WO2003051897A1

WO2003051897A1 - Nucleoside analog libraries and compounds

Info

Publication number: WO2003051897A1
Application number: PCT/US2002/040365
Authority: WO
Inventors: Haoyun An; Jean-Luc Girardet; Kanda Ramasamy; Yili Ding; Zhi Hong
Original assignee: Ribapharm Inc.
Priority date: 2001-12-17
Filing date: 2002-12-17
Publication date: 2003-06-26
Also published as: AU2002357876A1; WO2003051897B1

Abstract

Nucleoside analog libraries are prepared in a combinatorial library approach. In some preferred aspects, diverse heterocyclic bases and/or diverse nucleoside substituents are prepared using solid phase-coupled nucleosides in a series of at least two modification reactions. Particularly preferred compounds include various pyrazolopyrimidine, pyrrolidinopyrimidinone, and benzimidazole nucleoside analogs generated using contemplated libraries, which may be useful in the treatment of various conditions, particularly viral infections and neoplastic diseases.

Description

NUCLEOSIDE ANALOG LIBRARIES AND COMPOUNDS

Priority Claim This application claims priority to US 60/342,452 filed December 17, 2001.

Field of The Invention

The field of the invention is combinatorial nucleoside libraries and related compounds.

Background of The Invention Nucleosides and related compounds interact with many biological targets, and some nucleoside analogues have been used as antimetabolites for treatment of cancers and viral infections. After entry into the cell, many nucleoside analogues can be phosphorylated to monophosphates by nucleoside kinases, and then further phosphorylated by nucleoside monophosphate kinases and nucleoside diphosphate kinases to give nucleoside triphosphates. Once a nucleoside analogue is converted to its triphosphate inside the cell, it can be incorporated into DNA or RNA. Incorporation of certain unnatural nucleoside analogues into nucleic acid replicates or transcripts can interrupt gene expression by early chain termination, or by interfering with function of the modified nucleic acids. In addition, certain nucleoside analogue triphosphates are very potent, competitive inhibitors of DNA or RNA polymerases, which can significantly reduce the rate at which the natural nucleoside can be incorporated. Many anti-HIV nucleoside analogues fall into this category, including 3'-C-azido-3'-deoxythymidine, 2',3'- dideoxycytidine, 2',3 '-dideoxyinosine, and 2',3 '-didehydro-2',3 '-dideoxythymidine.

Various nucleoside analogues can also act in other ways, for example, causing apoptosis of cancer cells and/or modulating immune systems. In addition to nucleoside antimetabolites, a number of nucleoside analogues that show very potent anticancer and antiviral activities act through still other mechanisms. Some well-known nucleoside anticancer drugs are thymidylate synthase inhibitors such as 5-fluorouridine, and adenosine deaminase inhibitors such as 2- chloroadenosine. A well-studied anticancer compound, neplanocin A, is an inhibitor of S- adenosylhomocysteine hydrolase, which shows potent anticancer and antiviral activities.

Many of these nucleoside analogues that can inhibit tumor growth or viral infections are also toxic to normal mammalian cells, primarily because these nucleoside analogues lack adequate selectivity between the normal cells and the virus-infected host cells or cancer cells. l For this reason many otherwise promising nucleoside analogues fail to become therapeutics in the treatment of various diseases.

Selective inhibition of cancer cells or host cells infected by viruses has been an important subject for some time, and tremendous efforts have been made to search for more selective nucleoside analogues. In general, however, a large pool of nucleoside analogues is thought to be necessary in order to identify highly selective nucleoside analogues. Unfortunately, the classical method of synthesizing nucleosides and nucleotides having desired physiochemical properties, and then screening them individually, takes a significant amount of time to identify a lead molecule. Although thousands of nucleoside analogues were synthesized over the past decades, if both sugar and base modifications are considered, many additional analogues are still waiting to be synthesized.

During the last few years, combinatorial chemistry has been used to generate huge numbers of organic compounds, resulting in large compound libraries. If nucleosides could be made through a combinatorial chemistry approach, a large number of nucleoside analogues could be synthesized within months instead of decades, and large nucleoside libraries could be developed.

A combinatorial chemistry approach to nucleosides may also encourage a focus beyond previously addressed biological targets. For example, in the past nucleoside analogues were usually designed as potential inhibitors of DNA or RNA polymerases and several other enzymes and receptors, including inosine monophosphate dehydrogenase, protein kinases, and adenosine receptors. If a vast number of diversified nucleoside analogues could be created, their use may be far beyond these previously recognized biological targets, which would open a new era for the use of nucleoside analogues as human therapeutics.

The generation of combinatorial libraries of chemical compounds by employing solid phase synthesis is well known in the art. For example, Geysen, et al. (Proc. Natl. Acac. Sci. USA, 3998 (1984)) describes the construction of a multi-amino acid peptide library; Houghton, et al. (Nature, 354, 84 (1991)) describes the generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery; and Lam, et al. (Nature, 354, 82 (1991)) describes a method of synthesis of linear peptides on a solid support such as polystyrene or polyacrylamide resin. Although a combinatorial chemistry approach has proven to work well with many types of compounds, there are certain hurdles to the generation of nucleoside libraries. Among numerous other 'difficulties, most nucleoside analogues contain a sugar moiety and a nucleoside base, which are linked together through a glycosidic bond. The formation of the glycosidic bond can be achieved tlirough a few types of condensation reactions. However, most of the reactions do not give a very good yield of desired products, which may not be suitable to generations of nucleoside libraries. Moreover, the glycosidic bonds in many nucleosides are in labile to acidic condition, and many useful reactions in combinatorial chemistry approaches cannot be used in the generation of nucleoside analogue libraries. As a result, many researchers focused their attention to areas in pharmaceutical chemistry that appear to present easier access to potential therapeutic molecules, and there seems to be a lack of methods for generating libraries of nucleosides and nucleotides using solid phase synthesis. Therefore, there is still a need to provide methods for generation of nucleoside and nucleotide libraries.

