Nucleoside derivatives as inhibitors of rna-dependent rna viral polymerase

Description of the invention

The invention relates to nucleoside derivatives, a pharmaceutical composition containing them and their use. The compounds of the invention are inhibitors of RNA-dependent replication of RNA viruses and find use in the treatment of RNA-dependent viral infections. They are particularly useful as inhibitors of hepatitis C virus (HCV) NS5B polymerase, as inhibitors of viral replication, and as agents for the treatment of hepatitis C.

Hepatitis C (HCV) is a significant health problem. HCV causes a significant percentage of infections, estimated at 2-15% of the world's population, and leads to chronic liver diseases such as cirrhosis and hepatocellular carcinoma. According to the American Center for Disease Control, there are approximately 4.5 million people infected in the United States alone. According to the World Health Organization (WHO), there are over 200 million people infected in the world, and at least 3 to 4 million people are infected every year. After infection, about 20% of people clear the virus from the body, but the rest remain HCV carriers for the rest of their lives. 10-20% of those chronically infected develop liver-destroying cirrhosis or cancer. This viral disease is transmitted parenterally through contaminated blood and blood products, contaminated needles, or by sexual and vertical transmission from infected mothers and pregnant women to their offspring. The current treatment of HCV infection, limited to immunotherapy with either interferon-α alone or in combination with ribavirin nucleoside analogues, has limited clinical benefit. Moreover, no vaccine against HCV has been developed. Accordingly, there is an urgent need for improved therapeutic agents capable of effectively combating chronic HCV infection. The current knowledge on the treatment of HCV infection was reviewed based on the following publications: B. Dymock, et al., Novel approaches to the treatment of hepatitis C virus infection. Antiviral Chemistry & Chemotherapy. 11: 79-96 (2000); H. Rosen, et al., Hepatitis C virus: current understanding and prospects for future therapies, Molecular Medicine Today, 5: 393-399 (1999); D. Moradpour, et al., Current and evolving therapies for hepatitis C, European J. Gastroenterol. Hepatol. 11: 1189-1202 (1999); R. Bartenschlager, Candidate Targets for Hepatitis C Virus-Specific Antiviral Therapy, Intervirology, 40: 378-393 (1997); GM Lauer and BD Walker, Hepatitis C Virus Infection, N. Engl. J. Med., 345: 41-52 (2001); BW Dymock, Emerging therapies for hepatitis C virus infection, Emerging Drugs, 6: 13-42 (2001); and C. Crabb, Hard-Won Advances Spark Excitement about Hepatitis C, Science: 506-507 (2001).

Various attempts have been made to treat HCV, including inhibition of viral serine proteinase (NS3 protease), helicase, RNA-dependent RNA polymerase (NS5B), and vaccine development.

The HCV virion consists of an envelope within which is a positive strand of viral RNA with a genomic sequence of approximately 9,600 bases of single oligoribonucleotides encoding a polyprotein of approximately 3,010 amino acids. The protein product of the HCV gene consists of the structural proteins C, E1 and E2, as well as the nonstructural proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B. Nonstructural (NS) proteins are believed to constitute the catalytic system of viral replication. The NS3 protease liberates NS5B, an RNA-dependent RNA polymerase, from a polyprotein chain.

HCV NS5B polymerase is necessary for the synthesis of double-stranded RNA from single-stranded viral RNA, which serves as a template in the HCV replication cycle. Thus, NS5B polymerase is considered an essential component of the HCV replication complex [see K. Ishi, et al., Expression of Hepatitis C Virus NS5B Protein: Characterization of Its RNA Polymerase Activity and RNA Binding, Hepatology, 29: 1227-1235 (1999) and V. Lohmann, et al., Biochemical and Kinetic Analyzes of NS5B RNA-Dependent RNA Polymerase of the Hepatitis C Virus, Virology, 249: 108-118 (1998)]. Inhibition of HCV NS5B polymerase prevents the formation of HCV double-stranded RNA and therefore represents an interesting starting point for the development of HCV-specific antiviral therapies.

The nucleoside compounds of the present invention have been found to be potent inhibitors of RNA-dependent replication of RNA virus, and in particular of HCV replication. The 5'-triphosphate derivatives of these nucleoside compounds are inhibitors of RNA-dependent viral RNA polymerase, and in particular of HCV NS5B polymerase. Thus, the nucleoside compounds of the invention are useful in the treatment of RNA dependent RNA virus infections, and in particular HCV infection.

The present invention relates to nucleoside derivatives of the structural formula II with the indicated stereochemical configuration:

PL 207 405 B1

<img file="PL207405B1_D0001.tif" />

(AT)

NO¹¹ and their pharmaceutically acceptable salts; in which formula:

R¹ is C1-3 alkyl optionally substituted with hydroxyl or one to three fluoro substituents;

R² is hydroxy or C1-4 alkoxy;

R³ is hydrogen, halogen or hydroxy;

R⁵ is hydrogen, P3O9H4, P2O6H3, or PO3H2;

R⁸ is hydrogen;

R⁹ is hydrogen, methyl or halogen; and

R¹⁰ and r¹¹ each independently represent hydrogen, halogen, hydroxyl, amino, C1-4 alkylamino, di (C1-4alkyl) amino, or C3-6cycloalkylamino.

Preferably in the compound of structural formula II:

R¹ is methyl, fluoromethyl or hydroxymethyl;

R² is hydroxy or methoxy;

R³ is hydrogen, fluoro or hydroxy;

R⁵ is hydrogen or P3O9H4;

R⁸ is hydrogen;

R⁹ is hydrogen, methyl or halogen; and

R¹⁰ and r¹¹ each independently represent hydrogen, fluoro, hydroxy, or amino. Preferred compounds of structural formula II of the invention which are useful as inhibitors of RNA-dependent viral RNA polymerase are the following compounds:

4-amino-7- (2-C-methyl-eD-arabinofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

4-amino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

4-methylamino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

4-dimethylamino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

4-cyclopropylamino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

4-amino-7- (2-C-hydroxymethyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

4-amino-7- (2-C-fluoromethyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

4-amino-5-methyl-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

4-amino-5-bromo-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

4-amino-5-chloro-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

4-amino-5-fluoro-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

2,4-diamino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

2-amino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

2-amino-4-cyclopropylamino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

2-amino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidin-4 (3H) -one,

4-amino-7- (2-C-ethyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

4-amino-7- (2-C, 2-O-dimethyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidin-4 (3H) -one,

2-amino-5-methyl-7- (2-C, 2-O-dimethyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidin-4 (3H) -one,

4-amino-7- (3-deoxy-2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

4-amino-7- (3-deoxy-2-C-methyl-eD-arabinofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

4-amino-2-fluoro-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine, and

4-amino-7- (3-deoxy-3-fluoro-2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine;

PL 207 405 B1 and the corresponding 5'-triphosphates;

and their pharmaceutically acceptable salts.

Among these compounds, the following compounds are particularly preferred:

4-amino-7- (2-C-methyl-eD-arabinofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

4-amino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

4-amino-7- (2-C-fluoromethyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

4-amino-5-methyl-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

4-amino-5-bromo-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

4-amino-5-chloro-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine,

4-amino-5-fluoro-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine and

4-amino-7- (2-C, 2-O-dimethyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine, and corresponding 5'-triphosphates;

or a pharmaceutically acceptable salt thereof.

The following compounds are very preferred:

4-amino-7- (2-C-methyl-eD-arabinofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine or pharmaceutically acceptable salts thereof,

4-Amino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine or a pharmaceutically acceptable salt thereof.

4-amino-7- (2-C-fluoromethyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-tf] pyrimidine or pharmaceutically acceptable salts thereof,

4-amino-5-chloro-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine or pharmaceutically acceptable salts thereof,

4-Amino-5-bromo-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-tf] pyrimidine or a pharmaceutically acceptable salt thereof.

The nucleoside compounds of the present invention are useful as positive-sense single-stranded RNA-dependent viral RNA polymerase inhibitors, positive-sense single-stranded RNA-dependent RNA virus replication inhibitors, and / or for the treatment of positive-sense single-stranded RNA-dependent RNA virus infection. The positive-sense RNA dependent RNA virus is either the Flaviviridae virus or the Picornaviridae virus. In a subclass of this class, the Picornaviridae virus is rhinitis virus, polio virus, or hepatitis A virus. In a second subclass of this class, the Flaviviridae virus is a virus selected from the group consisting of hepatitis C virus, yellow fever virus, and Dengue virus. , West Nile virus, Japanese meningitis virus, Banzi virus, and Viral Diarrhea Virus (BVDV). In a subclass of this class, the Flaviviridae virus is hepatitis C.

The RNA-dependent RNA polymerase is a positive-sense, single-stranded, RNA-dependent RNA viral polymerase. Such an RNA polymerase is Flaviviridae virus polymerase or Picornaviridae virus polymerase. Picornaviridae virus polymerase is rhinitis virus polymerase, poliovirus polymerase, or hepatitis A polymerase. In the second subclass, Flaviviridae virus polymerase is selected from the group consisting of hepatitis C virus polymerase, yellow fever virus polymerase, Dengue virus polymerase, West Nile virus polymerase, Japanese meningitis virus polymerase, Banzi virus polymerase, and diarrhea virus polymerase. viral (BVDV).

The replication of an RNA-dependent RNA virus is the replication of a positive-sense RNA-dependent RNA virus. The replication of the positive-sense, single-stranded RNA-dependent RNA virus is either the replication of the Flaviviridae virus or the replication of the Picornaviridae virus. Picornaviridae virus replication is rhinitis virus replication, polio virus replication, or hepatitis A virus replication. Flaviviridae virus replication is selected from the group consisting of hepatitis C virus replication, yellow fever virus replication, Deng replication, West Nile virus replication, Japanese meningitis virus replication, Banzi virus replication, and viral diarrhea virus (BVDV) replication .

An RNA-dependent RNA virus infection is infection by a positive-sense, single-stranded RNA RNA virus. Such infection is either a Flaviviridae virus infection or a Picornaviridae virus infection. A Picornaviridae virus infection is a rhinitis infection, polio infection, or hepatitis A infection. In the second subclass of this class, Flaviviridae virus infection is selected from the group consisting of hepatitis C infection, yellow fever virus infection, Dengue virus infection, West Nile virus infection, Japanese meningitis virus infection PL 207 405 B1 semen, Japanese meningitis virus infection, Banzi and viral diarrhea virus (BVDV) infection.

As used herein, the term alkylamino refers to straight and branched chain alkylamines having the specified number of carbon atoms, e.g., methylamino, ethylamino, isopropylamino, t-butylamino.

The term halogen includes the halogen atoms of fluorine, chlorine, bromine and iodine.

The term 5'-triphosphate refers to a triphosphoric acid ester derivative of the 5'-hydroxyl group of a compound of the present invention, having the following structural formula III:

<img file="PL207405B1_D0002.tif" />

in which R.¹, R², R³, R⁵, R⁸, R⁹, R¹⁰ and r¹¹ have meanings as defined above, and R.⁴ and r⁷are H. The compounds of the present invention also include the pharmaceutically acceptable salts of the triphosphate esters and the pharmaceutically acceptable salts of the 5'-monophosphate and 5'-diphosphate derivatives of structures IV and V.

<img file="PL207405B1_D0003.tif" />

Also within the scope of the invention is a pharmaceutical composition which comprises a compound according to the present invention as an active ingredient and a pharmaceutically acceptable carrier.

The composition is useful for inhibiting RNA-dependent viral RNA polymer , inhibiting RNA-dependent RNA virus replication, and / or treating an infection with an RNA-dependent RNA virus.

The composition of the invention preferably inhibits NS5B polymerase, HCV replication, and is preferably useful for the treatment of HCV infection.

The invention also relates to the use of a derivative of the invention for the manufacture of a medicament for inhibiting RNA-dependent RNA viral polymerase or inhibiting replication of an RNA-dependent RNA virus in a mammal, for the manufacture of a medicament for the treatment of an RNA-dependent virus infection in a mammal, especially hepatitis C virus.

The composition of the invention or the medicament produced according to the use of the invention may be administered in combination with one or more agents useful for the treatment of HCV infection. Such anti-HCV agents include, but are not limited to, ribavirin,

PL 207 405 B1 levovirin, viramidine, thymosin alfa-1, interferon-α, pegylated interferon-α (peginterferon-α), combination of interferon-α and ribavirin, combination of peginterferon-α and ribavirin, combination of interferon-α and levovirin, and combination of peginterferon -α and levovirine. Interferon-α includes, but is not limited to, recombinant interferon-a2a (such as Roferon interferon available from Hoffmann LaRoche, Nutley, NJ), pegylated interferon-a2a (Pegasys ™), interferon-a2b (such as Intron-A interferon available from Schering Corp ., Kenilworth, NJ), pegylated interferon-α2b (PegIntron ™), recombinant consensus interferon (such as alphacon-1 interferon) and purified interferon-α. Amgen's recombinant consensus interferon is called Infergen®. Levovirin is the L-enantiomer of ribavirin, which has immunomodulatory activity similar to that of ribavirin. Viramidine is an analog of ribavirin disclosed in WO 01/60379 (ICN Pharmaceuticals). The individual components of the combination can be administered separately at different times during the therapy, or concurrently in separate or single combination forms, according to concurrent or alternating treatment regimens. The term administration should be interpreted accordingly. It should be understood that the range of combinations of the compounds or compositions of the present invention with other agents useful for treating HCV infection includes substantially any combination with any of the pharmaceutical compositions for treating HCV infection. When a compound of the present invention, or pharmaceutically acceptable salts thereof, is used in combination with a second anti-HCV therapeutic agent, the dose of each compound may be either the same as or different from the dose when the compound is used alone.

For the treatment of HCV infection, the compositions of the present invention may also be administered in combination with an HCV NS3 serine protease inhibitor agent. HCV NS3 serine protease is an essential viral enzyme and has been described as an excellent target for inhibiting HCV replication. HCV NS3 serine protease inhibitors, both substrate and non-substrate, are disclosed in WO 98/22496, WO 98/46630, WO 99/07733, WO 99/07734, WO 99/38888, WO 99/50230, WO 99/64442, WO 00/09543, WO 00/59929, and GB-2337262. The HCV NS3 protease as a target for the development of HCV replication inhibitors and for the treatment of HCV infection is discussed in BW Dymock, Emerging therapies for hepatitis C virus infection, Emerging Drugs, 6: 13-42 (2001).

Ribavirin, levovirin, and viramidine can exert their anti-HCV activities by modulating the intracellular pool of guanine nucleotides by inhibiting the intracellular enzyme inosine monophosphate dehydrogenase (IMPDH). IMPDH is a rate-limiting enzyme in the de novo biosynthetic pathway of guanine nucleotide biosynthesis. Ribavirin is readily phosphorylated intracellularly and the monophosphate derivative is an inhibitor of IMPDH. Thus, inhibition of IMPDH represents another useful target for the discovery of inhibitors of HCV replication. Accordingly, the compounds of the present invention may also be administered in combination with an IMPDH inhibitor such as VX-497, which is disclosed in WO 97/41211 and WO 01/00622 (Vertex); another IMPDH inhibitor, such as that disclosed in WO 00/25780 (Bristol-Myers Squibb); or mycophenolate mofetil [see AC Allison and EM Eugui, Agents Action, 44 (Suppl.): 165 (1993).

For the treatment of HCV infection, the compounds of the present invention may also be administered in combination with the antiviral agent, amantadine (1-aminoadamantane) [for a comprehensive description of this agent, see J. Kirschbaum, Anal. Profiles Drug Subs. 12: 1-36 (1983)].

By pharmaceutically acceptable is meant that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The compositions of the invention include compositions suitable for oral, rectal, topical, parenteral (including subcutaneous, intramuscular and intravenous), ocular (ophthalmic), pulmonary (nasal or buccal inhalation) or intranasal administration, although in each particular case the most appropriate route of administration is will depend on the nature of the active ingredient and the severity of the disease. They may conveniently be presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy.

In practical use, the compounds of structural formula II as the active ingredient can be combined in intimate admixture with a pharmaceutical carrier by conventional pharmaceutical compounding techniques. The carrier may take a wide variety of forms depending on the form of drug desired for administration, e.g., oral or parenteral (including intravenous). Any conventional pharmaceutical medium such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives,

Dyes and the like. For solid oral preparations, such as, for example, powders, hard and soft capsules and tablets, carriers such as starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents and the like can be used. Oral solids are preferred over liquid oral formulations.

Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, which obviously employs solid pharmaceutical carriers. If desired, tablets may be coated by standard aqueous or non-aqueous techniques. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of active compound in these compositions may, of course, be varied and may conveniently range between about 2% and about 60% by weight per unit. The amount of the active ingredient in such a composition is such that an effective dosage will be obtained. The active compounds can also be administered intranasally, for example in the form of liquid drops or spray.

