, 1H) , 4.26 (dt, 1H), 4.34 (m, 1H), 4.86 (hept, 1H), 5.65 (d, 1H), 6.14 (dd, 1H), 6.23 (s, 1H), 6.27 (s, 1H), 7.21 (m, 3H), 7.37 (t, 2H), 7.50 (d, 1H).

¹³ C NMR (126 MHz, DMSO) 5 19.62 (d), 21.25 (d), 49.61, 63.83, 67.92, 74.16 (d), 78.53, 102.42, 114.03 (d), 119.85 (d), 124.49, 129.58, 139.35 ( Dd), 150.26, 150.56(d), 162.87, 172.51(d).

Compound 26b

^{1 H NMR (500MHz, DMSO)} δ 3.62 (d, 1H), 3.80 (m, 2H), 4.15 (dd, 1H), 5.26 (s, 1H), 5.77 (d, 1H), 6.31 (d, 1H) , 6.41 (s, 1H), 7.33 (s, 1H), 7.36 (s, 1H), 7.73 (d, 1H).

¹³ C NMR (126 MHz, DMSO) 5 58.50, 58.62, 73.62 (d), 80.48, 87.01 (m), 94.50, 94.56, 114.92 (d), 140.04, 154.57, 165.42.

Compound 27d

^{1 H NMR (500MHz, D20)} δ 4.12 (d, 1H), 4.24 (ddd, 1H), 4.33 (m, 1H), 4.46 (dd, 1H), 6.09 (d, 1H), 6.39 (d, 1H) , 7.80 (d, 1H).

¹³ C NMR (126 MHz, D 20 ) δ 62.48 (d), 73.03 (d), 78.99 (d), 88.15 (d), 97.04, 113.71 (d), 140.63, 157.39, 166.21.

Biological instance Replicon analysis

The activity of a compound of formula I in inhibiting HCV RNA replication can be tested in a cellular assay designed to identify compounds that inhibit HCV functional cell replication cell lines (also known as HCV replicons). Suitable cell analysis is based on a bicistronic expression construct in a multi-target screening strategy, as described by Lohmann et al. (1999), Science, Vol. 285, pp. 110-113, by Krieger et al. (2001), Modifications are made as described in Journal of Virology 75: 4614-4624.

This analysis utilized the stably transfected cell line Huh-7 luc/neo (hereinafter referred to as Huh-Luc). This cell line has an RNA encoding a bicistronic expression construct comprising the wild type NS3-NS5B region of HCV type 1b translated from the internal ribosome entry site (IRES) of encephalomyocarditis virus (EMCV). The reporter gene portion (FfL-luciferase) and the selectable marker portion (neo ^R , neomycin phosphotransferase). The construction system is bordered by the 5' and 3' NTR (non-translated regions) of HCV type 1b. Continued culture of replicon cells in the presence of G418 (neo ^R ) depends on replication of HCV RNA. Replicon cells that express HCV RNA and that replicate autonomously and to a high degree, particularly encoding luciferase, are used to screen for antiviral compounds.

Replicon cells were plated in 384 well plates in the presence of test and control compounds added at various concentrations. In the cultivation for 3 days, by analysis of luciferase activity (using standard luciferase assay reagents and by mass and Perkin Elmer ViewLux ^TM ultraHTS microplate imager) measuring HCV replication. Replicon cells in control cultures have high luciferase performance without any inhibitor. The inhibitory activity of the compounds on luciferase activity was monitored on Huh-Luc cells to provide a dose-response curve for each test compound. EC ₅₀ values _were then calculated, which value represents the detected value of the luciferase activity by 50%, or more of the required amount of a compound of the ability to replicate genetically linked HCV replicon RNA of specific terms.

Enzyme analysis

As demonstrated in the replicon assay, the compounds of the invention are metabolized by the cellular kinase in the target tissue to the 5'-triphosphate. This triphosphate is believed to be an antiviral active. The enzyme assays described herein can be used to confirm that the compounds of the invention have antiviral activity as 5'-triphosphate metabolites.

Enzyme assay The inhibitory effect of the triphosphate compound was measured in the HCV NS5B-21 (21-amino acid C-terminally truncated form) SPA assay (scintillation proximity assay). This analysis was performed by assessing the amount of radiolabeled ATP incorporated into newly synthesized RNA by HCV NS5B-21 using a heterogeneous biotinylated RNA template.