Summary of the Invention The present invention is directed to nucleoside analog libraries and compounds represented in and derived from these libraries. In one aspect of the inventive subject matter, contemplated libraries and compounds include pyrazolopyrimidine nucleosides having a general structure according to Formula 1

Formula 1

wherein the substituents are described in the respective portion of the detailed description below.

In another aspect of the inventive subject matter, contemplated libraries and compounds include pyrrolidinopyrimidinone nucleosides having a general structure according to Formula 3

Formula 3

In a further aspect of the inventive subject matter, contemplated libraries and compounds include benzimidazole nucleosides having a general structure according to Formulae 7, 8, and 11

Formula 5A Formula 5B Formula 5E

In a still further aspect of the inventive subject matter, it is preferred that in all of the contemplated libraries and compounds the sugar portion in the nucleoside is a ribofuranose, a substituted ribofuranose, a carbocyclic ring system, and/or an arabinose, wherein the sugar is in a D-configuration or in an L-configuration.

Various objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of preferred embodiments of the invention.

Detailed Description

The term "nucleoside library" as used herein refers to a plurality of chemically distinct nucleosides, nucleotides, nucleoside analogs, and/or nucleotide analogs wherein at least some of the nucleosides, nucleotides, nucleoside analogs, and/or nucleotide analogs include, or have been synthesized from a common precursor. For example, a plurality of nucleosides, nucleotides, nucleoside analogs, and/or nucleotide analogs that were prepared using l'-azido or 1 '-amino ribofuranose as a building block/precursor is considered a nucleoside library under the scope of this definition. Therefore, the term "common precursor" may encompass a starting material in a first step in a synthesis as well as a synthesis intermediate (i.e., a compound derived from a starting material). In another example, at least one step in the synthesis of one of the nucleosides, nucleotides, nucleoside analogs, and/or nucleotide analogs is concurrent with at least one step in the synthesis of another one of the nucleosides, nucleotides, nucleoside analogs, and/or nucleotide analogs, and synthesis is preferably at least partially automated. In contrast, a collection of individually synthesized nucleosides, nucleotides, nucleoside analogs, and/or nucleotide analogs, and especially a collection of compounds not obtained from a nucleoside library, is not considered a nucleoside library because such nucleosides, nucleotides, nucleoside analogs, and/or nucleotide analogs will not have a common precursor, and because such nucleosides, nucleotides, nucleoside analogs, and/or nucleotide analogs are not concurrently produced.

It is further generally contemplated that the complexity of contemplated libraries is at least 20 distinct nucleosides, nucleotide, nucleoside analogs, and/or nucleotide analogs, more typically at least 100 distinct nucleosides, nucleotide, nucleoside analogs, and/or nucleotide analogs, and most typically at least 1000 distinct nucleosides, nucleotide, nucleoside analogs, and/or nucleotide analogs. Consequently, a typical format of a nucleoside library will include multi-well plates, or a plurality of small volume (i. e. , less than 1 ml) vessels coupled to each other. The term "library compound" as used herein refers to a nucleoside, nucleotide, nucleoside analog, and/or nucleotide analog within a nucleoside library.

As also used herein, the terms "heterocycle" and "heterocyclic base" are used interchangeably herein and refer to any compound in which a plurality of atoms form a ring via a plurality of covalent bonds, wherein the ring includes at least one atom other than a carbon atom. Particularly contemplated heterocyclic bases include 5- and 6-membered rings with nitrogen, sulfur, or oxygen as the non-carbon atom (e.g., imidazole, pyrrole, triazole, dihydropyrimidine). Further contemplated heterocylces may be fused (i.e., covalently bound) to another ring or heterocycle, and are thus termed "fused heterocycle" or "fused heterocyclic base" as used herein. Especially contemplated fused heterocycles include a 5-membered ring fused to a 6-membered ring (e.g., purine, pyrrolo[2,3-d]pyrimidine), and a 6-membered ring fused to another 6- membered or higher ring (e.g., pyrido[4,5-d]pyrimidine, benzodiazepine). Examples of these and further preferred heterocyclic bases are given below. Still further contemplated heterocyclic bases may be aromatic, or may include one or more double or triple bonds. Moreover, contemplated heterocyclic bases and fused heterocycles may further be substituted in one or more positions (see below).

As further used herein, the term "sugar" refers to all carbohydrates and derivatives thereof, wherein particularly contemplated derivatives include deletion, substitution or addition of a chemical group or atom in the sugar. For example, especially contemplated deletions include 2'-deoxy and/or 3'-deoxy sugars. Especially contemplated substitutions include replacement of the ring-oxygen with sulfur or methylene, or replacement of a hydroxyl group with a halogen, an amino-, sulfhydryl-, or methyl group, and especially contemplated additions include methylene phosphonate groups. Further contemplated sugars also include sugar analogs (i.e., not naturally occurring sugars), and particularly carbocyclic ring systems. The term " carbocyclic ring system" as used herein refers to any molecule in which a plurality of carbon atoms form a ring, and in especially contemplated carbocyclic ring systems the ring is formed from 3, 4, 5, or 6 carbon atoms. Examples of these and further preferred sugars are given below.