The tablets, pills, capsules and the like may also contain a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium hydrogen phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid; a lubricant such as magnesium stearate, and a sweetening agent such as sucrose, lactose or saccharin. When the dosage form is a capsule, it can, in addition to materials of the above type, contain a liquid carrier such as a fatty oil.

Various other materials may be present as coatings or to modify the physical form of the dosage unit. For example, tablets may be coated with shellac and / or sugar. A syrup or elixir may contain, in addition to the active ingredient, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a color and a flavoring such as cherry or orange flavor.

The compounds of structural formula II can also be administered parenterally. Solutions or suspensions of these active compounds can be prepared in water suitably mixed with a surfactant such as hydroxypropyl cellulose. Dispersions can be made in glycerin, liquid polyethylene glycols, and mixtures thereof in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile aqueous solutions or dispersions. In any event, the dosage form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerin and liquid polyethylene glycols), suitable mixtures thereof, and vegetable oils.

Any suitable route of administration may be employed to provide a mammal, especially a human, with an effective dose of a compound of the present invention. For example, oral, rectal, topical, parenteral, ocular, pulmonary, intranasal and the like can be used. The drug forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, sprays and the like. Preferably, compounds of structural formula II are administered orally.

For oral administration to humans, the dosage range is 0.01 to 1000 mg / kg body weight in divided doses. In one embodiment, the dosage range is 0.1 to 100 mg / kg body weight in divided doses. In another embodiment, the dosage range is 0.5 to 20 mg / kg body weight in divided doses. Compositions for oral administration are preferably in the form of tablets or capsules containing 1.0 to 1000 milligrams of active ingredient, especially 1.5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500. , 600, 750, 800, 900, and 1000 milligrams of active ingredient for symptomatic dose adjustment for the treated patient.

The effective dose of the active ingredient employed may vary depending upon the particular compound employed, the mode of administration, the condition being treated and the severity of the condition being treated. Such dosages can be readily adjusted by one skilled in the art. This dosing regimen can be adjusted to obtain the optimal therapeutic response.

The compounds of the present invention contain one or more asymmetric centers and can therefore occur as racemates and racemic mixtures, single enantiomers, mixtures of diastereoisomers and single diastereoisomers. The present invention thus includes nucleoside compounds having the β-D stereochemistry of a five-membered furanose ring,

As depicted in the following structural formula, that is, nucleoside compounds wherein the substituents on C-1 and C-4 of the five-membered furanose ring have the β stereochemical configuration (upward orientation as indicated by the bold bar).

<img file="PL207405B1_D0004.tif" />

Some of the compounds described herein contain olefinic double bonds, and unless otherwise indicated, include both E and Z geometric isomers.

Some of the compounds described herein may exist as tautomers, such as keto-enol tautomers. Compounds of structural formula II include single tautomers and mixtures thereof. An example of keto-enol tautomers falling within the scope of the compounds of the present invention is illustrated below:

<img file="PL207405B1_D0005.tif" />

Compounds of structural formula II can be separated into their individual diastereoisomers, for example by fractional crystallization from a suitable solvent, for example methanol or ethyl acetate or mixtures thereof, or by chiral chromatography using an optically active stationary phase.

Alternatively, each of the stereoisomers of a compound of structural formula II can be obtained by stereospecific synthesis using optically pure starting materials or reagents of known configuration.

The stereochemistry of the substituents at the C-2 and C-3 positions of the furanose ring of the compounds of the present invention of structural formula II is indicated by serpentine lines, which means that R¹, R², R³ and r⁴ they may independently be of the α (substituent down) or β (upward substituent) configuration. The stereochemistry notation in bold lines at the C-1 and C-4 positions of the furanose ring indicates that the substituent has the β configuration (substituent up).

<img file="PL207405B1_D0006.tif" />

PL 207 405 B1

The compounds of the present invention may be administered in the form of a pharmaceutically acceptable salt. The term pharmaceutically acceptable salt refers to salts prepared from pharmaceutically acceptable non-toxic inorganic or organic bases and inorganic or organic acids. The term pharmaceutically acceptable salt for a basic compound refers to non-toxic salts of the compounds of the invention which are generally prepared by reacting the free base with an appropriate organic or inorganic acid. Representative salts of the basic compounds of the present invention include the following non-limiting salts: acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetane, edisylate, estolate, esylate, fumarate, gluceptan, gluconate, glutamate, hydrocarbamate, hexylarsorbate hydrobromide, hydrochloride, hydroxylnaphthoate, iodide, isothioate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methyl nitrate, methyl sulfate, mucate, napsylate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pamoate (pamoate), palmitate, pantothenate, phosphate / diphosphate, polygalacturonate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, tartrate, and theoclanate. In addition, when the compounds of the invention have an acidic moiety, suitable pharmaceutically acceptable salts thereof include, but are not limited to, salts made from inorganic bases, including aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganese, manganese, potassium, sodium, zinc and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium and sodium salts. Salts prepared from pharmaceutically acceptable non-toxic organic bases include salts of primary, secondary and tertiary amines, cyclic amines, and basic ion exchange resins such as arginine, betaine, caffeine, choline, N, N-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylamino -ethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

Also, when a carboxylic acid (-COOH) group or an alcohol group is present in the compounds of the present invention, pharmaceutically acceptable esters of carboxylic derivatives such as methyl, ethyl or pivaloyloxymethyl, or acyl alcohol derivatives such as acetate or maleate may be used. These include ester and acyl groups known in the art to modify solubility or hydrolysis characteristics for use in sustained release or prodrug formulations.

Preparation of nucleoside derivatives according to the invention

The nucleoside derivatives of the present invention can be prepared according to the following synthetic methods well known in the art of nucleoside and nucleotide chemistry. A description of the synthetic methods used to prepare the compounds of the present invention can be found in the following: Chemistry of Nucleosides and Nucleotides, LB Townsend, ed., Vols. 1-3, Plenum Press, 1988.

A representative general method for preparing compounds of the present invention is outlined in Scheme 1 below. This scheme illustrates the synthesis of compounds of the present invention having Structural Formula 1-7 wherein the furanose ring has the β-D-ribo configuration. The substrate is a 3,5-bis-O-protected alkylfuranoside, such as methylfuranoside, of structural formula 1-1. The C-2 hydroxyl group is then oxidized with a suitable oxidant such as chromium trioxide or chromate, Dess-Martin periodinate, or by Swern oxidation to provide a C-2 ketone of structural formula 12. Addition to a 1-2 carbonyl double bond of a Grignard reagent such as an alkyl, alkenyl, or alkynylmagnesium halide (e.g. MeMgBr, EtMgBr, vinylMgBr, allylMgBr, and ethynylMgBr) or an alkyl, alkenyl or alkynyl lithium such as MeLi, in a suitable organic solvent such as tetrahydrofuran, diethyl ether, and the like, gives a C-2 tertiary alcohol of structural formula 1-3. A good leaving group (such as Cl, Br and I) is then introduced at the C-1 position of the (anomeric) furanose sugar derivative by treating furanoside of formula 1-3 with hydrogen halide in a suitable organic solvent such as hydrogen bromide in acetic acid to give an intermediate furanosyl halide 1-4. A sulfonate at the C-1 position, such as a methanesulfonate (MeSO2O-), trifluoromethanesulfonate (CF3SO2O-), or p-toluenesulfonate (-OTs) may also serve as a useful leaving group in the subsequent glycosidic (nucleoside) bond formation reaction. A nucleoside bond is constructed by treating an intermediate of structural formula 1-4 with a metal salt (such as lithium),

Sodium or potassium) of an appropriately substituted 1H-pyrrolo [2,3-d] pyrimidine 1-5, such as an appropriately substituted 4-halo-1H-pyrrolo [2,3-d] pyrimidine, which can be generated in situ by treatment with an alkali metal hydride (such as sodium hydride), an alkali metal hydroxide (such as potassium hydroxide), an alkali metal carbonate (such as potassium carbonate), or alkali metal hexamethyldisilazide (such as NaHMDS) in a suitable anhydrous organic solvent such as acetonitrile, tetrahydrofuran, 1-methyl-2-pyrrolidinone, or N, N-dimethylformamide (DMF). The displacement reaction can be catalyzed using a phase transfer catalyst such as TDA-1 or triethylbenzylammonium chloride in a two-phase (solid-liquid or liquid-liquid) system. Subsequently, any protective groups present on the protected nucleoside of structural formula 1-6 are cleaved using known deprotection methods such as those described in TW Greene and PGM Wuts, Protective Groups in Organic Synthesis, 3.^rd ed., John Wiley &amp; Sons, 1999. Optional introduction of an amino group at the 4-position of the pyrrolo [2,3-d] pyrimidine core is accomplished by treating the 4-halogen intermediate 1-6 with a suitable amine such as alcoholic ammonia or liquid ammonia. ammonia to form a primary amino group at the C-4 position (-NH2), an alkylamine to form a secondary amino group (-NHR), or a dialkylamine to form a tertiary amino group (-NRR '). Compound 7H-pyrrolo [2,3-d] pyrimidin-4 (3H) one can be obtained by hydrolyzing compound 1-6 with an aqueous base such as aqueous sodium hydroxide. Alcoholysis (e.g., methanolysis) of 1-6 gives an alkoxide at the C-4 position (-OR), while treatment with an alkyl mercaptide gives an alkylthio derivative at the C-4 position (-SR). Further chemical manipulations may be required to obtain the desired compounds of the present invention well known to those of ordinary skill in the art of organic / medical chemistry.

Scheme 1

<img file="PL207405B1_D0007.tif" />

<img file="PL207405B1_D0008.tif" />

PL 207 405 B1

In the following examples, reference is made to literature publications that contain details of the preparation of the final compounds or intermediates used in the preparation of the final compounds of the present invention. The nucleoside compounds of the present invention were prepared according to the procedures detailed in the examples below. The examples are not intended to limit the scope of the invention in any way and should be interpreted as such. It will be apparent to those skilled in the art of nucleoside and nucleotide synthesis that known variations on the conditions and methods set forth in the following preparative procedures can be used to prepare these and other compounds of the present invention. All temperatures are in degrees Celsius.

EXAMPLE 1

4-Amino-7- (2-C-methyl-eD-arabinofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine

<img file="PL207405B1_D0009.tif" />

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To chromium trioxide (1.57 g, 1.57 mmol) in dichloromethane (DCM) (10 ml) at 0 ° C was added acetic anhydride (145 mg, 1.41 mmol) and then pyridine (245 mg, 3.10 mmoles). The mixture was stirred for 15 min, then a solution of 7- [3,5-O- [1,1,3,3-tetrakis (1-methylethyl) -1,3-disiloxanediyl] -eD-ribofuranosyl] -7H-pyrrolo [ 2,3-d] -pyrimidin-4-amine [for preparation see J. Am. Chem. Soc. 105: 4059 (1983)] (508 mg, 1.00 mmol) in DCM (3 ml). The resulting solution was stirred for 2 h and then poured into ethyl acetate (10 mL), and then filtered through silica gel, using ethyl acetate as eluent. The combined filtrates were evaporated in vacuo, taken up in diethyl ether / THF (1: 1) (20 ml), cooled to -78 ° C and methylmagnesium bromide (3M, in THF) (3.30 ml, 10 mmol) was added dropwise. The mixture was stirred at -78 ° C for 10 min, then allowed to come to room temperature and saturated aqueous ammonium chloride solution (10 ml) was added and extracted with DCM (20 ml). The organic phase was evaporated in vacuo and the crude product was purified over silica gel using 5% methanol in dichloromethane as the eluent. Product containing fractions were combined and evaporated in vacuo. The resulting oil was taken up in THF (5 ml) and tetrabutylammonium fluoride (TBAF) was added on silica gel (1.1 mmol / g on silica gel) (156 mg). The mixture was stirred at room temperature for 30 minutes, filtered, and evaporated in vacuo. The crude product was purified over silica gel using 10% methanol in dichloromethane as the eluent. Product containing fractions were combined and evaporated in vacuo to give the desired compound (49 mg) as a colorless solid.

¹H NMR (DMSO-d6): δ 1.08 (s, 3H), 3.67 (m, 2H), 3.74 (m, 1H), 3.83 (m, 1H), 5.19 (m , 1H), 5.23 (m, 1H), 5.48 (m, 1H), 6.08 (1H, s), 6.50 (m, 1H), 6.93 (bs, 2H), 7 . 33 (m, 1H), 8.02 (s, 1H).

EXAMPLE 2

4-Amino-7- (2-C-methyl-eD-ribofuranosyl] -7H-pyrrolo [2,3-d] pyrimidine

<img file="PL207405B1_D0010.tif" />

Stage A: 3,5-Bis-O- (2,4-dichlorophenylmethyl) -1-O-methyl-αD-ribofuranose

A mixture of 2-O-acetyl-3,5-bis-O- (2,4-dichlorophenylmethyl) -1-O-methyl-αD-ribofuranose [for preparation see Helv. Chim. Acta 78: 486 (1995)] (52.4 g, 0.10 mol) in a methanolic solution

PL 207 405 B1

K2CO3 (500 mL, saturated at RT) was stirred at room temperature for 45 minutes and then concentrated under reduced pressure. The oily residue was suspended in CH2Cl2 (500 ml), washed with water (300 ml + 5 x 200 ml) and brine (200 ml), dried (Na2SO4), filtered and concentrated to give the title compound (49.0 g) as a colorless oil which was used without further purification in Step B below.

¹H NMR (DMSO-d6): δ 3.28 (s, 3H, OCH3), 3.53 (d, 2H, J5.4 = 4.5Hz, H-5a, H-5b), 3.72 ( dd, 1H, J3.4 = 3.6Hz, J3.2 = 6.6Hz, H-3), 3.99 (ddd, 1H, d J2.1 = 4.5Hz, J2, OH-2 = 9.6 Hz, H-2), 4.07 (m, 1H, H-4), 4.50 (s, 2H, CH2Ph), 4.52, 4.60 (2d, 2H, Jgem = 13 , 6 Hz, CH2Ph), 4.54 (d, 1H, OH-2), 4.75 (d, 1H, H-1), 7.32-7.45, 7.52-7.57 (2m , 10H, 2Ph).

¹³C NMR (DMSO-d6): δ 55.40, 69.05, 69.74, 71.29, 72.02, 78.41, 81.45, 103.44, 127.83, 127.95,

129,05, 129,28, 131,27, 131,30, 133,22, 133,26, 5 133,55, 133,67, 135,45, 135,92.

Step B: 3,5-Bis-O- (2,4-dichlorophenylmethyl) -1-O-methyl-αD-erythropentofuranos-2-ulose

To ice-cooled Dess-Martin periodinate (50.0 g, 118 mmol) in anhydrous CH2Cl2 (350 ml) under argon (Ar) was added dropwise over 0.5 h a solution of the compound from Step A (36.2 g, 75 mmol) in anhydrous CH2Cl2 (200 ml). The reaction mixture was stirred at 0 ° C for 0.5 h and then at room temperature for 3 days. The mixture was diluted with anhydrous Et2O (600 mL) and poured into an ice-cooled Na mixture₂S.₂ABOUT₃^ 5H₂O (180 g) in a saturated aqueous NaHCO3 solution (1400 ml). The layers were separated, and the organic layer was washed with sat. Aq. NaHCO3 (600 ml), water (800 ml) and brine (600 ml), dried (MgSO4), filtered and evaporated to give the title compound (34.2 g) as a colorless oil which was used without further purification in Step C below.

¹H NMR (CDCl3): δ 3.50 (s, 3H, OCH3), 3.79 (dd, 1H, J5a, 5b = 11.3Hz, J5a, 4 = 3.5Hz, H-5a), 3 , 94 (dd, 1H, J5b, 4 = 2.3Hz, H-5b), 4.20 (dd, 1H, J3.1 = 1.3Hz, J3.4 = 8.4Hz, H-3 ), 4.37 (ddd, 1H, H-4), 4.58, 4.59 (2d, 2H, Jgem = 13.0 Hz, CH2Ph), 4.87 (d, 1H, H-1), 4.78, 5.03 (2d, 2H, Jgem = 12.5Hz, CH2Ph), 7.19-7.25, 7.31-7.42 (2m, 10H, 2Ph).

¹³C NMR (DMSO-d6): δ 55.72, 59.41, 59.81, 69.98, 77.49, 78.00, 98.54, 127.99, 128.05, 129.33, 129 , 38, 131.35, 131.72, 133.51, 133.53, 133.85, 133.97, 134.72, 135.32, 208.21.