₅₀ is a measured value IC, in 100μl final volume of the reaction mixture at different concentrations of the test compound. The reaction was stopped by the addition of 0.5 M EDTA solution. The sample was transferred to a streptavidin precoated scintillation plate. The incorporated radioactivity was quantified using a scintillation counter (Wallac Microbeta Trilux).

Materials and suppliers

Biotinylated RNA template: has the following sequence: 5'-UUU UUU UUU UAG UCA GUC GGC CCG GUU UUC CGG GCC-3' and biotinylated at the 5'-introduction end of 83 μM in 10 mM Tris-HCl

device

Wallac Microbeta Trilux Perkin Elmer Life Sciences

method Analysis condition

The analysis should include an enzyme control (approximately four, containing 1 μl of DMSO instead of the inhibitor) and a background control containing all components except the template.

Compounds were serially diluted in DMSO to 100 x final desired assay concentration on separate dilution plates.

The reaction mixture having a sufficient number of pores to be used was constructed according to the following table and 90 μl/well was added to a 96-well polypropylene plate. 1 μl of the compound in DMSO from the dilution plate was added to each well, except that 1 μl of DMSO was added to the enzyme control wells and the background control wells.

Reaction mixture

An ATP mixture containing 1.5 μl/well of ³ H-ATP (45 Ci/mmol), 2.0 μl/well of 100 μM ATP, and 6.5 μl/well of H ₂ O was prepared and the reaction was started by adding 10 μl/well of the mixture. .

Incubate at 22 ° C for 120 min.

The reaction was stopped by the addition of 100 μl/well of 0.5 M EDTA (pH = 8.0).

185 μl/well was transferred to a streptavidin scintillation plate.

Plates were incubated overnight and the scintillation plates were read in Microbeta Trilux using the protocol Flash plates H3.

Processing of results

Calculation of suppression:

Background = no template reaction buffer.

IC ₅₀ was determined using Graphpad Prism. A compound concentration curve is plotted as a logarithmic versus inhibition %. The curve and nonlinear regression were fitted to the Log (inhibitor) versus reaction equation.

Wherein Y is inhibited by %, X is log (inhibitor), and the top and bottom values are the upper and lower limits of % inhibition.

Biological example 1

Inhibition of HCV replication exhibited by the compounds of the invention was tested in the above replicon assay. The compound showed micro-mole activity with cytotoxicity in the Huh-Luc cell line exceeding 50 μM. EC ₅₀ values are provided in Table 1.

Biological example 2

The nucleotides of Examples 3 and 27 were tested in the above enzyme assay and the IC ₅₀ values were determined to be 0.72 μM and 0.089 μM, respectively.

Comparative example 1

Sububuwe is sold in several countries for the treatment of HCV, mainly for genotypes 1 and 4. The structure of Sufu Buwei:

As can be seen, the fubucarb is different from the compound of Example 2 of the present invention except that it has a β-methyl group at the 2' position, and the compound of the present invention has a β-chloro substituent at this position. In the split phase III clinical trial reported in Lawitz et al., N. Eng. J. Med., 2013; 368: 1878-87 , the response rate in the Subu-Bubavirin group was genotype 3 infection. The number of patients was lower than that of those infected with genotype 2 (56% vs 97%).

The antiviral activity of a commercially available compound of the compound of Sububuvir and Example 2 was compared in the genotype 3a transient replicon assay described in Kylefjord et al, J Virol. Methods 2014 195: 156-63.

EC complex bouvet speed for the ₅₀ genotype 3a based 0.230μM +/- 0.067, n = 11, compared with Example 2 Compound EC ₅₀ of 0.072μM +/- 0.024, n = 9. It is expected that the compound of the present invention will have a better efficacy than that of Sububuwei by a factor of three, which can significantly improve the viral response rate in the clinic.

Maintaining the efficacy of the compounds of the invention in a transient replicon of genotype 3a with a thorny S282T mutation (giving HCV nucleoside meritabine resistance) is improved several times over the speed-recovery, in which the complex Wei 0.48μM having the EC ₅₀ (n = 1) and examples of the compound having 2 0.13μM the EC ₅₀ (n = 1). Similarly, a L159F/L320F double mutant produced by exposure to nucleoside meritabine and conferring resistance to valproate was prepared in genotype 3a transient replicon (Tong et al. 2013 J. Infect. Dis., 209(5), 668-75), as described by Kylefjord et al., supra. In the double mutant, the complex speed of 0.190 Bouvet having EC ₅₀ (n = 1), the compound of Example 2 of the display EC 0.062 ₅₀ (n = 1).