The term "nucleoside" refers to all compounds in which a heterocyclic base is covalently coupled to a sugar, and an especially preferred coupling of the nucleoside to the sugar includes a

Cl'-(glycosidic) bond of a carbon atom in a sugar to a carbon- or heteroatom (typically nitrogen) in the heterocyclic base. The term "nucleoside analog" as used herein refers to all nucleosides in which the sugar is not a ribofuranose and/or in which the heterocyclic base is not a naturally occurring base (e.g., A, G, C, T, I, etc.). Similarly, the term "nucleotide" refers to a nucleoside to which a phosphate group is coupled to the sugar. Likewise, the term "nucleotide analog" refers to a nucleoside analog to which a phosphate group is coupled to the sugar.

It should further be particularly appreciated that the terms nucleoside, nucleotide, nucleoside analog, and/or nucleotide analog also includes all prodrug forms of a nucleoside, nucleotide, nucleoside analog, and/or nucleotide analog, wherein the prodrug form may be activated/converted to the active drug/nucleoside, nucleotide, nucleoside analog, and/or nucleotide analog in one or more than one step, and wherein the activation/conversion of the prodrug into the active drug/nucleoside, nucleotide, nucleoside analog, and/or nucleotide analog may occur intracellularly or extracellularly (in a single step or multiple steps). Especially contemplated prodrug forms include those that confer a particular specificity towards a diseased or infected cell or organ, and exemplary contemplated prodrug forms are described in "Prodrugs" by Kenneth B. Sloan (Marcel Dekker; ISBN: 0824786297), "Design of Prodrugs" by Hans Bundgaard (ASIN: 044480675X), or in copending US application number 09/594410, filed 06/16/2000, all of which are incorporated by reference herein. Particularly suitable prodrug forms of the above compounds may include a moiety that is covalently coupled to at least one of the C2'-OH, C3'-OH, and C5'-OH, wherein the moiety is preferentially cleaved from the compound in a target cell (e.g., Hepatocyte) or a target organ (e.g., liver). While not limiting to the inventive subject matter, it is preferred that cleavage of the prodrug into the active form of the drug is mediated (at least in part) by a cellular enzyme, and particularly receptor, transporter and cytochrome-associated enzyme systems (e.g., CYP-system).

Especially contemplated prodrugs comprise a cyclic phosphate, cyclic phosphonate and/or a cyclic phosphoamidates, which are preferentially cleaved in a hepatocyte to produce contemplated compounds. There are numerous such prodrugs known in the art, and all of those are considered suitable for use herein. However, especially contemplated prodrug forms are disclosed in WO 01/47935 (Novel Bisamidate Phosphonate Prodrugs), WO 01/18013 (Prodrugs For Liver Specific Drug Delivery), WO 00/52015 (Novel Phosphorus-Containing Prodrugs), and WO 99/45016 (Novel Prodrugs For Phosphorus-Containing Compounds), all of which are incorporated by reference herein. Consequently, especially suitable prodrug forms include those targeting a hepatocyte or the liver.

Still further particularly preferred prodrugs include those described by Renze et al. in

Nucleosides Nucleotides Nucleic Acids 2001 Apr-M;20(4-7):931-4, by Balzarini et al. in Mol Pharmacol 2000 Nov;58(5):928-35, or in U.S. Pat. No. 6,312,662 to Erion et al., U.S. Pat. No. 6,271,212 to Chu et al, U.S. Pat. No. 6,207,648 to Chen et al, U.S. Pat. No. 6,166,089 and U.S. Pat. No. 6,077,837 to Kozak, U.S. Pat. No. 5,728,684 to Chen, and published U.S. Patent Application with the number 20020052345 to Erion, all of which are incorporated by reference herein. Alternative contemplated prodrugs include those comprising a phosphate and/or phosphonate non-cyclic ester, and an exemplary collection of suitable prodrugs is described in U.S. Pat. No. 6,339,154 to Shepard et al, U.S. Pat. No. 6,352,991 to Zemlicka et al., and U.S. Pat. No. 6,348,587 to Schinazi et al. Still further particularly contemplated prodrug forms are described in FASEB J. 2000 Sep;14(12): 1784-92, Pharm. Res. 1999, Aug 16:8 1179-1185, and Antimicrob Agents Chemother 2000, Mar 44:3 477-483, all of which are incorporated by reference herein. The terms "alkyl" and "unsubstituted alkyl" are used interchangeably herein and refer to any linear, branched, or cyclic hydrocarbon in which all carbon-carbon bonds are single bonds. The terms "alkenyl" and "unsubstituted alkenyl" are used interchangeably herein and refer to any linear, branched, or cyclic alkyl with at least one carbon-carbon double bond. Furthermore, the terms "alkynyl" and "unsubstituted alkynyl" are used interchangeably herein and refer to any linear, branched, or cyclic alkyl or alkenyl with at least one carbon-carbon triple bond. The terms "aryl" and "unsubstituted aryl" are used interchangeably herein and refer to any aromatic cyclic alkenyl or alkynyl. The term "alkaryl" is employed where an aryl is covalently bound to an alkyl, alkenyl, or alkynyl.

The term "substituted" as used herein refers to a replacement of an atom or chemical group (e.g., H, NH₂, or OH) with a functional group, and particularly contemplated functional groups include nucleophilic groups (e.g., -NH₂, -OH, -SH, -NC, etc.), electrophilic groups (e.g., C(O)OR, C(X)OH, etc.), polar groups (e.g., -OH), non-polar groups (e.g., aryl, alkyl, alkenyl, alkynyl, etc.), ionic groups (e.g., -Ntfe^"1"), and halogens (e.g., -F, -CI), and all chemically reasonable combinations thereof. Thus, the term "functional group" as used herein refers to a nucleophilic group (e.g., -NH₂, -OH, -SH, -NC, -CN etc.), an electrophilic group (e.g., C(O)OR, C(X)OH, C(Halogen)OR, etc.), a polar group (e.g., -OH), a non-polar group (e.g., aryl, alkyl, alkenyl, alkynyl, etc.), an ionic group (e.g., -NH₃ ), and a halogen.