Step C: 3,5-Bis-O- (2,4-dichlorophenylmethyl) -2-C-methyl-1-O-methyl-αD-ribofuranose

To a solution of MeMgBr in anhydrous Et₂O (0.48 M, 300 mL) at -55 ° C, a solution of the compound from Step B (17.40 g, 36.2 mmol) in anhydrous Et2O (125 mL) was added dropwise. The reaction mixture was allowed to warm to -30 ° C and stirred for 7 h at -30 ° C to -15 ° C, then poured into ice water (500 ml) and the mixture was vigorously stirred at room temperature for 0.5 h. The mixture was filtered. through a pad of celite (10 x 5 cm) that was thoroughly washed with Et2O. The organic layer was dried (MgSO4), filtered and concentrated. The residue was dissolved in hexanes (-30 ml), applied to a silica gel column (10 x 7 cm, pre-packed with hexanes) and eluted with hexanes and hexanes / EtOAc (9/1) to give the title compound (16.7 g) in the form of a colorless syrup.

¹H NMR (CDCl3): δ 1.35 (d, 3H, JMe, OH = 0.9Hz, 2C-Me), 3.33 (q, 1H, OH), 3.41 (d, 1H, J3, 4 = 3.3Hz), 3.46 (s, 3H, OCH3), 3.55 (d, 2H, J5.4 = 3.7Hz, H-5a, H-5b), 4.18 (apparent q, 1H, H-4), 4.52 (s, 1H, H-1), 4.60 (s, 2H, CH2Ph), 4.63, 4.81 (2d, 2H, Jgem = 13.2 Hz, CH2Ph), 7.19-7.26, 7.34-7.43 (2m, 10H, 2Ph).

¹³C NMR (CDCl3): δ 24.88, 55.45, 69.95, 70.24, 70.88, 77.06, 82.18, 83.01, 107.63, 127.32, 129.36 , 130.01, 130.32, 133.68, 133.78, 134.13, 134.18, 134.45, 134.58.

Step D: 4-Chloro-7- [3,5-bis-O- (2,4-dichlorophenylmethyl] -2-C-methyl-eD-ribofuranosyl] -7H-pyrrolo [2,3-cflpyrimidine

To a solution of the compound from step C (9.42 g, 19 mmol) in dry dichloromethane (285 ml) at 0 ° C, HBr (5.7M acetic acid solution, 20 ml, 114 mmol) was added dropwise. The resulting solution was stirred at 0 ° C for 1 h and then at room temperature for 3 h, evaporated in vacuo and co-evaporated with anhydrous toluene (3 x 40 ml). The oily residue was dissolved in anhydrous acetonitrile (50 ml) and added to a solution of 4-chloro-1H-pyrrolo [2,3-d] pyrimidine sodium salt in acetonitrile [generated in situ from 4-chloro-1H-pyrrolo [2,3- d] pyrimidines [for preparation see: J. Chem. Soc: 131 (1960)] (8.76 g, 57 mmol) in anhydrous acetonitrile (1000 ml) and NaH (60% in mineral oil, 2.28 g, 57 mmol), after 4 hours of vigorous stirring at room temperature ]. The combined mixture was stirred at room temperature for 24 h, and then evaporated to dryness. The residue was suspended in water (250 ml) and extracted with EtOAc (2 x 500 ml). The combined extracts were washed with brine (300 mL, dried over Na2SO4, filtered and evaporated.

The product was purified on a silica gel column (10 x 10 cm) using ethyl acetate / hexane (1: 3 and 1: 2) as eluent. Product containing fractions were combined and evaporated in vacuo to give the desired product (5.05 g) as a colorless foam.

¹H NMR (CDCl₃): δ 0.93 (s, 3H, CH₃), 3.09 (s, 1H, OH), 3.78 (dd, 1H, J₅,_s= 10.9 Hz, J.₅'₄ = 2.5 Hz, H-5 ') 3.99 (dd, 1H, J₅„₄ = 2.2 Hz, H-5), 4.23-4.34 (m, 2H, H-3 ', H-4'), 4.63, 4.70 (2d, 2H, J_gem '= 12.7 Hz, CH2Ph), 4.71, 4.80 (2d, 2H, Jgem = 12.1Hz, CH2Ph), 6.54 (d, 1H, J5.6 = 3.8Hz, H -5), 7.23-7.44 (m, 10H, 2Ph).

¹³C NMR (CDCl3): δ 21.31, 69.10, 70.41, 70.77, 79.56, 80.41, 81.05, 91.11, 100.57, 118.21, 127.04 , 127.46, 127.57, 129.73, 129.77, 130.57, 130.99, 133.51, 133.99, 134.33, 134.38, 134.74, 135.21, 151 , 07, 151.15 152.47.

Step E: 4-Chloro-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-cf] -pyrimidine

Boron trichloride (1M in dichloromethane, 88 mL, 88 mmol) was added dropwise to a solution of the compound from Step D (5.42 g, 8.8 mmol) in dichloromethane (175 mL) at -78 ° C. The mixture was stirred at -78 ° C for 2.5 h, then at -30 ° C to -20 ° C for 3 h. The reaction was quenched by adding methanol / dichloromethane (1: 1) (90 ml) and the resulting mixture was stirred at -15 ° C for 30 min, then neutralized with an aqueous ammonia solution at 0 ° C and stirred at room temperature for 15 minutes. . The precipitate was filtered off and washed with CH2Cl2 / MeOH (1/1, 250 ml). The combined filtrates were evaporated, and the residue was purified by flash chromatography on silica gel, eluting with CH2Cl2 and a gradient of CH2Cl2: MeOH (99: 1, 98: 2, 95: 5 and 90:10) to give the desired compound (1.73 g) in colorless foam which after MeCN treatment turned to an amorphous precipitate.

¹H NMR (DMSO-d6): δ 0.64 (s, 3H, CH3), 3.61-3.71 (m, 1H, H-5 '), 3.79-3.88 (m, 1H, H-5), 3.89-4.01 (m, 2H, H-3 ', H-4'), 5.15-5.23 (m, 3H, 2'-OH, 3'-OH, 5'-OH), 6.24 (s, 1H, H-1 '), 6.72 (d, 1H, J5.6 = 3.8Hz, H-5), 8.13 (d, 1H, H-6), 8.65 (s, 1H, H-2).

¹³C NMR (DMSO-d6): δ 20.20, 59.95, 72.29, 79.37, 83.16, 91.53, 100.17, 117.63, 128.86, 151.13, 151 , 19, 151.45.

Step F: 4-Amino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-cf] -pyrimidine

To compound from Step E (1.54 g, 5.1 mmol) was added methanolic ammonia solution (saturated at 0 ° C; 150 ml). The mixture was heated in a stainless steel autoclave at 85 ° C for 14 h, then cooled and evaporated in vacuo. The crude mixture was purified on a silica gel column with CH2Cl2 / MeOH (9/1) as eluent to give the title compound as a colorless foam (0.8 g) which was separated as an amorphous solid after MeCN treatment. The amorphous precipitate was recrystallized from methanol / acetonitrile; mp 222 ° C.

¹H NMR (DMSO-d6): δ 0.62 (s, 3H, CH3), 3.57-3.67 (m, 1H, H-5 '), 3.75-3.97 (m, 3H, H-5, H-4 ', H-3'), 5.00 (s, 1H, 2'-OH), 5.04 (d, 1H, J₃oh3 '= 6.8 Hz, 3'-OH), 5.06 (t, 1H, J_5Oh₅'5 = 5.1 Hz, 5'-OH), 6.11 (s, 1H, H-1'), 6.54 (d, 1H, J5.6 = 3.6 Hz, H-5), 6.97 (br s, 2H, NH2), 7.44 (d, 1H, H-6), 8.02 (s, 1H, H-2).

¹³C NMR (DMSO-d6): δ 20.26, 60.42, 72.72, 79.30, 82.75, 91.20, 100.13, 103.08, 121.96, 150.37, 152 , 33, 158.15.

LC-MS: Found: 279.10 (MH⁺); calcd for C12H16N4O4 + H⁺: 279,11.

EXAMPLE 3

4-Amino-7- (2-C-ethyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine

<img file="PL207405B1_D0011.tif" />

Stage A: 3,5-Bis- (2,4-dichlorophenylmethyl) -2-C-ethyl-1-O-methyl-α-D-rvbofuranose

EtMgBr (3.0 M, 16.6 mL) was slowly added to diethyl ether (300 mL) at -78 ° C and then the compound from Step B of Example 2 (4.80 g, 10.0 mmol) in anhydrous was added dropwise. Et2O (100 mL). The reaction mixture was stirred at -78 ° C for 15 min, allowed to warm to -15 ° C and stirred for an additional 2 h, and then poured into a stirred mixture of water (300 ml).

PL 207 405 B1 and Et2O (600 mL). The organic phase was separated, dried (MgSO4), and evaporated in vacuo. The crude product was purified over silica gel using ethyl acetate / hexane (1: 2) as the eluent. Product containing fractions were combined and evaporated in vacuo to give the desired product (3.87 g) as a colorless oil.

Step B: 4-Chloro-7- [3,5-bis-O- (2,4-dichlorophenylmethyl) -2-C-ethyl-eD-ribofuranosyl] -7H-pyrrolo [2,3-dl-pyrimidine

To a solution of the compound from step A (1.02 mg, 2.0 mmol) in dichloromethane (40 ml) was added dropwise at 0 ° C HBr (5.7 M in acetic acid) (1.75 ml, 10.0 mmol). . The resulting solution was stirred at room temperature for 2 h, evaporated in vacuo and evaporated twice from toluene (10 ml). The oily residue was dissolved in acetonitrile (10 ml) and added to a vigorously stirred mixture of 4-chloro-1H-pyrrolo [2,3-d] pyrimidine (307 mg, 2.0 mmol), potassium hydroxide (337 mg, 6.0 mmol). ) and tris [2- (2-methoxyethoxy) ethyl] amine (130 mg, 0.4 mmol) in acetonitrile (10 ml). The resulting mixture was stirred at room temperature overnight, and then poured into a stirred mixture of saturated ammonium chloride (100 ml) and ethyl acetate (100 ml). The organic layer was separated, washed with brine (100 mL), dried over MgSO4, filtered, and evaporated in vacuo. The crude product was purified on silica gel using ethyl acetate / hexane (1: 2) as the eluent to give the desired product (307 mg) as a colorless foam.

Step C: 4-Chloro-7- (2-C-ethyl-eD-ribofuranosyl 1-7H-pyrrolo [2,3-d1-pyrimidine

To a solution of the compound from Step B (307 mg, 0.45 mmol) in dichloromethane (8 mL) was added boron trichloride (1M in dichloromethane) (4.50 mL, 4.50 mmol) at -78 ° C. The mixture was stirred at -78 ° C for 1 h, then at -10 ° C for 3 h. The reaction was quenched by adding methanol / dichloromethane (1: 1) (10 ml), stirred at -15 ° C for 30 h. minutes, and neutralized by adding aqueous ammonium hydroxide solution. The mixture was evaporated under reduced pressure and the resulting oil was purified over silica gel using methanol / dichloromethane (1: 9) as eluent. Product containing fractions were combined and evaporated in vacuo to give the desired product (112 mg) as a colorless foam.

Stage D: lAmino-ZTC-ethyl-eD-ribofuranosylolTH-pyrrololZS-dlpyrimidine

To compound from Step C (50 mg, 0.16 mmol) was added a saturated solution of ammonia in methanol (4 ml). The mixture was stirred at 75 ° C for 72 h in a closed container, cooled and evaporated in vacuo. The crude mixture was purified over silica gel using methanol / dichloromethane (1: 9) as eluent. Product containing fractions were combined and evaporated in vacuo to give the desired product (29 mg) as a colorless powder.

¹HNMR (200 MHz, DMSO-d6): δ 0.52 (t, 3H), 1.02 (m, 2H), 4.01-3.24 (m, 6H), 5.06 (m, 1H) , 6.01 (s, 1H), 6.51 (d, 1H), 6.95 (s br, 2H), 6.70 (d, 1H), 7.99 (s, 1H).

LC-MS: Found: 295.2 (M + H⁺); calcd for C13H18N4O4 + H⁺: 295,14.

EXAMPLE 4

2-Amino-7- (2-C-methyl-β-D-ribofuranosyl) -7H-pyrrolo [2,3-d1-pyrimidin-4 (3H) -one

<img file="PL207405B1_D0012.tif" />

Step A: 2-Amino-4-chloro-7- [3,5-bis-O- (2,4-dichlorophenylmethyl) -2-C-methyl-eD-ribofuranosylWH-pyrrole-O-rflpyrimidine

To an ice-cooled solution of the product of step C from Example 2 (1.27 g, 2.57 mmol) in CH 2 Cl 2 (30 ml), HBr (5.7M solution in acetic acid; 3 ml) was added dropwise. The reaction mixture was stirred at room temperature for 2 h, concentrated under reduced pressure and evaporated with toluene (2 x 15 ml). The resulting oil was dissolved in acetonitrile (MeCN) (15 ml) and added dropwise to a well-stirred mixture of 2-amino-4-chloro-7H-pyrrolo [2,3-d1-pyrimidine [for preparation see Heterocycles 35: 825 (1993)] (433 mg , 2.57 mmol), KOH (85%, powdered) (0.51 g, 7.7 mmol), tris- [2- (2-methoxyethoxy) ethylamine (165 0.51 mmol) in acetonitrile (30 ml) . The resulting mixture was stirred at room temperature for 1h, filtered and evaporated. The residue was purified on a silica gel column using 5/1, 3/1 and 2/1 hexanes / EtOAc as eluent to give the title compound as a colorless foam (0.65 g).

Step B: 2-Amino-4-chloro-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d-pyrimidine To a solution of the product from step A (630 mg, 1.0 mmol) in CH₂CI₂ (20 mL) Boron trichloride (1M in CH 2 Cl 2) (10 mL, 10 mmol) was added at -78 ° C. The mixture was stirred at -78 ° C for 2 h, then at -20 ° C for 2.5 h. The reaction was quenched with CH2Cl2 / MeOH (1: 1) (10 mL), stirred at -20 ° C for 0 hours. , 5 h, and neutralized at 0 ° C with aqueous ammonia solution. The precipitate was filtered off, washed with CH2Cl2 / MeOH (1: 1) and the combined filtrates were evaporated in vacuo. The residue was purified on a silica gel column with CH2Cl2 / MeOH 50/1 and 20/1 as eluent to give the title compound as a colorless foam (250 mg).

Step C: 2-Amino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d1-pyrimidin-4 (3H) -one Mixture of the product from Step B (90 mg, 0.3 mmol ) in aq. NaOH solution (2N, 9 ml) was heated at reflux for 5 h, then neutralized at 0 ° C with 2N aq. HCl solution and evaporated to dryness. Purification on a silica gel column with CHaCl2 / MeOH 5/1 as the eluent afforded the title compound as a white solid (70mg).

¹H NMR (200 MHz, CD3OD): δ 0.85 (s, 3H), 3.79 (m, 1H), 3.90-4.05 (m, 3H), 6.06 (s, 1H), 6 , 42 (d, J = 3.7 Hz, 1H), 7.05 (d, J = 3.7 Hz, 1H).

EXAMPLE 5

2-Amino-4-cyclopropylamino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d1-pyrimidine

<img file="PL207405B1_D0013.tif" />

2-Amino-4-chloro-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine solution (example 4, step B) (21 mg, 0.07 mmol) in cyclopropylamine (0.5 mL) was heated to 70^aboutC for two days, then evaporated to an oily residue and purified on a silica gel column with CH2Cl2 / MeOH 20/1 as eluent to give the title compound as a white solid (17mg).

¹H NMR (200 MHz, CD3CN): δ 0.61 (m, 2H), 0.81 (m, 2H), 0.85 (s, 3H), 2.83 (m, 1H), 3.74- 3.86 (m, 1H), 3.93-4.03 (m, 2H), 4.11 (d, J = 8.9Hz, 1H), 6.02 (s, 1H), 6.49 (d, J = 3.7 Hz, 1H), 7.00 (d, J = 3.7 Hz, 1H).

EXAMPLE 6

4-Amino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-cflpyrimidine-5-carbonitrile

<img file="PL207405B1_D0014.tif" />

This compound was prepared according to the procedures described by Y. Murai et al. in Heterocycles 33: 391-404 (1992).

EXAMPLE 7

4-Amino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d1-pyrimidine-5-carboxamide

PL 207 405 B1

<img file="PL207405B1_D0015.tif" />

This compound was prepared according to the procedures described by Y. Murai et al. in Heterocycles 33: 391-404 (1992).

EXAMPLE 8

5'-triphosphate derivatives

The nucleoside 5'-triphosphates of the present invention were prepared according to the general procedures described in Chem. Rev. 100: 2047 (2000).

EXAMPLE 9

Purification and purity analysis of 5'-triphosphate derivatives

The triphosphate derivatives were purified by anion exchange chromatography (AX) using a 30 x 100 mm Mono Q column (Pharmacia) with a 50 mM Tris buffering system, pH 8. Elution gradients were typically from 40 mM NaCl to 0.8 M NaCl in two volumes columns at a flow rate of 6.5 ml / min. The appropriate fractions from the anion exchange chromatography were collected and desalted by reverse phase (RP) chromatography using a Luna C18 250 x 21 mm column (Phenomenex) at a flow rate of 10 ml / min. Elution gradients were typically from 1% to 95% methanol over 14 minutes at a constant concentration of 5 mM triethylammonium acetate (TEAA).