Compounds of Example 2 were further evaluated to evaluate the antiviral activity of genotypes 1-6 (wild type and various clinically relevant mutant strains) against HCV. The results of the evaluation and the corresponding values ₅₀ and the average speed Bouvet multiplexing of genotypes with EC summarized in Table 2 and 3.

In addition to AVG, EC ₅₀ data (all expressed begin μM) system to provide a geometric mean, an arithmetic system which EC ₅₀ provides mean +/- SEM.

* The chimeric replicon contains the GT NS5B gene in the con1 background.

References: Con1 (Lohmann et al., 2003); H77 (Blight et al., 2003); GT2a (Wakita et al., 2005); GT3a (Kylefjord et al., 2013); GT4-6 (Wong et al., 2012); L159F/L320F (Tong et al., 2013).

In addition to AVG, EC ₅₀ data (all expressed begin μM) system to provide a geometric mean, an arithmetic system which EC ₅₀ provides mean +/- SEM.

* The chimeric replicon contains the GT NS5B gene in the con1 background.

References: Con1 (Lohmann et al., 2003); H77 (Blight et al., 2003); GT2a (Wakita et al., 2005); GT3a (Kylefjord et al., 2013); GT4-6 (Wong et al., 2012); L159F/L320F (Tong et al., 2013).

From the two, it was shown that the compound of Example 2 of the present invention was significantly more effective against HCV GT3a in the wild-type strain and the two clinically relevant mutant strains than the quick-bubbo. Improve while maintaining good results for other genotypes.

Triphosphate formation analysis

To estimate the ability of the compounds of the invention to generate antiviral active triphosphate species, triphosphate formation assays were performed. Each compound was tested in triplicate in the analysis.

Fresh human tiled hepatocytes (Biopredic, France) in a 12-well plate were used. Each well was tiled with 0.76 x 10 ⁶ cells and incubated with compound 10 μM DMSO solution (0.1% DMSO) in a 1 mL incubation medium at 37 °C for 6-8 hours in a CO ₂ incubator. The incubation was stopped by washing each well twice with 1 mL of ice-cold Hank's equilibrium solution (pH 7.2), followed by the addition of 0.5 mL of ice-cold 70% methanol. Immediately after the addition of methanol, the cell layer was detached from the bottom of the well by a cell scraper and pumped up and down 5-6 times using an automatic pipette. The cell suspension was transferred to a glass vial and stored at -20 °C overnight.

Samples each consisting of different amounts of protein, free nucleoside and monophosphate, diphosphate and triphosphate were then vortexed and centrifuged at 14,000 rpm for 10 minutes at 10 ° C in Eppendorf centrifuge 5417R. The supernatant was transferred to a 2 mL glass vial with a gasket and subjected to bioassay.

Biological analysis

An internal standard (Indinavir) was added to each sample and the sample (10 μL injection volume) was analyzed on a two-column system coupled to a QTRAP 5000 mass spectrometer. The two-column system consists of two binary pumps X and Y, two switching valves and an autosampler. The two HPLC columns used were Synergy POLAR-RP 50*4.6 mm, 4 μm particles and BioBasic AX 50*2.1 mm 5 μm particles. The LC flow rate was 0.4-0.6 mL/min mL/min (higher flow rate was used in the improvement step).

The HPLC mobile phase of the POLAR-RP column consisted of 10 mmol/L ammonium acetate (mobile phase A) in 2% acetonitrile and 10 mmol/L ammonium acetate (mobile phase B) in 90% acetonitrile and HPLC flow of BioBasic AX column. The phase consisted of 10 mmol/L ammonium acetate (mobile phase C) in 2% acetonitrile and 1% ammonium hydroxide (mobile phase D) in 2% acetonitrile. Pump Y's HPLC gradient with 0% flow Phase B begins and remains for 2 min. During the loading phase, the mobile phase passes through the POLAR-RP and BioBasic AX columns, and the prodrugs, nucleosides, and internal standards are retained on the POLAR-RP column; nucleotides (monophosphates, diphosphates, and The triphosphate) was dissolved onto a BioBasic AX column and trapped there.