Contemplated Sugars It is contemplated that suitable sugars will have a general formula of C_nH _nO_n, wherein n is between 2 and 8, and wherein (where applicable) the sugar is in the D- or L-configura- tion. Moreover, it should be appreciated that there are numerous equivalent modifications of such sugars known in the art (sugar analogs), and all of such modifications are specifically included herein. For example, some contemplated alternative sugars will include sugars in which the heteroatom in the cyclic portion of the sugar is an atom other than oxygen (e.g., sulfur, carbon, or nitrogen) analogs, while other alternative sugars may not be cyclic but in a linear (open-chain) form. Suitable sugars may also include one or more double bonds. Still further specifically contemplated alternative sugars include those with one or more non-hydroxyl substituents, and particularly contemplated substituents include mono-, di-, and triphosphates (preferably as C₅' esters), alkyl groups, alkoxy groups, halogens, amino groups and amines, sulfur-containing substituents, etc. Particularly contemplated modifications include substituted ribofuranoses, wherein the substituent on the substituted ribofuranose is a -CR substituent on at least at one of the 2' and 3' carbon atom, with R being C_1-10 alkyl, alkenyl, alkynyl, aryl, heterocycle, CF₃, CF₂H, CC1₃, CC1₂H, CH₂OH, CN, COOR', and CONHR', and with R' being C_1-10 alkyl, alkenyl, alkynyl, aryl. It is still further contemplated that all contemplated substituents (hydroxyl substituents and non-hydroxyl substituents) may be directed in the alpha or beta position.

Numerous of the contemplated sugars and sugar analogs are commercially available. However, where contemplated sugars are not commercially available, it should be recognized that there are various methods known in the art to synthesize such sugars. For example, suitable protocols can be found in "Modern Methods in Carbohydrate Synthesis" by Shaheer H. Khan (Gordon & Breach Science Pub; ISBN: 3718659212), in U.S. PatNos. 4,880,782 and 3,817,982, in WO88/00050, or in EP199,451. An exemplary collection of further contemplated sugars and sugar analogs is depicted below, wherein all of the exemplary sugars may be in D- or L- configuration, and wherein at least one of the substituents may further be in either alpha or beta orientation.

X, Y,Z = O, S, Se,NH,NR, CH₂, CHR, P(O), P(0)OR

R = H,OH, NHR, halo, CH₂OH, COOH, N₃, alkyl, aryl, alkynyl, heterocycles, OR, SR, P(0)(OR)₂

OCOR, NHCOR, NHS0₂R, NH₂NH₂, amidine, substituted amidine, quanidine, substituted gyanidine

An especially contemplated class of sugars comprises alkylated sugars, wherein one or more alkyl groups (or other substituents, including alkenyl, alkynyl, aryl, halogen, CF₃, CHF₂, CC1₃, CHC1₂, N₃, NH₂, etc.) are covalently bound to sugar at the C'ι, C'₂,C'₃,C'₄, or C'₅ atom. In such alkylated sugars, it is especially preferred that the sugar portion comprises a furaiiose (most preferably a D- or L-ribofuranose), and that at least one of the alkyl groups is a methyl group. Of course, it should be recognized that the alkyl group may or may not be substituted with one or more substituents. One exemplary class of preferred sugars is depicted below:

in which B is hydrogen, hydroxyl, or a heterocyclic base, R is independently hydrogen, hydroxyl, substituted or unsubstituted alkyl (branched, linear, or cyclic), with R including between one and twenty carbon atoms.

Contemplated Heterocyclic Bases It is generally contemplated that all compounds in which a plurality of atoms (wherein at least one atom is an atom other than a carbon atom) form a ring via a plurality of covalent bonds are considered a suitable heterocyclic base. However, particularly contemplated heterocyclic bases have between one and three rings, wherein especially preferred rings include 5- and 6- membered rings with nitrogen, sulfur, or oxygen as the non-carbon atom (e.g., imidazole, pyrrole, triazole, dihydropyrimidine). Further contemplated heterocylces may be fused (i.e., covalently bound) to another ring or heterocycle, and are thus termed "fused heterocycle" as used herein. Especially contemplated fused heterocycles include a 5-membered ring fused to a 6- membered ring (e.g., purine, pyrrolo[2,3-d]pyrimidine), and a 6-membered ring fused to another 6-membered or higher ring (e.g., pyrido[4,5-d]pyrimidine, benzodiazepine). While cytidine heterocyclic bases are especially contemplated, an exemplary collection of alternative heterocyclic bases is depicted below, wherein all of the depicted heterocyclic bases may further include one or more substituents, double and triple bonds, and any chemically reasonable combination thereof. It should further be appreciated that all of the contemplated heterocyclic bases may be coupled to contemplated sugars via a carbon atom or a non-carbon atom in the heterocyclic base.