Mass spectra of purified triphosphates were measured using HPLC with on-line mass spectrometry on a Hewlett-Packard (Palo Alto, CA) MSD 1100 instrument. A Phenomenex Luna (C18 (2)) column, 150 x 2 mm, plus a pre-column was used for RP HPLC. 30 x 2 mm, particle size 3 μm. A linear gradient of 0 to 50% (15 min) acetonitrile in 20 mM TEAA (triethylammonium acetate) pH 7 was run with mass spectroscopy detection in negative ionization mode. Nitrogen gas and a pneumatic nebulizer were used to generate the electron beam. A molecular weight range of 150-900 was tested. Molecular weights were determined using an HP Chemstation analysis kit.

The purity of the purified triphosphates was measured using analytical RP and AX HPLC. RP HPLC on Phenomonx Luna or Jupiter (250 x 4.6 mm) column, 5-L.im particle size was typically carried out with a 2-70% acetonitrile gradient in 15 minutes in 100 mM TEAA, pH 7. AX HPLC was performed on the column 1.6 x 5mm Mono Q (Pharmacia). The triphosphates were eluted with a gradient of 0 to 0.4 M NaCl with a constant concentration of 50 mM Tris, pH 8. The purity of the triphosphates is generally&gt; 80%.

EXAMPLE 10

5-monophosphate derivatives

The nucleoside 5'-monophosphates of the present invention were prepared according to the general procedures described in Tetrahedron Lett. 50: 5065 (1967).

EXAMPLE 11

Characterization of 5'-triphosphate derivatives by mass spectroscopy

The mass spectra of the 5'-triphosphate compounds of the present invention were measured as described in Example 10. The table below shows the calculated and experimental masses for representative 5'-triphosphates prepared according to the procedures of Example 8. The example numbers correspond to the 5'-triphosphate matrix compound.

<td>Example</td><td>Calculated</td><td>Found</td>

<td> 1</td><td> 520,0</td><td> 519,9</td>

<td> 2</td><td> 520,0</td><td> 520,0</td>

<td> 3</td><td> 534,0</td><td> 534,0</td>

<td> 4</td><td> 536,0</td><td> 536,0</td>

PL 207 405 B1

EXAMPLE 12

4-Amino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine 5'-monophosphate

<img file="PL207405B1_D0016.tif" />

To the compound of Step F of Example 2 (14 mg, 0.05 mmol) (dried by evaporation with pyridine and several times with toluene) was added trimethyl phosphate (0.5 ml). The mixture was stirred overnight in a closed container. It was then cooled to 0 ° C and phosphorus oxychloride (0.0070 mL, 0.075 mmol) was added via syringe. The mixture was stirred for 3 h at 0 ° C, then quenched by the addition of tetraethylammonium bicarbonate (TEAB) (1M) (0.5 ml) and water (5 ml). The reaction mixture was purified and analyzed according to the procedure described in Example 10.

Electrospray mass spectrum (ES-MS): Found: 359.2 (MH⁺), calcd for C12H17N4O7P- H⁺: 359,1.

EXAMPLE 13

4-Amino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine 5'-diphosphate

To the compound of Step F of Example 2 (56 mg, 0.20 mmol) (dried by evaporation together with pyridine and several times with toluene) was added trimethyl phosphate (stored above the sieves) (1.0 ml). The mixture was stirred overnight in a closed container. It was then cooled to 0 ° C and phosphorus oxychloride (0.023 mL, 0.25 mmol) was added via syringe. The mixture was stirred for 2 h at 0 ° C, then tributylamine (0.238 ml, 1.00 mmol) and tributylammonium phosphate (made from phosphoric acid and tributylamine in pyridine, then azeotropically evaporated with pyridine and acetonitrile several times) (1.0 mmol in 3.30 ml acetonitrile). The mixture was stirred for an additional 30 minutes at 0 ° C, then the sealed vial was opened and the reaction was quenched with the addition of TEAB (IM) (1.0 mL) and water (5 mL). The reaction mixture was purified and analyzed according to the procedure described in Example 9.

ES-MS: Found: 439.0 (MH⁺), calcd for C12H18N4O10P2-H⁺: 439,04.

EXAMPLE 14

4-Amino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine 5'-triphosphate

<img file="PL207405B1_D0017.tif" />

To the compound of Step F of Example 2 (20 mg, 0.07 mmol) (dried by evaporation with pyridine and several times with toluene) was added trimethyl phosphate (stored above the sieves) (0.4 ml). The mixture was stirred overnight in a closed container. It was then cooled to 0 ° C and phosphorus oxychloride (0.0070 mL, 0.075 mmol) was added via syringe. The mixture was stirred for 3 h at 0 ° C, then tributylamine (0.083 ml, 0.35 mmol), tributylammonium pyrophosphate (127 mg, 0.35 mmol) and acetonitrile (stored above the sieves) (0.25 ml) were added. The mixture was stirred for an additional 30 minutes at 0 ° C, then the sealed vial was opened and the reaction was quenched with the addition of TEAB (1M) (0.5 ml) and water (5 ml). Clean the reaction mixture 18

The PL 207 405 B1 was determined and analyzed according to the procedure described in Example 9. ES-MS: Found: 519.0 (MH⁺), calcd for C12H19N4O13P3- H⁺: 519,01.

EXAMPLE 15

7- (2-C-methyl-eD-rvbofuranozvlol-7H-pyrrolo [2₁3-dlpyrvmidvn-4 (3H) -one

<img file="PL207405B1_D0018.tif" />

Aqueous sodium hydroxide (1M) was added to the compound of step E of Example 2 (59 mg, 0.18 mmol). The mixture was refluxed for 1 h1 cooled, neutralized with aq. HCl (2M) and evaporated in vacuo. The residue was purified on silica gel using dichloromethane / methanol (4: 1) as eluent. Product containing fractions were combined and evaporated in vacuo to give the desired product (53 mg) as a colorless oil.

¹H NMR (CD3CN): δ 0170 (s1 3H) 1 3134-4115 (overlapping m1 7H) 1 6116 (s1 1H) 1 6157 (d1 316

Hz1 1H) 1 7137 (d1 316 Hz1 1H) 18183 (s1 1H).

EXAMPLE 16

4-Amino-5-chloro-7- (2-C-methyl-eD-rvbofuranosyl) -7H-pyrrolo [2-3-pyrimidine

<img file="PL207405B1_D0019.tif" />

N-Chlorosuccinimide (01075 g10155 mmol) in DMF (015 ml) was added dropwise to a pre-cooled (0 ° C) solution of the compound from step F of Example 2 (140 mg10150 mmol) in DMF (215 ml). The solution was stirred at room temperature for 1 h and quenched by adding methanol (4 mL) and evaporated in vacuo. The crude product was purified over silica gel using methanol / dichloromethane (1: 9) as the eluent. Product containing fractions were combined and evaporated in vacuo to give the desired product (55 mg) as a colorless solid.

¹H NMR (CD3CN): δ 0180 (s1 3H) 1 3165-4114 (overlapping m1 7H) 1.5197 (s br1 2H) 1 6117 (s1 1H) 17151 (s1 1H) 18116 (s1 1H).

ES-MS: Found: 31510 (M + H⁺) 1 calcd for C12H15ClN4O4 + H⁺: 315109.

EXAMPLE 17

4-Amino-5-bromo-7- (2-C-methyl-eD-rvbofuranosyl) -7H-pyrrolo [2,3-pyrimidine

<img file="PL207405B1_D0020.tif" />

To a cooled (0 ° C) solution of the compound from step F of Example 2 (28 mg10110 mmol) in DMF (015 ml) was added dropwise N-bromosuccinimide (01018 g11010 mmol) in DMF (015 ml). The solution was stirred at 0 ° C for 20 minutes and then at room temperature for 10 minutes. Reactions

The mixture was quenched by the addition of methanol (4 ml) and evaporated in vacuo. The crude product was purified over silica gel using methanol / dichloromethane (1: 9) as the eluent. Product containing fractions were combined and evaporated in vacuo to give the desired product (13.0 mg) as a colorless solid.

¹H NMR (CD3CN): δ 0.69 (s, 3H), 3.46-4.00 (overlapping m, 7H), 5.83 (s br, 2H), 6.06 (s, 1H), 7.45 (s, 1H), 8.05 (s, 1H).

ES-MS: Found: 359.1 (M + H⁺), calcd for C12H15BrN4O4 + H⁺: 359,04.

EXAMPLE 18

2-Amino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine

<img file="PL207405B1_D0021.tif" />

A mixture of 2-amino-4-chloro-7- (2-C-methyl-pD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine (example 4, step B) (20 mg, 0.07 mmol) in EtOH (1.0 mL), pyridine (0.1 mL) and 10% Pd / C (6 mg) under H 2 (atmospheric pressure) were stirred overnight at room temperature. The mixture was filtered through a pad of celite which was washed thoroughly with EtOH. The combined filtrates were evaporated and purified on a silica gel column with CH2Cl2 / MeOH 20/1 and 10/1 as eluent to give the title compound as a white solid (16mg).

¹H NMR (200 MHz, CD3OD): δ 0.86 (s, 3H, 2'C-Me), 3.82 (dd, J5.4 = 3.6 Hz, J5 ', 5' = 12.7 Hz , 1H, H-5 '), 3.94-4.03 (m, 2H, H-5', H-4 '), 4.10 (d, J3.4 = 8.8Hz, 1H, H -3 '), 6.02 (s, 1H, H-1'), 6.41 (d, J5.6 = 3.8Hz, 1H, H-5), 7.39 (d, 1H, H -6), 8.43 (s, 1H, H-4). ES MS: 281.4 (MH⁺).

EXAMPLE 19

2-Amino-5-methyl-7- (2-C, 2-O-dimethyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-cf] pyrimidin-4 (3H) -one

<img file="PL207405B1_D0022.tif" />

Step A: 2-Amino-4-chloro-7- [3,5-bis-O- [2,4-dichlorophenylmethyl) -2-C-methyl-eD-ribofuranosyl] -5-methyl-7H-pyrrolo [2 , 3-d] pyrimidine

To an ice-cooled solution of the product of step C from Example 2 (1.57 g, 3.15 mmol) in CH2Cl2 (50 ml), HBr (5.7M solution in acetic acid; 3.3 ml) was added dropwise. The reaction mixture was stirred at 0 ° C for 1 h and then at room temperature for 2 h, concentrated in vacuo and evaporated with toluene (2 x 20 ml). The resulting oil was dissolved in MeCN (20 ml) and added dropwise to a solution of 2-amino-4-chloro-5-methyl-1H-pyrrolo [2,3-d] pyrimidine sodium salt in acetonitrile [generated in situ from 2-amino-4 -chloro-5-methyl-1H-pyrrolo [2,3-d] pyrimidine [for preparation see Liebigs Ann. Chem. 1984: 708-721] (1.13 g, 6.2 mmol) in anhydrous acetonitrile (150 ml), and NaH (60% in mineral oil, 248 mg, 6.2 mmol), after 2 h of vigorous stirring at room]. The combined mixture was stirred at room temperature for 24 h and then evaporated to dryness. The residue was suspended in water (100 ml) and extracted with EtOAc (300 + 150 ml). The combined extracts were washed with brine (100 ml), dried over Na2SO4, filtered and evaporated. The crude product was purified over a silica gel column (5 x 7 cm), eluting with ethyl acetate / hexane (0 to 30% EtOAc, 5% step gradient). Product containing fractions were combined and evaporated in vacuo to give the desired product (0.96 g) as a colorless foam.

Step B: 2-Amino-4-chloro-7- [3,5-bis-O- (2,4-dichlorophenylmethyl] -2-C, 2-O-dimethyl-eD-ribofuranosyl] -5-methyl-7H -pyrrolo [2,3-cf] pyrimidine

NaH (60% in mineral oil, 29 mg) was added to an ice-cooled mixture of the product from step A (475 mg, 0.7 mmol) in THF (7 ml) and stirred at 0 ° C for 0.5 h.

Mel (48 μθ) was added and the reaction mixture was stirred at room temperature for 24 h. The reaction was quenched with MeOH and the mixture was evaporated. The crude product was purified on a silica gel column (5 x 3.5 cm) using hexane / ethyl acetate as eluent. (9/1, 7/1, 5/1 13/1). Product containing fractions were combined and evaporated to give the desired compound (200 mg) as a colorless foam.

Step C: 2-Amino-7- [3,5-bis-O- (2,4-dichlorophenylmethyl) -2-C, 2-O-dimethyl-eD-ribofuranosyl] -5-methyl-7H-pyrrolo [2 , 3-dj-pyrimidine-4 (3H) -one

A mixture of the product from step B (200 mg, 0.3 mmol) in 1,4-dioxane (15 ml) and aqueous NaOH (2N, 15 ml) in a pressure bottle was heated to 135 ° C overnight. The mixture was then cooled to 0 ° C, neutralized with 2N aqueous HCl and evaporated to dryness. The crude product was suspended in MeOH, filtered, and the precipitate was washed thoroughly with MeOH. The combined filtrates were concentrated, and the residue was purified on a silica gel column (5 x 5 cm) using CH2Cl2 / MeOH (40/1, 30/1 and 20/1) as eluent to give the desired compound (150 mg) as a colorless foam.

Step D: 2-Amino-5-methyl-7- (2-C, 2-O-dimethyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d1-pyrimidin-4 (3H) -one

A mixture of the product from Step C (64 mg, 0.1 mmol) in MeOH (5 ml) and Et₃N (0.2 ml) and 10% Pd / C (24 mg) were hydrogenated on a Parr apparatus at 50 psi at room temperature for 1.5 days, then filtered through a pad of celite which was thoroughly washed with MeOH . The combined filtrates were evaporated and the residue was purified on a silica gel column (3 x 4 cm) with CH2Cl2 / MeOH (30/1, 20/1) as eluent to give 2-amino-5-methyl-7- (5-O-benzyl-). 2-C, 2-O-dimethyl-pD-ribofuranosyl) -7H-pyrrolo [2,3-d1-pyrimidin-4 (3H) -one. The compound (37 mg) was further hydrogenated in EtOH (2 ml) with 10% Pd / C and under an atmospheric hydrogen pressure. After being stirred for 2 days at room temperature, the reaction mixture was filtered through celite, the filtrate was evaporated and the crude product was purified on a silica gel column (1 x 7 cm) with CH2Cl2 / MeOH (30/1, 20/1 and 10/1) as eluent to obtain the title compound (12mg) after lyophilization.

¹H NMR (200 MHz, CD3OD): δ 0.81 (s, 3H, 2'C-Me), 2.16 (d, JH-6, C5-Me = 1.3 Hz, 3H, C5-Me) , 3.41 (s, 3H, 2'-OMe), 3.67 (dd, J5'4 '= 3.4 Hz, J5'5 "= 12.6 Hz, 1H, H-5'), 3 , 81-3.91 (m, 3H, H-5, H-4 ', H-3'), 6.10 (s, 1H, H-1 '), 6.66 (d, 1H, H- 6). ES MS: 323.3 (MH)⁺

EXAMPLE 20

4-Amino-5-methyl-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d1-pyrimidine

<img file="PL207405B1_D0023.tif" />

Step A: 4-Chloro-7- [3,5-bis-O- (2,4-dichlorophenylmethyl) -2-C-methyl-8-D-ribofuranosyl] -5-methyl-7H-pyrrolo [2,3 -d1pyrimidine

To an ice-cooled solution of the product from step C of Example 2 (1.06 g, 2.1 mmol) in CH2Cl2 (30 ml), HBr (5.7M solution in acetic acid; 2.2 ml) was added dropwise. The reaction mixture was stirred at 0 ° C for 1 h and then at room temperature for 2 h, concentrated in vacuo and evaporated with toluene (2 x 15 ml). The resulting oil was dissolved in MeCN (10 ml) and added dropwise to a solution of 4-chloro-5-methyl-1H-pyrrolo [2,3-d-1-pyrimidine sodium salt in acetonitrile [generated in situ from 4-chloro-5-methyl-7H-pyrrolo [2,3-d] pyrimidines [for preparation see J. Med. Chem. 33: 1984 (1990) 1 (0.62 g, 3.7 mmol) in dry acetonitrile (70 ml), and NaH (60% in mineral oil, 148 mg, 3.7 mmol), after 2 h of vigorous stirring at room temperature 1. The combined mixture was stirred at room temperature for 24 h and then evaporated to dryness. The residue was suspended in water (100 ml) and extracted with EtOAc (250 + 100 ml). The combined extracts were washed with brine (50 ml), dried over Na2SO4, filtered and evaporated. The crude product was purified over a silica gel column (5 x 5 cm) eluting with a gradient of hexane / ethyl acetate (9/1, 5/1, 3/1). Product containing fractions were combined and evaporated in vacuo to give the desired product (0.87 g) as a colorless foam.