In the next step, the fluid is switched from the POLAR-RP column to MS and the mobile phase C is switched from the pump X to the BioBasic AX column. Compounds on the POLAR-RP column were eluted with a gradient of 0% B up to 100% B over approximately 2 minutes and analyzed in either positive or negative mode using various reaction monitoring modes (MRM). In the final step, the fluid of the BioBasic AX column was switched to MS and the phosphate was eluted with a gradient of up to 50% D for about 7 minutes and analyzed in positive or negative mode using MRM. During the final step, the two columns are improved.

The triphosphate concentration of each compound was then determined by comparison to a standard curve. A standard curve was prepared by analyzing a standard sample having a known concentration of triphosphate. The standard is allowed to flow in the same matrix as the test sample. Due to changes in the degree of phosphorylation of the hepatocyte donors, an internal reference compound is required in each round of analysis to enable the results of the different rounds to be ranked to each other.

Throughout the specification and the accompanying claims, the words "comprise" and variations (eg, "comprises" or "comprising") are understood to include the integers, unless the context requires otherwise. A step, integer group, or group of steps, but does not exclude any other integer, step, integer group, or group of steps.

All documents (including patents and patent applications) cited herein are hereby incorporated by reference in their entirety.

Claims (15)

A compound represented by Formula I: Where: B line nucleobase: Wherein R ⁷ and R ⁸ are both H; R ¹ is R ² is H; R ¹⁴ is phenyl, which is optionally substituted by 1, 2 or 3 R ²² ; one of R ¹⁵ and R ^{15 '} is H and the other is C ₁ -C ₃ alkyl; R ¹⁶ is C ₁ -C ₈ alkyl; each R ²² is independently selected from halo, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₁ -C ₆ haloalkyl, benzene Base, hydroxy C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, C ₁ -C ₆ alkylcarbonyl, C ₃ -C ₆ cycloalkylcarbonyl, carboxy C ₁ -C ₆ alkyl, side oxygen Base, OR ²⁰ , SR ²⁰ , N(R ²⁰ ) ₂ , CN, NO ₂ , C(O)OR ²⁰ , C(O)N(R ²⁰ ) ₂ and NHC(O)R ^{20 are} each independently R ²⁰ H, C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₃ -C ₇ cycloalkyl, C ₁ -C ₆ hydroxyalkyl or C ₃ -C ₇ cycloalkyl C ₁ - C ₃ alkyl; R ²⁴ lines H; R ³³ are each independently selected from H and lines C ₁ -C ₆ alkyl; Department of the U-O; or a pharmaceutically acceptable salt and / or solvate thereof. The compound of claim 1, wherein R ¹⁴ is phenyl substituted by 1 or 2 R ²² , wherein each R ²² is independently selected from halo, C ₁ -C ₆ alkyl, C ₂ -C ₆ Alkenyl and OR ²⁰ and R ²⁰ is C ₁ -C ₆ alkyl. The compound of claim 1, wherein one of R ¹⁵ and R ^15' is H and the other is methyl. The compound of any one of claims 1 to 3, wherein R ¹⁶ is methyl, ethyl, propyl or isopropyl. The compound of claim 4, wherein R ¹⁶ is isopropyl. The compound of any one of claims 1 to 3, wherein R ¹⁶ is 2-ethylbutyl, 2-pentyl, 2-butyl, isobutyl, and third pentyl. The compound of claim 6 wherein R ¹⁶ is 2-ethylbutyl. The compound of claim 1, which is: Or a pharmaceutically acceptable salt and/or solvate thereof. The compound of claim 1, which is: Or a pharmaceutically acceptable salt and/or solvate thereof. The compound of claim 1, which is: Or a pharmaceutically acceptable salt and/or solvate thereof. The compound of claim 1, which is: Or a pharmaceutically acceptable salt and/or solvate thereof. A compound according to any one of claims 1 to 3 and 8 to 11 for use as a medicament. Use of a compound according to any one of claims 1 to 11 for the preparation of a medicament for the treatment or prevention of hepatitis C virus infection. A pharmaceutical composition comprising a compound according to any one of claims 1 to 11 together with a pharmaceutically acceptable adjuvant, diluent or carrier. A pharmaceutical composition comprising a compound according to any one of claims 1 to 11, further comprising one or more additional additional antiviral agents.