Contemplated Solid Phases

It is generally contemplated that all known types of solid phases are suitable for use herein, so long as contemplated nucleosides (or sugar, or heterocyclic base) can be coupled to such solid phases, and so long as the coupled nucleoside (or sugar, or heterocyclic base) will remain coupled to the solid phase during at least one chemical reaction on the nucleoside (or sugar, or heterocyclic base). Especially contemplated solid phases (i.e., solid supports) include Merrifield resins, ArgoGel (available from Argonaut, San Francisco, CA), Sasrin resin (a polystyrene resin available from Bachem Bioscience, Switzerland), TentaGel S AC, TentaGel PHB, or TentaGel S NH₂ resin (polystyrene-polyethylene glycol copolymer resins available from Rappe Polymere, Tubingen, Germany). Alternatively, contemplated solid supports may also include glass, as described in U. S. Pat. No. 5,143,854. Another preferred solid support comprises a "soluble" polymer support, which may be fabricated by copolymerization of polyethylene glycol, polyvinylalcohol, or polyvinylalcohol with polyvinyl pyrrolidine or derivatives thereof (e.g., see Janda and Hyunsoo (1996) Methods Enzyrnol. 267:234-247; Gravert and Janda (1997) Chemical Reviews 97:489-509; and Janda and Hyunsoo, PCT publication No. _Λ WO 96/03418).

Consequently, it should be recognized that there are numerous methods of coupling nucleosides, sugars, or heterocyclic bases to solid phases that may be appropriate, and a particular method will generally depend on the particular type of solid phase and/or type of sugar. Thus, all of such known methods are contemplated suitable for use herein, and exemplary suitable solid phase coupling reactions are described, for example, in "Organic Synthesis on Solid Phase - Supports, Linkers, Reactions" by Florencio Zaragoza Dorwald et al. John Wiley & Sons; ISBN: 3527299505, or in "Solid-Phase Synthesis and Combinatorial Technologies" by Pierfausto Seneci, John Wiley & Sons; ISBN: 0471331953.

Contemplated Combinatorial Reactions

It is generally contemplated that all known types of combinatorial reactions and/or reaction sequences may be used in conjunction with the teaching presented herein so long as such combinatorial reactions between a substrate and at least two distinct reagents will result in at least two distinct products. Contemplated combinatorial reactions and/or reaction sequences may therefore be performed sequentially, in parallel, or in any chemically reasonable combination thereof. It is still further contemplated that suitable combinatorial reactions and/or reaction sequences may be performed in a single compartment or multiple compartments. Preferred combinatorial reactions and/or reaction sequences include at least one step in which a substrate or reaction intermediate is coupled to a solid phase (which may include the wall of the reaction compartment or a solid or soluble polymers), and that the solid phase is physically separated from another substrate on another solid phase. While not limiting to the inventive subject matter, it is generally preferred that contemplated solid phase synthesis is at least partially automated. There are numerous methods and protocols for combinatorial chemistry known in the art, and exemplary suitable protocols and methods are described in "Solid-Phase Synthesis and Combinatorial Technologies" by Pierfausto Seneci (John Wiley & Sons; ISBN: 0471331953) or in "Combinatorial Chemistry and Molecular Diversity in Drug Discovery" by Eric M. Gordon and James F. Kerwin (Wiley-Liss; ISBN: 0471155187).

Contemplated Libraries and Nucleosides

The inventors discovered that nucleoside analog libraries can be prepared in various combinatorial library approaches, including libraries in which diverse heterocyclic bases and/or diverse nucleoside substituents are prepared from precursor nucleosides (or modified sugars) that are derivatized in subsequent and/or parallel modification reactions.

Pyrazolopyrimidine Libraries

The inventors discovered that pyrazolopyrimidine libraries can be synthesized by coupling a (e.g., commercially available) pyrazolopyrimidine to a sugar using coupling methods well lαiown in the art. The so generated nucleoside analog is then derivatized with a leaving group, preferably a halogen (e.g., bromine viaNBS) to the resulting derivatized pyrazolopyrimidine nucleoside analog, which may then be subjected to a reaction with a reagent (e.g., a nucleophilic reagent) that replaces the halogen with a desired substituent as depicted in exemplary synthetic Scheme 1 below.

Scheme I

Further diversification can be achieved by replacing the proton on the NH in the pyrazole ring with a second set of substituents, and/or by replacing at least one of the carbonyl oxygens in the pyrimidine portion of the base with a leaving group (e.g., TPS-C1) that is subsequently replaced with a third set of substituents. Alternatively, pyrazolopyrimidine nucleoside analogs may be prepared from coupling an appropriately substituted pyrazolopyrimidine with a suitable sugar to yield the corresponding N-substituted an/or carbonyl-substituted nucleoside analog.

With respect to the sugar, it should be appreciated that the particular nature of the sugar is not limiting to the inventive subject matter. Consequently, numerous alternative sugars are also appropriate, and particularly contemplated alternative sugars include various substituted ribofuranoses, carbocyclic ring systems with 5 or 6 carbon atoms, and arabinose, wherein the sugar is in D- or L-configuration. Further suitable sugars include those described in the section entitled "Contemplated Sugars", and it is especially contemplated that where the sugar has a C₂' and/or C₃' substituent other than a hydroxyl group, such alternative sugars may include hydrogen, a halogen, or an azide group in at least one of these positions (in either alpha or beta orientation). Moreover, the coupling of the heterocyclic base to the sugar may be in a position other than the exposition, and it is especially contemplated that where the sugar is a pentose or hexose, alternative positions include the C₂' and C₃'-position. There are numerous protocols lαiown in the art to couple a heterocyclic base to a sugar, and all of such methods are considered suitable for use herein (see e.g., "Modern Methods in Carbohydrate Synthesis" by Shaheer H. Khan (Gordon & Breach Science Pub; ISBN: 3718659212).

Consequently, the nature of protecting groups for the sugar may vary considerably, and while it is particularly contemplated that suitable protection groups include benzyl-, acetyl-, and TBDMS groups, numerous alternative protection groups are also considered suitable. Among other groups, a collection of appropriate alternative protection groups and their reactions is described in Protective Groups in Organic Synthesis by Peter G. M. Wuts, Theodora W. Greene, John Wiley & Sons; ISBN: 0471160199.