PL 207 405 B1

Step B: 4-Chloro-5-methyl-7- (2-C-methyl-β-D-ι'vbofuranosyl) -7H-pyrrolo [2-3-cflpyrimidine

Boron trichloride (1M in dichloromethane, 1.910 ml, 1.910 mmol) was added dropwise to a solution of the compound from step A (0187 g 1019 mmol) in dichloromethane (30 ml) at -78 ° C. The mixture was stirred at -78 ° C for 215 h1 then at -30 ° C to -20 ° C for 3 h. The reaction was quenched by adding methanol / dichloromethane (1: 1) (9 ml) and the resulting mixture was stirred at -15 ° C for 30 minutes and then neutralized with aqueous ammonia at 0 ° C and stirred at room temperature for 15 minutes. The precipitate was filtered off and washed with CH2Cl2 / MeOH (1/11 of 50 ml). The combined filtrates were evaporated and the residue was purified on a silica gel column (5 x 5 cm) using a gradient of CH2Cl2 and CH2Cl2 / MeOH (40/1 and 30/1) as eluent1 to give the desired compound (0122 g) as a colorless foam.

Step C: 4-Amino-5-methyl-7- (2-C-methyl-β-D-ribofuranosyl) -7H-pyrrolo [2l3-dl-pyrimidine

Methanolic ammonia solution (saturated at 0 ° C; 40 ml) was added to the compound of step B (012 g10164 mmol). The mixture was heated in a stainless steel autoclave at 100 ° C for 14 hl then cooled and evaporated in vacuo. The crude mixture was purified on a silica gel column (5 x 5 cm) using a CH2Cl2 / MeOH gradient (50/11 30/11 20/1) as the eluent to afford the title compound as a white solid (0112 g).

¹H NMR (DMSO-d6): δ 0160 (s1 3H1 2'C-Me) 1 2126 (s1 3H1 5C-Me) 1 3152-3161 (m1 1H1 H-5 ') 1 3170-3188 (m1 3H1 H-51 H-4'1 H-3 ') 1 5100 (s1 1H1 2'-OH) 1 4191-4199 (m1 3H1 2'-OH1 3'-OH1 5'-OH) 1 6104 (s1 1H1 H-1') 1 6148 (br s1 2H1 O NH2) 1 7112 (s1 1H1 H-6) 1 7194 (s1 1H1 H-2). ES MS: 29512 (MH⁺).

EXAMPLE 21

4-Amino-7- (2-C-methyl-β-D-ribofuranosyl) -7H-pyrrolo [2l3-dl-pyrimidine-5-carboxylic acid

<img file="PL207405B1_D0024.tif" />

The compound of Example 6 (01035 g10111 mmol) was dissolved in a mixture of aqueous ammonia solution (4 mL of 30% by weight and saturated methanolic ammonia solution (2 mL) 1 and a solution of H2O2 in water (2 mL of 35% by weight) was added. at room temperature for 18 h. Solvent was removed under reduced pressure1 and the resulting residue was purified by HPLC on a reverse phase column (Altech Altima C-181 10x 299mm1 A = water1 B = acetonitrile1 10 to 60% B in 50 min1 flow 2 ml / min) 1 to give the compound title (01015 g1 41%) as a white solid.

¹H NMR (CD3OD): δ 0185 (s1 3H1 Me) 1 3161 (m 1 1H) 1 3182 (m 1 1H) 3199-4186 (m 1 2H) 1 6126 (s 1 1H) 1 8110 (s 1 2H) 8122 (s 1 1H);

¹³C NMR (CD3OD): 201131 611371 731791 801421 841011 931001 1021661 1121071 1301071 1511401 1521741 1591121 169130.

HRMS (FAB) Calculated for C13H17N4O6⁺ 325111481 Found 32511143.

EXAMPLE 22

4-Amino-7- (2-C-vinyl-β-D-ribofuranosyl-7H-pyrrolo [2l3-dlpyrimidine

<img file="PL207405B1_D0025.tif" />

PL 207 405 B1

Stage A: 3,5-Bis-O- (2,4-dichlorophenylmethyl) -2-C-vinyl-1-O-methyl-α -D-ribofuranose

Cerium chloride heptahydrate (50 g, 134.2 mmol) was ground finely in a heated mortar and transferred to a round bottom flask equipped with a mechanical stirrer. The flask was heated under high vacuum at 160 ° C overnight. The vacuum was removed with argon and the flask was cooled to room temperature. Anhydrous THF (300 mL) was added to the flask via a cannula. The resulting suspension was stirred at room temperature for 4 h and then cooled to -78 ° C. Vinylmagnesium bromide (1M in THF, 120 ml, 120 mmol) was added and stirring was continued at -78 ° C for 2 h. A solution of 3,5-bis-O- (2,4-dichlorophenylmethyl) was added dropwise to this suspension with continued stirring. ) -1-O-methyl-αD-erythro-pentofuranose-2-ulose (14 g, 30 mmol) [from Example 2, Step B] in anhydrous THF (100 ml). The reaction was stirred at -78 ° C for 4 h. The reaction was quenched with saturated aqueous ammonium chloride solution and allowed to come to room temperature. The mixture was filtered through a pad of celite and the residue was washed with Et2O (2 x 500 mL). The organic layer was separated and the aqueous layer was extracted with Et2O (2 x 200 mL). The combined organic layers were dried over anhydrous Na2SO4 and concentrated to a viscous oil. The oil was purified by flash chromatography (SiO2, 10% EtOAc in hexanes). The title compound (6.7 g, 13.2 mmol) was obtained as a light yellow oil.

Step B: 4-Chloro-7- [3,5-bis-O- (2,4-dichlorophenylmethyl) -2-C-vinyl-eD-ribofuranosyl-7H-pyrimidine [2.3-cflpyrimidine

To a solution of the compound from step A (6.4 g, 12.6 mmol) in dry dichloromethane (150 ml) at -20 ° C, HBr (30% AcOH solution, 20 ml, 75.6 mmol) was added dropwise. The resulting solution was stirred between -10 ° C and 0 ° C for 4 h, evaporated in vacuo and co-evaporated with anhydrous toluene (3 x 40 ml). The oily residue was dissolved in dry acetonitrile (100 ml) and to the solution was added 4-chloro-1H-pyrrolo [2,3-d] pyrimidine sodium salt (5.8 g, 37.8 mmol) in acetonitrile (prepared in situ as described in example 2) at -20 ° C. The resulting mixture was allowed to come to room temperature and stirred at room temperature for 24 h. The mixture was then evaporated to dryness, taken up in water and extracted with EtOAc (2 x 300 ml). The combined extracts were dried over Na2SO4, filtered and evaporated. The crude mixture was purified by flash chromatography (SiO2, 10% EtOAc in hexanes) and the title compound isolated as a white foam (1.75 g).

Step C: 4-Amino-7- [3,5-bis-O- (2,4-dichlorophenylmethyl) -2-C-vinyl-eD-ribofuranosyl-7H-pyrimidine [2,3-cflpyrimidine

Compound from Step B (80, mg) was dissolved in a minimum amount of 1,4-dioxane and placed in a stainless steel bomb. The bomb was cooled to -78 ° C and liquid ammonia was added. The bomb was sealed and heated at 90 ° C for 24 h. The ammonia was allowed to evaporate and the residue was concentrated to give a white solid which was used in the next step without further purification.

Step D: 4-Amino-7- (2-C-vinyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-cflpyrimidine

Boron trichloride (1M in dichloromethane) was added dropwise to a solution of the compound from Step C (60 mg) in dichloromethane at -78 ° C. The mixture was stirred at -78 ° C for 2.5 h, then at -30 ° C to -20 ° C for 3 h. The reaction was quenched by adding methanol / dichloromethane (1: 1) and the resulting mixture was stirred at - 15 ° C for 0.5 h, then neutralized with an aqueous ammonia solution at 0 ° C and stirred at room temperature for 15 min. The precipitate was filtered off and washed with methanol / dichloromethane (1: 1). The combined filtrates were evaporated and the residue was purified by flash chromatography (SiO2, 10% methanol in EtOAc containing 0.1% triethylamine). Product containing fractions were evaporated to afford the title compound as a white solid (10mg).

¹H NMR (DMSO-d6): δ 3.6 (m, 1H, H-5 '), 3.8 (m, 1H, H-5), 3.9 (md, 1H, H-4' ), 4.3 (t, 1H, H-3 '), 4.8-5.3 (m, 6H, CH = CH2, 2'-OH, 3'-OH, 5'-OH) 6.12 (s, 1H, H-1 '), 6.59 (d, 1H, H-5), 7.1 (br s, 1H, NH2), 7.43 (d, 1H, H-6), 8 . 01 (s. 1H, H -2).

ES-MS: Found: 291.1 (MH); calcd for C13H16N4O4 -H^-: 291,2.

EXAMPLE 23

4-Amino-7- (2-C-hydroxymethyl-eD-ribofuranosyl-7H-pyrrolo [2,3-dlpyrimidine

PL 207 405 B1

<img file="PL207405B1_D0026.tif" />

Step A: 4-Chloro-7- [3,5-bis-O- (2,4-dichlorophenylmethyl) -2-C-hydroxymethyl-eD-ribofuranosyl 1-7H-pyrrolo [2,3-d1-pyrimidine

To a solution of the compound of Example 23, Step B (300 mg, 0.48 mmol) in 1,4-dioxane (5 ml) was added N-methylmorpholine N-oxide (300 mg, 2.56 mmol) and osmium tetroxide (4% solution in water, 0.3 ml). The mixture was stirred in the dark for 14 h. The precipitate was removed by filtration through a pad of celite, diluted with water (3 x), and extracted with EtOAc. The EtOAc layer was dried over Na2SO4 and concentrated in vacuo. The oily residue was taken up in dichloromethane (5 ml) and stirred over NalO4 on silica gel (3 g, 10% NalO4) for 12 h. The silica gel was removed by filtration and the residue was evaporated and taken up in absolute ethanol (5 ml). The solution was cooled in an ice bath and sodium borohydride (300 mg, 8 mmol) was added in small portions. The resulting mixture was stirred at room temperature for 4 h and then diluted with EtOAc. The organic layer was washed with water (2 x 20 ml), brine (20 ml) and dried over Na2SO4. The solvent was evaporated and the residue was purified by flash chromatography (SiO2, 2: 1 hexanes / EtOAc) to give the title compound (160 mg, 0.25 mmol) as white flakes.

Step B: 4-Amino-7- [3,5-bis-O- (2,4-dichlorophenylmethyl) -2-C-hydroxymethyl-eD-ribofuranosyl 1-7H-pyrrolo [2,3-d1-pyrimidine

Compound from Step A (150 mg, 0.23 mmol) was dissolved in a minimum amount of 1,4-dioxane (10 mL) and placed in a stainless steel bomb. The bomb was cooled to -78 ° C and liquid ammonia was added. The bomb was sealed and heated at 90 ° C for 24 h. The ammonia was allowed to evaporate and the residue was concentrated to give a white solid which was used in the next step without further purification.

Step C: 4-Amino-7- (2-C-hydroxymethyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d1-pyrimidine

Compound from step B (120 mg, 0.2 mmol) was dissolved in 1: 1 methanol / dichloromethane, 10% Pd-C was added and the suspension was stirred under an atmosphere of H 2 for 12 h. The catalyst was removed by filtration through a celite pad and washed copiously with methanol . The combined filtrates were evaporated in vacuo, and the residue was purified by flash chromatography (SiO2, 10% methanol in EtOAc containing 0.1% triethylamine) to give the title compound (50 mg) as a white powder.

¹H NMR (CD3OD): δ 3.12 (d, IH, CH2 '), 3.33 (d, 1H, CH2), 3.82 (m, 1H, H-5'), 3.99-4, 1 (m, 2H, H-4 ', H-5), 4.3 (d, 1H, H-3'), 6.2 (s, 1H, H-1 '), 6.58 (d, 1H, H-5), 7.45 (d, 1H, H-6), 8.05 (s, 1H, H-2). LC-MS: Found: 297.2 (M + H⁺); calcd for C12H16N4O5 + H⁺: 297,3.

EXAMPLE 24

4-Amino-7- (2-C-fluoromethyl-eD-ribofuranosyl) -7H-pyrrolo [2.3-d1-pyrimidine

<img file="PL207405B1_D0027.tif" />

Step A: 4-Chloro-7- [3,5-bis-O- (2,4-dichlorophenylmethyl) -2-C-fluoromethyl-eD-ribofuranosyl 1-7H-pyrrolo [2,3-cf1-pyrimidine

To a solution of the compound of Example 24, Step A (63 mg, 0.1 mmol) in dry dichloromethane (5 ml) under argon, were added 4-dimethylaminopyridine (DMAP) (2 mg, 0.015 mmol) and triethylamine (62 μ (

PL 207 405 B1

0.45 mmol). The solution was cooled in an ice bath and p-toluenesulfonyl chloride (30 mg, 0.15 mmol) was added. The reaction was stirred at room temperature overnight, washed with NaHCO3 (2 x 10 mL), water (10 mL), brine (10 mL), dried over Na2SO4 and concentrated in vacuo to give a pink solid. The precipitate was dissolved in anhydrous THF (5 ml) and cooled in an ice bath. Tetrabutylammonium fluoride (1M solution in THF, 1 mL, 1 mmol) was added and the mixture was stirred at room temperature for 4 h. The solvent was removed in vacuo, the residue was taken up in dichloromethane, and washed with NaHCO3 (2 x 10 mL), water (10 mL), and brine (10 mL). The dichloromethane layer was dried over anhydrous Na2SO4, concentrated in vacuo, and purified by flash chromatography (SiO2, 2: 1 hexanes / EtOAc) to give the title compound (20 mg) as a white solid.

Step B: 4-Amino-7- [3,5-bis-O- (2,4-dichlorophenylmethyl) -2-C-fluoromethyl-β-D-rvbofuranoyl-1-ZH-pyrrolorel-cflpyrimidine

Compound from Step A (18 mg, 0.03 mmol) was dissolved in a minimum amount of 1,4-dioxane and placed in a stainless steel bomb. The bomb was cooled to -78 ° C and liquid ammonia was added. The bomb was sealed and heated at 90 ° C for 24 h. The ammonia was allowed to evaporate and the residue was concentrated to give a white solid which was used in the next step without further purification.

Step C: 4-Amino-7- (2-C-fluoromethyl-β-D-rvbofuranoyl) -7H-pyrrolo [2,3-d1-pyrimidine

The compound from Step B (16 mg) was dissolved in 1: 1 methanol / dichloromethane, 10% Pd-C was added, and the suspension was stirred under an atmosphere of H 2 for 12 h. The catalyst was removed by filtration through a celite pad and washed copiously with methanol. The combined filtrates were evaporated in vacuo and the residue was purified by flash chromatography (SiO2, 10% methanol in EtOAc containing 0.1% triethylamine) to give the title compound (8 mg) as a white powder.

¹H NMR (DMSO-c6): δ 3.6-3.7 (m, 1H, H-5, 3.8-4.3 (m, 5H, H-5, H-4 ', 5H-3 ', CH2) 5.12 (t, 1H, 5'-OH), 5.35 (d, 1H, 3'-OH), 5.48 (s, 1H, 2'-OH), 6.21 ( s, 1H, H-1 '), 6.52 (d, 1H, H-5), 6.98 (br s, 2H, NH2), 7.44 (d, 1H, H-6), 8, 02 (s, 1H, H -2).

¹⁹F NMR (DMSO-c6): δ -230.2 (t).

ES-MS: Found: 299.1 (M + H⁺), calcd for C12H15FN4O4 + H⁺: 3 299,27.

EXAMPLES 25 and 26

4-Amino-7- (3-deoxy-2-C-methyl-β-D-rvbofuranoyl) -7H-pyrrolo [2,3- &lt;C1-pyrimidine and 4-amino-7- (3-deoxy] -C-methyl -pD-arabinofuranosyl ^ H-pyrrolo ^^ - Clpyimidine

<img file="PL207405B1_D0028.tif" />

Stage A: 7- [2,5-Bis-O- (tert-butyl dimethylsilyl) -β-D-rvbofuranoyl 1-7H-pyrrolo [2.3-d1-pyrimidvna

LT-JOd-bis-O-itt-butyldimethylsilol-eD-ribofuranosol-TH-pyrroleJ ^^ - Cpyrimidine

Silver nitrate (6.36 g, 38.8 mmol) was added to a stirred solution of tubercidin (5.0 g, 18.7 mmol) in a mixture of pyridine (7.5 ml) and DMF (18.5 ml). This mixture was stirred at room temperature for 2 h. It was cooled in an ice bath and THF (37.4 ml) and tert-butyldimethylsilyl chloride (5.6 g, 37 mmol) were added and the mixture was stirred at room temperature for 2 h. filtered through a pad of celite and washed with THF. The filtrate and washes were diluted with ether containing a small amount of chloroform. The organic layer was washed successively with sodium bicarbonate and water (3 x 50 mL), dried over anhydrous sodium sulfate, and concentrated. Pyridine was removed by co-evaporation with toluene and the residue was purified by flash chromatography on silica gel using 5-7% MeOH in CH2Cl2 as eluent; yield 3.0 g.

Stage B: T-IZ ^ d-Bis-O-iieii-butyldimethylsilyl] -eD-ribofuranosyl] J-Z-fdi-iZ-methoxyphenylphenylene -lmethyllamino ^ H-pyrrole ^^ - dlpyrimidine and 7-i3,5-bis-O- ( terf-butyldimethylsilyl) -eD-ribofuranosylHddi-M-methoxyphenylphenylmethyllamino ^ H-pyrrolore ^ -dlpyrimidine

To a solution of the mixture of compounds from step A (3.0 g, 6.0 mmol) in anhydrous pyridine (30 ml) was added 4,4'-dimethoxytrityl chloride (2.8 g, 8.2 mmol) and the reaction mixture was stirred at temperature. room until the next day. The mixture was then triturated with aqueous pyridine and extracted with ether. The organic layer was washed with water, dried over anhydrous sodium sulfate, and concentrated to give a yellow foam (5.6 g). The residue was purified by silica gel flash chromatography using 20-25% EtOAc in hexanes as the eluent. The appropriate fractions were collected and concentrated to give 2 ', 5'-bis-O- (tert-butyldimethylsilyl) - and 3', 5'-bis-O- (tert-butyldimethylsilyl) protected nucleosides as a colorless foam (2.2 g and 1.0 g).

Step C: 7- [2,5-Bis-O- (t [beta] -butyldimethylsilyl] -3-O-tosyl-eD-ribofuranosyl)] - 4- [di- (4-methoxy-phenyl) phenylmethyl] amino- 7H-pyrrolo [2,3-d] pyrimidine

To an ice-cooled solution of 2 ', 5'-bis-O - ((e / Y-butyldimethylsilyl) -protected nucleoside from step B (2.0 g, 2.5 mmol) in pyridine (22 ml) was added p-toluenesulfonyl chloride (1.9 g, 9.8 mmol) The reaction mixture was stirred at room temperature for four days It was then triturated with aqueous pyridine (50%, 10 mL) and extracted with ether (3 x 50 mL) containing a small amount of CH2Cl2 ( 10 ml) The organic layer was washed with sodium bicarbonate and water (3 x 30 ml). The organic layer was dried over anhydrous Na2SO4 and concentrated. Pyridine was removed by evaporation together with toluene (3 x 25 ml). The residual oil was filtered through a pad of silica gel using hexane: ethyl acetate (70:30) as eluent; yield 1.4 g.

Step D: 4- [di- (4-methoxyphenyl) phenylmethyl] amino-7- [3-O-tosyl-3-D-ribofuranosyl-7H-pyrrolo [2,3-d] pyrimidine

A solution of the compound from Step C (1.0 g, 1.1 mmol) and THF (10 mL) was stirred with tetrabutylammonium fluoride (1M solution in THF, 2.5 mL) for 0.5h. The mixture was cooled and diluted with ether (50 ml). The solution was washed with water (3 x 50 mL), dried over anhydrous Na2SO4, and concentrated to an oil. The residue was purified by passing through a silica gel pad using hexane: ethyl acetate (1: 1) as eluent; yield 780 mg.

Step E: 4-Amino-7- (3-deoxy-2-C-methyl-3-D-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine and 4-amino-7- (3-deoxy- 2-C-methyl-3-D-arabinofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine

CH solution₃MgI (3.0 M ether solution, 3.0 mL) in anhydrous toluene (3.75 mL) was cooled in an ice bath. Thereto was added a solution of the compound from Step D (500 mg, 0.8 mmol) in dry toluene (3.7 ml). The resulting mixture was stirred at room temperature for 3.5 h. It was cooled and quenched with aq. NH 4 Cl and extracted with ether (50 mL, containing 10 mL CH 2 Cl 2). The organic layer was separated and washed with brine (2 x 30 ml) and water (2 x 25 ml), dried over anhydrous Na2SO4 and concentrated to give an oil which was purified by flash chromatography on silica gel using 4% MeOH in CH₂CI₂to give 2-C- &amp; lt; - &amp; gt; - methyl compound (149 mg) and 2-Cp-methyl compound (34 mg). These derivatives were treated separately with 80% acetic acid and the reaction mixture was stirred at room temperature for 2.5 h. The acetic acid was removed by repeated evaporation together with ethanol and toluene. The residue was partitioned between chloroform and water. The aqueous layer was washed with chloroform and concentrated. The evaporation residue was purified over silica gel using 5-10% MeOH in CH2Cl2 as the eluent to give the desired compounds as white solids.

4-Amino-7- (3-deoxy-2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine (9.0 mg):

¹H NMR (DMSO-6₆): δ 0.74 (s, 3H, CH₃), 1.77 (dd, 1H, H-3 '), 2.08 (t, 1H, H-3), 3.59 (m, 1H, H-5'), 3.73 (m, 1H , H-5), 4.15 (m, 1H, H-4 '), 5.02 (t, 1H, OH-5'), 5.33 (s, 1H, OH-2 '), 6, 00 (s, 1H, H-1 '), 6.54 (d, 1H, H-7), 6.95 (br s, 2H, NH2), 7.47 (d, 1H, H-8), 8.00 (s, 1H, H -2); ES-MS: 263.1 [MH].

4-Amino-7- (3-deoxy-2-C-methyl-3-D-arabinofuranosyl] -7H-pyrrolo [2,3-d] pyrimidine (15 mg):

¹H NMR (DMSO-6₆): δ 1.23 (s, 3H, CH₃), 2.08 (ddd, 2H, H-3 'and 3), 3.57 (m, 2H, H-5' and 5), 4.06 (m, 1H, H-4), 5.10 (s, 1H, OH-2 '), 5.24 (t, 1H, OH-5'), 6.01 (s, 1H, H-1 '), 6.49 (d, 1H, H-7 ), 6.89 (br s, 2H, NH2), 7.35 (d, 1H, H-8), 8.01 (s, 1H, H-2).

ES-MS: 265.2 [M + H].

EXAMPLE 27

4-Amino-7- (2,4-C-dimethyl-3-D-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine

<img file="PL207405B1_D0029.tif" />

PL 207 405 B1

Step A: 5-Deoxy-1,2-O-isopropylidene-D-xylfuranose

1,2-O-Isopropylidene-D-xylfuranose (38.4 g, 0.2 mol), 4-dimethylaminopyridine (5 g), triethylamine (55.7 ml, 0.4 mol) was dissolved in dichloromethane (300 ml) . P-Toluenesulfonyl chloride (38.13 g, 0.2 mol) was added and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was then poured into saturated aqueous sodium bicarbonate solution (500 ml) and the two layers were separated. The organic layer was washed with an aqueous citric acid solution (20%, 200 ml), dried (Na2SO4) and evaporated to give a solid (70.0 g). The precipitate was dissolved in dry THF (300 ml) and added portionwise over 30 min. LiAlH4 (16.0 g, 0.42 mol). The mixture was stirred at room temperature for 15 h. Ethyl acetate (100 ml) was added dropwise over 30 minutes and the mixture was filtered through a bed of silica gel. The filtrate was concentrated and the resulting oil was chromatographed on silica gel (EtOAc / hexane 1/4) to give the product as a solid (32.5 g).

Step B: 3,5-Bis-O- (2,4-dichlorophenylmethyl] -1-O-methyl-4-methyl-αD-ribofuranose

Chromium oxide (50 g, 0.5 mol), acetic anhydride (50 ml, 0.53 mol) and pyridine (100 ml, 1.24 mol) were added to dichloromethane (1 L) in an ice-water bath, and the mixture was stirred for 15 minutes. 5-deoxy-1,2-O-isopropylidene-D-xylfuranose (32 g, 0.18 mol) in dichloromethane (200 ml) was added, and the mixture was stirred at the same temperature for 30 minutes. The reaction solution was diluted with ethyl acetate (1 L) and filtered through a bed of silica gel. The filtrate was concentrated to give a yellow oil. The oil was dissolved in 1,4-dioxane (1 L) and formaldehyde (37%, 200 mL). The solution was cooled to 0 ° C and solid KOH (50 g) was added. The mixture was stirred at room temperature overnight and then extracted with ethyl acetate (6 x 200 ml). After concentration, the residue was chromatographed on silica gel (EtOAc) to give the product as an oil (1.5 g). The oil was dissolved in 1-methyl-2-pyrrolidinone (20 ml) and 2,4-dichlorophenylmethyl chloride (4 g, 20.5 mmol) and NaH (60%, 0.8 g) were added. The mixture was stirred overnight and diluted with toluene (100 ml). The mixture was then washed with saturated aqueous sodium bicarbonate solution (3 x 50 ml), dried (Na2SO4) and evaporated. The residue was dissolved in methanol (50 ml) and HCl in dioxane (4 M, 2 ml) was added. The solution was stirred overnight and evaporated. The residue was chromatographed on silica gel (EtOAc / hexane: 1/4) to give the desired product as an oil (2.01 g).

Step C: 3,5-Bis-O- (2,4-Dichilorophenylmethyl) -2,4-di-C-methyl-1-O-methyl-αD-ribofuranose Product (2.0 g, 4.0 mmol) from step B and Dess-Martin periodinane (2.0 g) in dichloromethane (30 ml) were stirred overnight at room temperature and then concentrated under reduced pressure. The residue was triturated with ether (50 ml) and filtered. The filtrate was washed with a solution of Na2S2O3-5H2O (2.5 g) in a saturated aqueous sodium bicarbonate solution (50 ml), dried (MgSO4), filtered and evaporated. The residue was dissolved in anhydrous Et2O (20 mL) and added dropwise to a solution of MeMgBr in Et2O (3M, 10 mL) at -78 ° C. The reaction mixture was allowed to warm to -30 ° C and stirred at -30 ° C to -15 ° C for 5 h, then poured into saturated aqueous ammonium chloride solution (50 ml). The two layers were separated and the organic layer was dried (MgSO4), filtered and concentrated. The residue was chromatographed on silica gel (EtOAc / hexane: 1/9) to give the title compound as a syrup (1.40 g).

Step D: 4-Chloro-7- [3,5-bis-O- (2,4-dichlorophenylmethyl) -2,4-di-C-methyl-eD-ribofuranosyl] -7H-pyrrolo [2,3-d ] pyrimidine

To compound from Step C (0.70 g, 1.3 mmol) was added HBr (5.7 M acetic acid solution, 2 ml). The resulting solution was stirred at room temperature for 1 h, evaporated in vacuo and co-evaporated with anhydrous toluene (3 x 10 ml). 4-Chloro-1H-pyrrolo [2,3-d] pyrimidine (0.5 g, 3.3 mmol) and powdered KOH (85%, 150 mg, 2.3 mmol) were mixed in 1-methyl-2-pyrrolidinone (5 ml) for 30 minutes and the mixture was evaporated with toluene (10 ml). The resulting solution was poured into the above bromosaccharide residue and the mixture was stirred overnight. The mixture was diluted with toluene (50 ml), washed with water (3 x 50 ml) and concentrated under reduced pressure. The residue was chromatographed on silica gel, eluting with EtOAc / hexane (15/85) to give a solid (270mg).

Step E: 4-Amino-7- (2,4-C-dimethyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine

Compound from step D (270 mg) was dissolved in dioxane (2 ml) in a stainless steel autoclave and liquid ammonia (20 g) was added. The mixture was heated at 100 ° C for 15 h, then cooled and evaporated. The residue was chromatographed on silica gel (EtOAc) to give a solid (200 mg). The precipitate (150 mg) and Pd / C (10%, 150 mg) in methanol (20 ml) was shaken under an atmosphere of H 2 0.207 MPa (30 psi) for 3 h, filtered and evaporated. The residue was chromatographed on silica gel (MeOH / CH2Cl2: 1/9) to give the desired product as a solid (35 mg).

PL 207 405 B1 ¹H NMR (DMSO-d6): δ 0165 (s1 3H) 1 1118 (s1 3H) 1 3143 (m1 2H) 1 4106 (d1 1H1 J613 Hz) 1 4187 (s1 1H) 1 5126 (br1 1H) 1 5108 (d1 1H1 J613 Hz) 1 5125 (t1 1H1 J 310 Hz) 1 6117 (s1 1H) 1 6154 (d1 1H1 J315 Hz) 1 6197 (s1 br1 2H) 1 7154 (d1 1H1 J 314 Hz) 1 8102 (s1 1H).

¹³C NMR (DMSO-d6): δ 181191 211321 651381 731001 791331 841801 901661 991091 1021411 1211901 1491581 1511481 157138.

LC-MS: Found: 29511 (M + H⁺); calcd for C13H18N4O4 + H⁺: 29511.

EXAMPLE 28

4-Amino-7- (3-deoxy-3-fluoro-2-C-methyl-β-D-ribofuranosyl-7H-pyrrolo [2,3-d] pyrimidine

<img file="PL207405B1_D0030.tif" />

Stage A: 3-Deoxy-3-fluoro-1-O-methyl-5-O-toluoyl-αD-ribofuranose

1,2-O-Isopropylidene-D-xylfuranose (9.0 g, 50 mmol) and p-toluoyl chloride (7.0 mL, 50 mmol) in pyridine (50 mL) were stirred for 30 minutes. Water (10 ml) was added and the mixture was concentrated under reduced pressure. The residue was dissolved in toluene (500 ml) and the solution was washed with water (200 ml) and saturated aqueous sodium bicarbonate solution (200 ml). The two layers were separated and the organic layer was evaporated. The residue was dissolved in methanol (100 ml) and HCl in dioxane (4M, 10 ml) was added. The mixture was stirred at room temperature overnight and then evaporated under reduced pressure. The resulting oil was chromatographed on silica gel (EtOAc / hexane: 1/1) to give an oil (10.1 g). The oil was dissolved in dichloromethane (100 ml) and diethylaminosulfur trifluoride (DAST) (5.7 ml) was added. The mixture was stirred overnight and then poured into a saturated aqueous solution of sodium bicarbonate (100 ml). The mixture was extracted with toluene (2 x 50 ml) and the combined organic layers were concentrated. The residue was chromatographed on silica gel (EtOAc / hexane: 15/85) to give the title compound as an oil (1.50 g).

Stage B: 3-Deoxv-3-fluoro-2-C-methyl-1-O-methyl-5-O-toluoyl-αD-ribofuranose

The product from Step A (1.0 g, 3.5 mmol) and Dess-Martin periodinate (2.5 g) in dichloromethane (20 ml) were stirred overnight at room temperature and then concentrated under reduced pressure. The residue was triturated with diethyl ether (50 ml) and filtered. The filtrate was washed with a Na solution₂S.₂ABOUT₃^ 5H₂O (12.5 g) in a saturated aqueous sodium bicarbonate solution (100 ml), dried (MgSO4), filtered and evaporated. The residue was dissolved in anhydrous THF (50 ml). TiCl4 (3 mL) and methylmagnesium bromide in diethyl ether (3M, 10 mL) were added at -78 ° C and the mixture was stirred at -50 to -30 ° C for 2 h. The mixture was poured into saturated aqueous sodium bicarbonate solution (100 ml) and filtered through celite. The filtrate was extracted with toluene (100 ml) and evaporated. The residue was chromatographed on silica gel (EtOAc / hexane: 15/85) to give the title compound as an oil (150 mg).

Stage C: 4zAmjnozZz (3deoxy-3-fluoro-2-C-methyl-eD-ribgfuranosyl)_with7Hj_:pyroJoI2₁3-d] irymL dyna

The product of step B (150 mg, 0.5 mmol) was dissolved in HBr (30%) in acetic acid (2 ml). After one hour, the mixture was evaporated under reduced pressure and evaporated with toluene (10 ml). 4-Chloro-1H-pyrrolo [2,3-d] pyrimidine (0.5 g, 3.3 mmol) and powdered KOH (85%, 150 mg, 2.3 mmol) were stirred in DMF (3 mL) for 30 minutes and the mixture was evaporated with toluene (2 ml). The resulting solution was poured into the above bromosaccharide and the mixture was stirred overnight. The mixture was diluted with toluene (50 ml), washed with water (3 x 50 ml) and concentrated under reduced pressure. The residue was chromatographed on silica gel (EtOAc / hexane: 15/85) to give an oil (60 mg). The oil was dissolved in dioxane (2 ml) in a stainless steel autoclave and liquid ammonia (20 g) was added. The mixture was heated at 85 ° C for 18 h, then cooled and evaporated. The residue was chromatographed on silica gel (methanol / dichloromethane: 1/9) to afford the title compound as a solid (29mg).