Furthermore, it is contemplated that at least some of the reactions may be carried out while the nucleoside is coupled to a solid phase. Suitable solid phases and methods of coupling the solid phase to the nucleoside will at least in part depend on the particular sugar and position of coupling. Therefore, it is contemplated that all known solid phases are suitable for use in conjunction with the teachings presented herein, and exemplary suitable solid phases are described, for example, in Organic Synthesis on Solid Phase - Supports, Linkers, Reactions; by Florencio Zaragoza Dorwald et al. John Wiley & Sons; ISBN: 3527299505, or in Solid-Phase Synthesis and Combinatorial Technologies by Pierfausto Seneci, John Wiley & Sons; ISBN: 0471331953. Preferred solid phases, however, include Merrifield resins, ArgoGel (available from Argonaut, San Francisco, CA), Sasrin resin (a polystyrene resin available from Bachem Bioscience, Switzerland), TentaGel S AC, TentaGel PHB, or TentaGel S NH₂ resin

(polystyrene-polyethylene glycol copolymer resins available from Rappe Polymere, Tubingen, Germany).

Similarly, it is contemplated that the chemical nature of contemplated heterocyclic bases need not be restricted to the heterocyclic base in Scheme 1 above, and suitable alternative heterocyclic bases include various substituted pyrazolopyrimidines. Thus an exemplary alternative heterocyclic base may have the structure

wherein R_ls R₂, and R₃ are independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, a substituted alkynyl, NH₂, NHNH₂, NHR', NHOH, NHOR', NHNHCONH₂, NHNHCONHNH₂, SR', CN, C(NH)NH₂, guanidine, hydroxyguanidine, mercaptoguanidine, COR', OR', CR', OH, and SH; wherein R' is selected from the group consisting of hydrogen, an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, a substituted alkynyl, an aryl, a substituted aryl.

It is further contemplated that suitable halogens may be introduced into the heterocyclic base using various protocols well lαiown in the art. For example, Br may be introduced using NBS following standard procedures as described elsewhere (e.g., Veliz and Beal in J. Org.

Chem. 2001, 66, 8592-8598). Depending on the particular configuration of suitable heterocyclic bases, it should be recognized that contemplated halogens may be introduced at various positions. However, it is generally preferred that the halogen is introduced into the pyrazole ring system.

With respect to a first set of substituents, it is contemplated that all substituents are appropriate that can replace the halogen in the pyrazole portion of the heterocyclic base, and particularly preferred substituents include nucleophilic reagents (e.g., numerous primary and secondary amines as depicted in Scheme 1 above). Further especially preferred reagents include reagents that can participate in a Heck reaction (e.g., R-C≡≡CH), Suzuki reaction (e.g., arylB(OH)₂), or Stille reaction (e.g., arylSnBu₃). Reaction conditions for all such reactions are well lαiown in the art (see e.g., Can. J. Chem. (2000), Vol. 78 (7): 957-962, or Tetrahedron (2001) Vol., 57(14): 2787-2789).

Thus, especially preferred reagents include R-C≡CH, wherein R is alkyl, alkenyl, alkynyl, aryl, and alkaryl, all of which may further be substituted, and ArSnBu3, wherein R is aryl and aralkyl, both of which may further be substituted. It is generally contemplated that almost all such reagents are commercially available. However, where a particular reagent is not commercially available, it should be recognized that such reagents can be prepared from commercially available precursors without expenditure of undue experimentation (see e.g., Advanced Organic Chemistry: Structure and Mechanisms (Part A) by Francis A. Carey, Richard J. Su dberg; Plenum Pub Corp; ISBN: 0306462435; or Advanced Organic Chemistry : Reactions and Synthesis (Part B) by Francis Carey, Richard J. Sundberg; Plenum Pub Corp; ISBN: 0306434571, or Compendium of Organic Synthetic Methods, Volume 9, by Michael B. Smith, John Wiley & Sons; ISBN: 0471145793).

Similarly, it is contemplated that the second and third set of substituents may vary considerably, however, it is generally preferred that the second and third set of substituents will include nucleophilic reagents, and particularly nucleophilic reagents with a NH₂ group. For example, where the second reagent is a reagent that replaces a proton of the pyrazole-NH group, particularly contemplated reagents include an electrophilic center. In another example, where the third reagent is a reagent that replaces a TPS-Ieaving group, it is contemplated that suitable reagents generally include all nucleophilic reagents, and especially contemplated reagents include nitrogen-containing nucleophiles (e.g. , RNH₂, NH₂OH, NH₂NHCONH₂), various thiols (e.g., RSH), and various alcohols (e.g., R-OH).

Consequently, a pyrazolopyrimidine library may comprise a plurality of compounds according to Formula 1 A, wherein a first compound of the plurality of compounds has a first set of substituents A, Ri, R₂, and R₃ wherein a second compound of the plurality of compounds has a second set of substituents A,

and R₃

Formula 1A

wherein A is a protected or unprotected sugar bound to a solid phase, R_ls R₂, and R₃ are independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, a substituted alkynyl, NH₂, NHNH₂, NHR', NHOH, NHOR', NHNHCONH₂, NHNHCONHNH₂, SR', CN, C(NH)NH₂, guanidine, hydroxyguanidine, mercaptoguanidine, COR', OR', CR', OH, and SH; wherein R' is selected from the group consisting of hydrogen, an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, a substituted alkynyl, an aryl, a substituted aryl; and wherein not all of the substituents A, Ri, R₂, and R₃ in the first set are the same as the substituents A, R_1? R₂, and R₃ in the second set.