PL 207 405 B1 ¹H NMR (DMSO-d6): δ 0.81 (s, 3H), 3.75 (m, 2H), 4.16 (m, 1H), 5.09 (dd, 1H, J53.2, 7, 8 Hz), 5.26 (br, 1H), 5.77 (s, 1H), 6.15 (d, 1H, J2.9Hz), 6.59 5 (d, 1H, J3.4Hz) , 7.02 (s br, 2H), 7.39 (d, 1H, J3.4Hz), 8.06 (s, 1H).

¹³C NMR (DMSO-d6): 19.40, 59.56, 77.24, 79.29, 90.15, 91.92, 99.88, 102.39, 121.17, 149.80, 151, 77, 157.47.

19F NMR (DMSO-d6): δ 14.66 (m).

ES-MS: Found: 283.1 (M + H⁺); calcd for C12H15FN4O3 + H +: 283.1.

EXAMPLE 29

4-Amino-7- (2-C, 2-O-dimethyl-eD-rvbofuranosyl) -7H-pyrrolo [2,3-c] pyrimidine

<img file="PL207405B1_D0031.tif" />

Step A: 4-chloro-7- | 3,5-bis-O- (2,4-dichlorophenylmethyl] -2-C_Ł2-O-dimethyl-β-D-rvbofuranozvlo] ^ H-pyrrole ^^ - dl-pyrimidine

To a cooled (0 ° C) solution of the compound of Example 2, Step D (618 mg, 1.0 mmol) in THF (8 ml) was added methyl iodide (709 mg, 5.0 mmol) and NaH (60% in mineral oil ) (44 mg, 1.1 mmol). The resulting mixture was stirred overnight at room temperature and then poured into a stirred mixture of saturated aqueous ammonium chloride (50 ml) and dichloromethane (50 ml). The organic layer was washed with water (50 ml), dried (MgSO4) and evaporated in vacuo. The resulting crude product was purified over silica gel using an ethyl acetate / hexane mixture as the eluent. Product containing fractions were combined and evaporated in vacuo to give the desired product (735 mg) as a colorless foam.

Step B: 4-amino-7- | 3,5-bis-O- (2,4-dichlorophenylmethyl) -2-C, 2-O-dimethyl-β-D-rvbofuranoyl] yB-pyrrole ^^ - dl-pyrimidine

To compound from Step A (735 mg, 1.16 mmol) was added methanolic ammonia solution (saturated at 0 ° C) (20 mL). The mixture was heated in a stainless steel autoclave at 80 ° C overnight, then cooled and the contents evaporated in vacuo. The crude mixture was purified over silica gel using ethyl acetate / hexane as eluent. Product containing fractions were combined and evaporated in vacuo to give the desired product (504 mg) as a colorless foam.

Step C: 4-Amino-7- (2-C, 2-O-dimethyl-β-D-rvbofuranoyl) -7H-pyrrolo [2,3-d1-pyrimidine

A mixture of the product from step C (64 mg, 0.1 mmol), MeOH (5 ml), Et 3 N (0.2 ml) and 10% Pd / C (61 mg) was hydrogenated on a Parr apparatus at 50 psi at room temperature to next day. The mixture was filtered through celite, evaporated in vacuo and filtered through a pad of silica gel using 2% methanol in dichloromethane as eluent. The desired product was collected and evaporated in vacuo. The compound was redissolved in methanol (10 ml) and 10% Pd / C (61 mg) was added. The mixture was hydrogenated on a Parr apparatus at 55 psi at room temperature for 2 weeks. The mixture was filtered through celite, evaporated in vacuo and purified on silica gel using 10% methanol in dichloromethane as eluent. Product containing fractions were combined and evaporated in vacuo to give the desired product (110 mg) as a colorless foam.

¹H NMR (DMSO-d6): δ 0.68 (s, 3H), 3.40 (s, 3H), 3.52-3.99 (overlapping m, 4H), 4.92 (d, 1H) , 5.07 (t, 1H), 6.26 (s, 1H), 6.55 (d, 1H), 7.00 (s br, 2H), 7.46 (d, 1H), 8.05 (s, 1H).

LC-MS: Found: 293.1 (MH⁺); calcd for C12H16N4O4-H⁺: 293,12.

EXAMPLE 30

4-MethylQamjnQ-7- (2-CmethylQ-gD-fishQfuranQzYlQ) -7H-pirQlo | 23rd [1-pyrimidine

PL 207 405 B1

NHMe

ΗΟ \ Ό

<img file="PL207405B1_D0032.tif" />

ch₃

HO OH

The compound of Step E from Example 2 (200 mg, 0.67 mmol) was added to methylamine (5 ml condensed in a stainless steel small autoclave) and heated to 85 ° C for 48 h, then cooled and evaporated in vacuo. The crude mixture was purified over silica gel with ethanol as eluent to afford the title compound which was isolated as an amorphous solid after MeCN treatment. The amorphous precipitate was dissolved in water and lyophilized to give a colorless powder (144 mg).

¹H NMR (DMSO-d6): δ 0.63 (s, 3H, CH3), 3.32 (s, 3H, N CH3), 3.58-3.67 (m, 1H, H-5 '), 3.79-3.39 (m, 3H, H-5, H-4 ', H-3'), 5.03 (s, 1H, 2'-OH), 5.04-5.11 (1H , 3'-OH, 1H, 5'-OH), 6.14 (s, 1H, H-1 '), 6.58 (d, 1H, J5.6 = 3.6Hz, H-5), 7.46 (d, 1H, H-6), 7.70 (br s, 1H, NH), 8.14 (s, 1H, H-2).

LC-MS: Found: 295.1 (MH⁺); calcd for C13H18N4O4 + H⁺: 294,3.

EXAMPLE 31

4-Dimethylamino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-cflpyrimidine

NMe₂

HO OH

The compound of Step E from Example 2 (200 mg, 0.57 mmol) was added to methylamine (5 ml condensed in a stainless steel small autoclave) and heated at 85 ° C for 48 h, then cooled and evaporated in vacuo. The crude mixture was purified over silica gel with ethanol as eluent to afford the title compound which was isolated as an amorphous solid after MeCN treatment. The amorphous precipitate was dissolved in water and lyophilized to give a colorless powder (164 mg).

¹H NMR (DMSO-d6): δ 0.64 (s, 3H, CH3), 3.29 (s, 3H, N CH3), 3.32 (s, 3H, N CH3), 3.50-3, 55 (m, 1H, H-5 '), 3.77-3.97 (m, 3H, H-5, H-4', H-3 '), 5.04 (s, 1H, 2'- OH), 5.05-5.11 (1H, 3'-OH, 1H, 5'-OH), 5.21 (s, 1H, H-1 '), 6.69 (d, 1H, J5, 6 = 3.6 Hz, H-5), 7.55 (d, 1H, H-5), 8.13 (s, 1H, H-2).

LC-MS: Found: 309.3 (MH⁺); calcd for C14H20N4O4 + H⁺: 308,33.

EXAMPLE 32

4-Cyclopropylamino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-cflpyrimidine

HO OH

PL 207 405 B1

The compound of Step E from Example 2 (200 mg, 0.67 mmol) was added to the cyclopropylamine (5 ml condensed in a stainless steel small autoclave) and heated to 85 ° C for 48 h, then cooled and evaporated in vacuo. The crude mixture was purified over silica gel with ethanol as eluent to afford the title compound which was isolated as an amorphous solid after MeCN treatment. The amorphous precipitate was dissolved in water and lyophilized to give a colorless powder (148 mg).

¹H NMR (DMSO-d6): δ 0.51-0.58 (m, 2H), 0.64 (s, 3H, CH3), 0.74-0.076 (m, 2H), 3.62-3, 67 (m, 1H, H-5 '), 3.79-3.82 (m, 3H, H-5), 3.92-3.96 (m, H-4', H-3 '), 5.03 (s, 1H, 2'-OH), 5.05-5.10 (1H, 3'-OH, 1H, 5'-OH), 6.15 (s, 1H, H-1 ') , 7.48 (d, 1H, J5.6 = 3.6Hz, H-5), 7.59 (d, 1H, H-6), 8.13 (s, 1H, H-2).

LC-MS: Found: 321.1 (MH⁺); calcd for C15H20N4O4 + H⁺: 320,3.

EXAMPLE 33

4-Amino-7- (3-C-methyl-eD-xylfuranosyl) -7H-pyrrolo [2,3-d] pyrimidine

<img file="PL207405B1_D0033.tif" />

Me OH

Step A: 7- [2,5-Bis-O- (tf-butyldimethylsilyl) -eD-ribofuranosyl)] - 4 - [(4-methoxyphenyl) diphenylmethyl] amino-7H-pyrrolo [2,3-d] pyrimidine and 7- [3,5-bis-O- (te / Y-butyldimethylsilyl) -eD-ribofuranosyl] -4 - [(4-methoxyphenyl) diphenylmethyl] amino-7H-pyrrolo [2,3-d] pyrimidine

To a solution of the mixture of compounds from step A of examples 26 and 27 (0.32 g, 0.65 mmol) in anhydrous pyridine (6 ml) was added monomethoxytrityl chloride (0.30 g, 0.98 mmol) and the reaction mixture was stirred at temperature. room until the next day. The mixture was then concentrated and the residue partitioned between CH2Cl2 (70 mL) and water (20 mL). The organic layer was washed with water and brine, dried (Na2SO4) and concentrated. The residue was purified on a silica gel column using 5-13% EtOAc in hexanes as the eluent. The appropriate fractions were collected and concentrated to give 2 ', 5'-bis-O- (te / t-butyldimethylsilyl) - and 3', 5'-bis-O- (te / t-butyldimethylsilyl) -protected nucleosides as colorless foams (343 mg and 84 mg, respectively).

Step B: 7- [2,5-Bis-O- (t [beta] -butyldimethylsilyl) -eDe ('yfrp-pentofuranos-3-ulosyl] -4 - [(4-methoxyphenyl) diphenylmethyl] amino-7H-pyrrolo [ 2,3-d] pyrimidine

To a well-stirred suspension of chromium trioxide (91 mg, 0.91 mmol) in CH2Cl2 (4 mL) at 0 ° C was added pyridine (147 g, 1.82 mmol) followed by acetic anhydride (86 g, 0.91 mmol) . The mixture was stirred at room temperature for 0.5 h. Then 2 ', 5'-bis-O- (te / t-butyldimethylsilyl) protected nucleoside from step A (343 mg 0.45 mmol) in CH2Cl2 (2.5 ml) was added and the mixture was stirred at room temperature for 2 h. The mixture was then poured into ice cold EtOAc (10 mL) and filtered through a short column of silica gel using EtOAc as eluent. The filtrate was evaporated and the residue was purified on a silica gel column with hexanes and hexanes / EtOAc (7/1) as eluent to give the title compound (180 mg).

Step C: 7- [2,5-Bis-O- (te / Y-butyldimethylsilyl) -3-C-methyl-eD-ribofuranosyl) -4 - [(4-methoxyphenyl) diphenylmethyl] amino-7H-pyrrolo [2 , 3-d] pyrimidine and 7- [2,5-bis-O- (te / Y-butyldimethylsilyl) -Cmethyl-eD-xylfuranosyl) -4 - [(4-methoxyphenyl) diphenylmethyl] amino-7H-pyrrolo [2 , 3-d] pyrimidine

To a mixture of MeMgBr (3.0 M ether solution; 0.17 ml, 0.5 mmol) in dry hexane (1.5 ml) at room temperature was added dropwise a solution of the compound from Step B (78 mg, 0.1 mmol) in anhydrous hexane (0.5 ml). After being stirred at room temperature for 2 h, the reaction mixture was poured into ice water (10 mL) and diluted with EtOAc (20 mL) then filtered through Celite which was then washed thoroughly with EtOAc. The layers were separated and the organic layer was washed with brine, dried (Na2SO4) and concentrated. The residue was purified on a silica gel column using 8 to 25% EtOAc in hexanes as eluent to give the 3-C-methyloxy- isomer (60 mg) and the 3-C-methyl rib- isomer (20 mg).

Step D: 4-Amino-7- (3-C-methyl-β-D-xylfuranosyl] -7H-pyrrolo [2,3-d] pyrimidine

PL 207 405 B1

To an ice-cold solution of the 3-C-methylxyl isomer from step C (60 mg, 0.08 mmol) in THF (2 mL) was added TBAF (1M in THF; 0.32 mL, 0.32 mmol). The reaction mixture was stirred at room temperature for 5 h, then diluted with CH 2 Cl 2 (50 ml), washed with water (3 x 15 ml), dried, and evaporated. The residue was dissolved in dioxane (0.3 ml) and 80% acetic acid (3 ml) was added. The reaction mixture was stirred at room temperature for 1 day and then evaporated. The residue was co-evaporated with dioxane, taken up in water (50 ml) and washed with CH2Cl2 (2 x 10 ml). The aqueous layer was concentrated and then lyophilized. The residue was purified on a silica gel column with CH2Cl2 / MeOH (20/1 and 10/1) as eluent to give the title compound as a white fluffy solid (10 mg) after lyophilization.

¹H NMR (CD3CN): δ 1.28 (s, 3H, CH3), 3.56 (br s, 1H, OH), 3.78 (m, 3H, H-4 ', H-5', H- 5), 4.10 (br s, 1H, OH), 4.44 (d, 1H, J2.1 '= 3.9Hz, H-2'), 5.58 (d, 1H, H-1 '), 5.85 (br s, 2H, NH2), 6.15 (br s, 1H, OH), 6.48 (d, 1H, J5.6 = 3.7Hz, H-5), 7 , 23 (d, 1H, H-6). 8.11 (s, 1H, H-2). ES-MS: 281 [MH1⁺

EXAMPLE 34

4-Amino-7- (3-C-methyl-eD-ribofuranosyl 1-7H-pyrrolo [2,3-d1-pyrimidine

<img file="PL207405B1_D0034.tif" />

HÓ

The ribo-isomer (20 mg) from Step C of Example 31 was deprotected using the procedure described in Step D of Example 31 to afford the title compound (4 mg).

¹H NMR (CD3CN): δ 1.43 (s, 3H, CH3), 3.28 (br s, 1H, OH), 3.58 (m, 2H, H-5 ', H-5), 3, 99 (m, 1H, H-4 '), 4.10 (br s, 1H, OH), 4.62 (d, 1H, J2'1' = 8.1 Hz, H-2 '), 5, 69 (d, 1H, H-1 '), 5.88 (br s, 3H, OH, NH2), 6.45 (br s, 1H, OH), 6.51 (d, 1H, J5.6 = 3.7Hz, H-5), 7.19 (d, 1H, H-6), 8.12 (s, 1H, H-2).

ES-MS: 281 [MH1⁺

EXAMPLE 35

2,4-Diamino-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-cf1-pyrimidine

<img file="PL207405B1_D0035.tif" />

A mixture of the product of step B from example 4 (24 mg) in aqueous ammonia (30%, 10 ml) was heated in a stainless steel autoclave at 100 ° C overnight, then cooled and evaporated. The residue was purified on a silica gel column with CH2Cl2 / MeOH (10/1 15/1) as eluent to afford the title compound (15mg).

¹H NMR (DMSO-d6): δ 0.68 (s, 3H, CH3), 3.48-3.58 (m, 1H, H-5 '), 3.68-3.73 (m, 2H, H -5, H-4 '), 3.84 (m, 1H, H-3'), 4.72 (s, 1H, 2'-OH), 4.97-5.03 (m, 2H, 3 '-OH, 5'-OH), 5.45 (br s, 2H, NH2), 6.00 (s, 1H, H-1', 6.28 (d, 1H, J = 3.7Hz, H-5), 6.44 (br s, 2H, NH2) 6.92 (d, 1H, J = 3.7Hz, H-6).

ES MS: 294.1 (MH⁺).

EXAMPLE 36

4-Amino-2-fluoro-7- (2-C-methyl-eD-ribofuranosyl-7H-pyrrolo [2,3-d1-pyrimidine

PL 207 405 B1

<img file="PL207405B1_D0036.tif" />

To a solution of HF / pyridine (70%, 2 ml) diluted with pyridine (1 ml) at -30 ° C was added the compound of Example 36 (60 mg, 0.2 mmol) in 0.5 ml of pyridine followed by tert-nitrite. butyl (36 g, 0.3 mmol). Stirring was continued at -25 ° C for 5 minutes. The solution was then poured into ice-water (5 ml), neutralized with 2N aqueous NaOH, and evaporated to dryness. The residue was purified on a silica gel column with CH2Cl2 / MeOH (20/1 and 10/1) as eluent to afford the title compound.

EXAMPLE 37

4-Amino-5-fluoro-7- (2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine

<img file="PL207405B1_D0037.tif" />

Step A: 4-Acetylamino-7- (2,3,5-tri-O-acetyl-2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine

To a solution of the compound of Step F of Example 2 (280 mg, 1.00 mmol) in pyridine was added acetic anhydride (613 mg, 6.0 mmol). The resulting solution was stirred overnight at ambient temperature, evaporated in vacuo and the resulting crude mixture was purified over silica gel using ethyl acetate / hexane as eluent. Fractions containing the desired product were combined and evaporated in vacuo to give the desired product.

Step B: 4-Acetylamino-5-bromo-7- (2,3,5-tri-O-acetyl-2-C-methyl-eD-ribofuranosyl) -7H-pyrrolo [2,3-d] pyrimidine

To a cooled (0 ° C) solution of the compound from Step A (460 mg, 1.00 mmol) in DMF was added N-bromosuccinimide (178 mg, 1.0 mmol) in DMF. The resulting solution was stirred at 0 ° C for 30 minutes, then at room temperature for an additional 30 minutes. The reactions were quenched by adding methanol and evaporated in vacuo. The resulting crude mixture was purified over silica gel using ethyl acetate / hexane as the eluent. Fractions containing the desired product were combined and evaporated in vacuo to give the desired product.

Step C: 4-Amino-5-1 ^ uoro-7- (2-C-methyl-β-D-ribofuranosyl-7H-pyrrolo [2,3-d] pyrimidine

Butyllithium (2M in hexane) (0.5 mL, 1.00 mmol) was added to a cooled (-78 ° C) solution of the compound from Step B (529 mg, 1.00 mmol) in THF. The resulting solution was stirred at -78 ° C for 30 min. and then quenched by adding N-fluorobenzenesulfonimide (315 mg, 1.00 mmol) in THF. The resulting solution was allowed to come to ambient temperature very slowly and then poured into a stirred mixture of saturated aqueous ammonium chloride solution and dichloromethane. The organic phase was evaporated in vacuo and treated with ammonium hydroxide at 55 ° C in a closed container overnight. The resulting crude mixture was purified over silica gel using a dichloromethane / methanol mixture as eluent. Fractions containing the desired product were combined and evaporated in vacuo to give the desired product.

PL 207 405 B1

BIOLOGICAL TESTS

Assays used to evaluate the inhibition of HCV NS5B polymerase and HCV replication are described below.

The efficacy of compounds of the invention as inhibitors of HCV NS5B RNA-dependent RNA polymerase (RdRp) was assessed in the following tests.

A. HCV NS5B Polymerase Inhibition Assay

This assay was used to evaluate the ability of the nucleoside derivatives of the invention to inhibit the enzymatic activity of the hepatitis C virus (HCV) RNA-dependent RNA polymerase (NS5B) on a heteromeric RNA template.

Procedure:

Composition of the Assay Buffer: (50L total per reaction) mM Tris, pH 7.5

LM EDTA mM DTT mM MgCl2 mM KCl

0.4 U / Ll RNAsin (Promega, 40 units / L stock) 0.75 Lg t500 (500-nt RNA generated using T7 sequence transcription from the NS2 / 3 region of the HCV genome)

1.6 Lg of purified HCV NS5B (21 amino acid cut form from C-terminus)

LM A, C, U, GTP (mixture of nucleoside triphosphates))

[alpha^-32Pl-GTP or [alpha^-33Pl-GTP

Compounds were tested at various concentrations up to a final concentration of 100 LM.

The appropriate volume of reaction buffer was prepared by adding both the enzyme and the t500 matrix. The nucleoside derivatives of the invention were pipetted into 96 wells per plate. A mixture of nucleoside triphosphate (NTP's) containing radiolabeled GTP was prepared and pipetted into 96 wells on the plate. The reaction was initiated by the addition of the enzyme-matrix reaction solution and left at room temperature for 1-2h.

The reaction was stopped by the addition of 20 L 0.5M EDTA, pH 8.0. A blank run was also performed in which the stop solution was added to the NTPs prior to the addition of the reaction buffer.

1l of the quenched reaction mixture was placed on DE 81 filter disks (Whatman) and allowed to dry for 30 minutes. Filters were rinsed with 0.3 M ammonium formate, pH 8 (150 ml / wash until cpm in 1 ml washes was less than 100, typically 6 washes). Filters were read after adding 5 ml of scintillation fluid in a scintillation counter.

The percent inhibition was calculated according to the following equation:

% inhibition = [1- (cpm test reaction - cpm blank) / (cpm control reaction - cpm blank) 1x 100, where cpm is the number of pulses per minute.

Representative compounds tested in the HCV NS5B polymerase assay exhibited an IC50 of less than 100 micromolar.

B. HCV RNA Replication Inhibition Test:

Compounds of the invention were also evaluated for their effect on hepatitis C RNA replication in cultures of hepatoma cells (HuH-7) containing the HCV subgenomic replicon. Details of the test are described below. This replicon assay is a modification of the assay described by V. Lohmann, F. Korner, J.O. Koch, U. Herian, L. Theilmann, and R. Bartenschlager, Replication of a Sub-genomic Hepatitis C Virus RNAs in a Hepatoma Cell Line, Science 285: 110 (1999).

Protocol:

The Ribonuclease protection Scintillation Proximity based-plate Assay-SPA was used. 10,000-40,000 cells were plated in 100-200 µl of medium containing 0.8 mg / ml G418 in a 96-well cytostar plate (Amersham). Test compounds were added to the cells at various concentrations up to 100 LM in 1% DMSO from 0 to 18 h and then cultured for 24-96 h. Cells were fixed (20 minutes, 10% formalin), filtered (20 minutes, 0.25% Triton X-100 / PBS), and hybridized (overnight at 50 °) with a single-stranded labeled probe. ³³The P RNA, complementary to the (+) strand of NS5B (or other genes) contained in the RNA of the viral genome. Cells were washed, treated with RNAse, washed, heated to 65 ° C, and counted in a Top-Count. Inhibition of replication was read as a decrease in cpm.

Human hepatoma HuH-7 cells, which were selected for the content of the subgenomic replicon, carry the cytoplasmic RNA consisting of the 5 'region of HCV that has not been subjected to

NTRs, a neomycin selectable marker, EMCV IRES (internal ribosome binding sites), and HCV nonstructural proteins from NS3 to NS5B followed by a 3'NTR.

Representative compounds tested in the replication assay exhibited an EC50 of less than 100 micromolar.

The nucleoside derivatives of the invention were also assessed for cell toxicity and antiviral specificity in the screening assays described below.

Screening tests:

The ability of the nucleoside derivatives of the invention to inhibit human DNA polymerase was assessed in the following tests.

a. Inhibition of human DNA polymerase alpha and beta.

Reaction Conditions:

pl - volume of reaction

Composition of the Reaction Buffer:

mM Tris-HCl, pH 7.5

200 μg / ml, bovine serum albumin

100 mM KCl mM β-mercaptoethanol mM MgCl2

1.6 pM dA, dG, dC, dTTP a-³³P-dATP

Enzyme and matrix:

0.05 mg / ml fish seed DNA template

0.01 U / pl DNA polymerase α or β

Preparation of DNA template from fish seed

Add 5 µl of 1M MgCl₂ up to 500 μl of activated fish semen DNA (USB 70076);

Heat to 37 ° C and add 30 μl of exonuclease III (65 U / pl-GibcoBRL 18013-011);

Incubate 5 min at 37 ° C;

Quench the reaction by heating to 65 ° C for 10 min;

Distribute 50-100 µl to Bio-spin 6 chromatography columns (Bio-Rad 732-6002) equilibrated with 20 mM Tris-HCl, pH 7.5;

Centrifuge 1000 xg for 4 minutes.

Collect the supernatant and determine the concentration by measuring absorbance at 260 nm.

The template DNA was diluted to the appropriate volume of 20 mM Tris-HCl, pH 7.5, and the enzyme was diluted to the appropriate volume of 20 mM Tris-HCl, containing 2 mM β-mercaptoethanol and 100 mM KCl. The matrix and enzyme were pipetted into microcentrifuge tubes or into a 96-well plate. An enzyme-free blank and a test compound-free control were also prepared using the enzyme dilution buffer or solvent for the test compounds as appropriate. The reaction was initiated using the reaction buffer, the composition of which is as shown above. The reaction was incubated for 1h at 37 ° C. The reaction was quenched by adding 20 µl of 0.5M EDTA. 50 µl of the reaction solution from the stopped reaction was placed on Whatman DE81 filter plates and air dried. The filter plates were rinsed several times with 150 ml of 0.3M ammonium formate, pH 8, until the cpm in 1 ml of the washings was &lt;100. The plates were washed twice with 150 ml of pure ethanol and once with 150 ml of anhydrous ether, dried and counted with 5 ml of scintillation fluid.

The percentage of inhibition was calculated according to the following equation:

% Inhibition = [1- (cpm of reaction test - cpm of blank) / (cpm of control reaction - cpm of blank) 1 x 100.

b. Inhibition of human gamma DNA polymerase

The ability to inhibit human gamma DNA polymerase was measured in a reaction that contained 0.5 ng / pl of the enzyme; 10 µM dATP, dGTP, dCTP, and TTP; 2 pCi / per reaction [a-³³P1-dATP, and 0.4 pg / pl activated fish semen DNA (purchased from US Biochemical) in a buffer containing 20 mM Tris pH 8, 2 mM β-mercaptoethanol, 50 mM KCl, 10 mM MgCl₂, and 0.1 pg / pl BSA. The reaction was run for 1 hour at 37 ° C and was quenched by the addition of 0.5 M EDTA to a final concentration of 142 mM. Product formation was quantified by binding on anion exchange filters and in a scintillation counter. Test compounds were tested up to 50 pM.

The percentage of inhibition was calculated according to the following equation:

% Inhibition = [1- (cpm of reaction tested - cpm of blank) / (cpm of control reaction - cpm of blank)] x 100.

The ability of the nucleoside derivatives of the invention to inhibit HIV infectivity and dissemination was measured by the following tests.

c. HIV infectivity test

Assays were performed using variant HeLa Magi cells expressing both CXCR4 and CCR5 selected for low β-galactosidase (β-gal) background expression. Cells were infected for 48h and production (β-gal) from the linked HIV-1 LTR promoter was assessed using a chemiluminescent substrate (Galactolight Plus, Tropix, Bedford, MA). Inhibitors were titrated (in duplicate) in duplicate serial dilutions starting at 100 µM; percent inhibition for each concentration was calculated relative to infection control.

d. Inhibition of HIV spread

The ability of the compounds of the invention to inhibit human immunodeficiency virus (HIV) dissemination was measured using the method described in US Patent No. 5,413,999 (Macy 9, 1995) and JPVacca, et al. Proc. Natl. Acad. Sci., 91: 4096-4100 (1994).

The nucleoside derivatives of the invention were also tested for cytotoxicity against hepatoma (HuH-7) cell cultures containing the HCV subgenomic replicon in the MTS cell-based assay described below. The HuH-7 cell line is described in H. Nakabayashi, et al., Cancer Res., 42: 3858 (1982).

e. Cytotoxicity test:

Cell cultures were prepared on appropriate media at a concentration of approximately 1.5 x 10⁵cells / ml for 3-day suspension cultures and 5.0 x 10⁴ cells / ml for adherent cultures for 3-day incubation. 99 µl of cell culture was transferred to a 96-well tissue culture plate and 1 µl of a 100-fold concentration of the final test compound in DMSO was added. Plates were incubated for a specified time at 37 ° C and 5% CO 2. Following incubation, 20 μl of CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS) (Promega) was added to each well, and plates were incubated at 37 ° C and 5% CO2 for an additional time up to 3 h. The plates were shaken to mix well and the absorbance was read at 490 nm in a plate reader. A standard curve for suspension cultures was constructed with a known cell number just prior to the addition of the MTS reagent. Metabolically active cells reduce MTS to formazan. Formazan absorbs at 490 nm. The absorbance at 490 nm in the presence of the test compound was compared to the absorbance of cells to which nothing was added. Reference: Cory, AH et al, Use of an aqueous soluble tetrazolium / formazan assay for cell growth assays in culture, Cancer Commun. 3: 207 (1991).

The following tests were used to measure the activity of the compounds of the invention against other RNA-dependent RNA viruses:

a. Assessment of the in vivo antiviral activity of compounds against rhinovirus (cytopathic effect inhibition test)

Test conditions are described in Sidwell and Huffman, Use of disposable microtissue culture plates for antiviral and interferon induction studies. Appl. Microbiol. 22: 797-801 (1971).

Viruses

The type 2 rhinovirus (RV-2), HPG strain with KB cells and medium (0.1% NaHCO₃, without antibiotics) according to Sidwell and Huffman's article. The virus, obtained from ATCC, was derived from a throat swab of an adult male with subacute febrile upper respiratory disease.

Rhinovirus type 9 (RV-9) strain 211 and rhinovirus type 14 (RV-14) strain Tow were also obtained from the American Type Culture Collection (ATCC)) in Rockville, MD. RV-9 was obtained from a human throat lavage, and RV-14 was from a throat swab from a young adult with upper respiratory disease. In both cases, the HeLa Ohio-1 cell line (Dr. Fred Hayden, Univ. Of VA), which is human cervical epithelial carcinoma cells, was used. MEM (Eagle's minimum essential medium) with the addition of 5% Fetal Bovine Serum (FBS) and 0.1% NaHCO3 was used as the growth medium. The antiviral test medium for all three viruses was MEM with 5% FBS, 0.1% NaHCO₃, 50 µg gentamicin / ml and 10 mM MgCl₂.

The highest concentration of compounds according to the invention used in the tests was 2000 µg / ml. The virus was added to the assay plate approximately 5 minutes after the test compound. Appropriate controls have also been prepared. Test plates were incubated in humidified air and 5% CO2 at 37 ° C. In control cells, morphological changes indicative of cytotoxicity were searched for microscopically. For the determination of ED50 (50% of the effective dose) and CC50 (50% of the cytotoxic concentration)

Regression analysis of viral CPE data and toxicity control data was used. The selectivity index (SI) was calculated according to the equation: SI = CC50 + ED50.

b. In Vitro Assessment of Antiviral Activity of Compounds in relation to Dengue, Banzi and Yellow Viruses

Febry (CPE Inhibition Test)

Details of the test are contained in the Sidwell and Huffman reference cited above.

Viruses:

Dengue virus type 2, New Guinea strain, was obtained from the Center for Disease Control. Two African green monkey (Vero) kidney cell lines were used to grow the virus and perform antiviral testing (MA-104). Both yellow fever virus strain 17D from infected mouse brain and Banzi virus strain H 336 from feverish South African boy serum were obtained from ATCC. Vero cells were used for both viruses and for the test.

Cells and Media:

MA-104 cells (BioWhittaker, Inc., Walkersville, MD) and Vero cells (ATCC) were used in medium 199 with 5% FBS and 0.1% NaHCO3 without antibiotics.

The test medium for Dengue, Yellow Fever and Banzi viruses was MEM, 2% FBS, 0.18% NaHCO3 with 50 µg gentamicin / ml.

Testing of the antiviral properties of the test compounds was carried out according to the Sidwell and Huffman guidelines, analogous to the antiviral tests carried out for rhinovirus. For each virus, a corresponding reading of the cytopathic effect (CPE) was obtained after 5-6 days.

c. In Vitro Assessment of Compounds' Antiviral Activity Against West Nile Virus (CPE Inhibition Test)

Details of the test are contained in the Sidwell and Huffman reference cited above. West Nile virus, New York isolate, isolated from cow brain, was obtained from the Center for Disease Control. Vero cells were grown and used as described above. The test medium was MEM, 1% FBS, 0.1% NaHCO₃ with 50 μg gentamicin / ml.

Testing of the antiviral properties of the compounds of the invention was performed according to the Sidwell and Huffman guidelines, similar to the antiviral tests performed for rhinovirus. The appropriate reading of the cytopathic effect (CPE) was obtained after 5-6 days.

d. In vitro evaluation of the antiviral activity of compounds against rhinoviruses, yellow fever viruses, Dengue, Banzi and West Nile viruses (Neutral Red Uptake Assay)

After performing the above CPE inhibition assays, an additional method for detecting cytopathic lesions was used, described in Microtiter Assay for Interferon: Microspectrophotometric Quantitation of Cytopathic Effect, Appl. Environ. Microbiol. 31: 35-38 (1976). A model EL309 microplate reader was used to read the test plates. ED values₅₀ and CD₅₀ calculated as described above.

Example of a pharmaceutical preparation

As an example of an oral composition of a compound of the present invention, 50 mg of the compound of Example 1 or Example 2 were formulated with sufficiently comminuted lactose to a total of 580 to 590 mg for filling size 0 hard gelatin capsules.