Thus, contemplated pyrazolopyrimidine nucleosides may have a structure according to Formula IB

. Formula IB

wherein A is a sugar, and Rj, R₂, and R₃ are independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, a substituted alkynyl, NH₂, NHNH₂, NHR', NHOH, NHOR', NHNHCONH₂, NHNHCONHNH₂, SR', CN, C(NH)NH₂, guanidine, hydroxyguanidine, mercaptoguanidine, COR', OR', CR', OH, and SH; and wherein R' is selected from the group consisting of hydrogen, an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, a substituted alkynyl, an aryl, a substituted aryl.

Pyrrolidinopyrimidinone Nucleoside Libraries

The inventors further discovered that pyrrolidinopyrimidinone nucleoside libraries can be generated using a procedure as outlined in Scheme 2 below, in which a 5-alkynyl substituted uracil nucleoside (or population of distinct alkynyl substituted uracil nucleosides) is bound to a solid phase and converted to the corresponding protected 5-alkynyl substituted uracil. The so protected and bound 5-alkynyl substituted uracil is further reacted with a leaving group (e.g., nitrotriazole) to replace at least one carbonyl oxygen with the leaving group. In a further reaction, the leaving group is then replaced by a nucleophilic reagent (e.g., primary or secondary amine) to yield the corresponding alkynyl substituted N-substituted cytidine. The triple bond of the alkynyl substituent may then be employed as a'reactant with the nitrogen in the N-substituent to form the pyrrolidino ring, and subsequent deprotection and cleavage from the solid support will then yield the disubstituted pyrrolidinopyrimidinone nucleoside.

Scheme 2

With respect to the sugar, it should be appreciated that the particular nature of the sugar is not limiting to the inventive subject matter. Consequently, numerous alternative sugars are also appropriate, and particularly contemplated alternative sugars include various substituted ribofuranoses, carbocyclic ring systems with 5 or 6 carbon atoms, and arabinose, wherein the sugar is in a D-configuration or in an L-configuration. Further suitable sugars include those described in the section entitled "Contemplated Sugars", and it is especially contemplated that where the sugar has a C₂' and/or C₃' substituent other than a hydroxyl group, such alternative sugars may include hydrogen, a halogen, SH, SCH₃, O-alkyl, O-alkenyl, O-alkynyl, or an azide group in at least one of these positions (in either alpha or beta orientation). Moreover, the coupling of the heterocyclic base to the sugar may be in a position other than the exposition, and it is especially contemplated that where the sugar is a pentose or hexose, alternative positions include the C₂' and C₃ '-position.

Furthermore, the solid phase and methods of coupling the solid phase to the nucleoside will at least in part depend on the particular sugar and position of coupling. Therefore, it is contemplated that all known solid phases are suitable for use in conjunction with the teachings presented herein, and exemplary suitable solid phases are described, for example, in Organic Synthesis on Solid Phase - Supports, Linkers, Reactions; by Florencio Zaragoza Dorwald et al. John Wiley & Sons; ISBN: 3527299505, or in Solid-Phase Synthesis and Combinatorial Technologies by Pierfausto Seneci, John Wiley & Sons; ISBN: 0471331953. Preferred solid phases, however, include Merrifield resins, ArgoGel (available from Argonaut, San Francisco, CA), Sasrin resin (a" polystyrene resin available from Bachem Bioscience, Switzerland), TentaGel S AC, TentaGel PHB, or TentaGel S NH resin (polystyrene-polyethylene glycol copolymer resins available from Rappe Polymere, Tubingen, Germany).

With respect to contemplated heterocyclic bases, it should be appreciated that numerous 5-alkynyl substituted uracil nucleosides are commercially available, or can be prepared from commercially available uracil following procedures well known in the art (see e.g., Brancale et al. in Antiviral Chemistry & Chemotherapy (2000) 11 :383— 393). Especially preferred 5-alkynyl substituents include alkynyls coupled to hydrogen, an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, and a substituted alkynyl, an aryl and a substituted aryl. Moreover, it should be appreciated that contemplated heterocyclic bases need not be limited to a 5-substituted uracil, and suitable heterocyclic bases include various 5- and 6-membered rings, fused heterocyclic bases, and heterocyclic bases with heteroatoms other than nitrogen. Exemplary heterocyclic bases are depicted in the section "Contemplated Heterocyclic Bases" above.

Preferred NH₂-containing reagents include hydroxylamine, NH₃, primary amines (e.g., R- NH₂, R-NHNH₂, R-O-NH₂, RSO₂NH₂), and secondary amines (e.g., R^NH), wherein R, R¹, and R² are independently an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, a substituted alkynyl, an aryl or a substituted aryl. However, it should be appreciated that numerous alternative amines are also contemplated. Consequently, the exchange of the amine for the leaving group will transform the substituted uracil into an N-substituted cytidine. Moreover, further contemplated reagents need not necessarily be limited to NH₂-containing reagents, and particularly contemplated alternative reagents include numerous nucleophilic reagents, including R-OH, R-SH, and Grignard reagents.

Therefore, contemplated nucleoside libraries may comprise a plurality of nucleosides according to Formula 3 A, wherein a first compound of the plurality of compounds has a first set of substituents A, Ri andR₂ wherein a second compound of the plurality of compounds has a second set of substituents A, Ri and R₂

Formula 3 A

wherein A is a protected or unprotected sugar (e.g., a ribofuranose, a substituted ribofuranose, a carbocyclic ring system, and an arabinose, wherein the sugar is in a D- configuration or in an L-configuration) that is covalently bound to a solid phase, wherein _\ and i

R₂ are independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, and a substituted alkynyl, an aryl and a substituted aryl, and wherein not all of the substituents A, R_ls R₂, and R₃ in the first set are the same as the substituents A, Ri, R₂, and R₃ in the second set.

Where a 2'-substituted ribofuranose is particularly preferred, contemplated nucleoside libraries may comprise a plurality of nucleosides according to Formula 4 A, wherein a first compound of the plurality of compounds has a first set of substituents Ri, R₂, and R₃ wherein a second compound of the plurality of compounds has a second set of substituents R_1; R₂ and R₃ ^~

Formula 4A

wherein Ri and R2 are independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, and a substituted alkynyl, an aryl and a substituted aryl, wherein R₃ is selected from the group consisting of hydrogen, OH, OCH₃, SH, SCH₃, 0-alkyl, O-alkenyl, O-alkynyl, alkyl, (and especially CH ) and wherein • is a solid phase, and wherein not all of the substituents A, R_l5 R₂, and R₃ in the first set are the same as the substituents A, R_ls R₂, and R₃ in the second set.

Thus, contemplated compounds may also have a structure according to Formula 3B wherein A is a protected or unprotected sugar, and wherein Ri and R₂ are independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, and a substituted alkynyl, an aryl and a substituted aryl

Formula 3B

and especially contemplated compounds may also have a structure according to

Formula 4B

Formula 4B

wherein Ri and R are independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, and a substituted alkynyl, an aryl and a substituted aryl, and wherein R₃ is selected from the group consisting of hydrogen, OH, OCH₃, SH, SCH₃, O-alkyl, O-alkenyl, O-alkynyl.

Benzimidazole Nucleoside Libraries

The inventors still further discovered that benzimidazole nucleoside libraries can be prepared using a procedure as outlined in Scheme 3 below, in which a halogenated benzimidazole nucleoside (preferably halogenated with a chlorine and a bromine) is bound to a solid phase. In one reaction, the chlorine is substituted with a first reagent comprising a nucleophilic group, and in a further reaction, the bromine is replaced by a second reagent (preferably in a Heck or Stille type reaction).

πucleophile

X = CI, Br, I R-i = amine or sulfide

P (0) reaction

R₂ = alkyl, alkenyl, alkynyl, aryl

Scheme 3 Alternatively, contemplated benzimidazole nucleosides may also be substituted with a halogen in a first position and an amino group in a second position as depicted in Scheme 4 below.

,

nucleophile

Ri = amine or sulfide R₂

reductive amination or similar prcosss

R₂ = alkyl, alkenyl, alkynyl, aryl

Scheme 4

Here, a benzimidazole nucleoside that is substituted with a chlorine and an amino group is first coupled to a solid phase and then reacted with a first reagent (preferably a nucleophile) in which the chlorine atom is replaced with the first reagent. In a further reaction (preferably reductive amination) with a second reagent, the amino group is converted into a secondary amine.

With respect to the number and/or position of the halogen(s) and/or amino group(s) in the nucleosides of Schemes 3 and 4, it should be appreciated that various alternative numbers and/or positions are also appropriate, and exemplary suitable numbers/positions are depicted below.

X = CI, Br, 1 X = CI, Br, 1 X ^ = CI, Br, 1 X = CI, Br, 1

It is still further contemplated that that the particular nature of the sugar is not limiting to the inventive subject matter. Consequently, numerous alternative sugars are also appropriate, and particularly contemplated alternative sugars include various substituted ribofuranoses, carbocyclic ring systems with 5 or 6 carbon atoms, and arabinose, wherein the sugar is in D- configuration or L-configuration. Further suitable sugars include those described in the section entitled "Contemplated Sugars", and it is especially contemplated that where the sugar has a C₂' and/or C₃' substituent other than a hydroxyl group, such alternative sugars may include hydrogen, a halogen, SH, SCH , O-alkyl, O-alkenyl, O-alkynyl, or an azide group in at least one of these positions (in either alpha or beta orientation). Moreover, the coupling of the heterocyclic base to the sugar may be in a position other than the exposition, and it is especially contemplated that where the sugar is a pentose or hexose, alternative positions include the C₂' and C₃'-position.

Consequently, where protecting groups are employed in the synthesis of benzimidazole libraries, it is contemplated that the nature of protecting groups for the sugar may vary- considerably, and while it is particularly contemplated that suitable protection groups include benzyl-, acetyl-, and TBDMS groups, numerous alternative protection groups are also considered suitable. Among other groups, a collection of appropriate alternative protection groups and their reactions is described in Protective Groups in Organic Synthesis by Peter G. M. Wuts, Theodora W. Greene, John Wiley & Sons; ISBN: 0471160199.

Furthermore, it is contemplated that at least one of the reactions involved in the synthesis of contemplated benzimidazole libraries may be performed while the nucleoside is covalently bound to a solid phase. The solid phase and methods of coupling the solid phase to the nucleoside will at least in part depend on the particular sugar and position of coupling. Therefore, it is contemplated that all known solid phases are suitable for use in conjunction with the teachings presented herein, and exemplary suitable solid phases are described, for example, in Organic Synthesis on Solid Phase - Supports, Linkers, Reactions; by Florencio Zaragoza Dorwald et al. John Wiley & Sons; ISBN: 3527299505, or in Solid-Phase Synthesis and Combinatorial Technologies by Pierfausto Seneci, John Wiley & Sons; ISBN: 0471331953. Preferred solid phases, however, include Merrifield resins, ArgoGel (available from Argonaut, San Francisco, CA), Sasrin resin (a polystyrene resin available from Bachem Bioscience, Switzerland), TentaGel S AC, TentaGel PHB, or TentaGel S NH₂ resin (polystyrene- polyethylene glycol copolymer resins available from Rappe Polymere, Tubingen, Germany).

With respect to contemplated heterocyclic bases, it should be appreciated that the benzimidazole heterocyclic base may be modified with one or more substituents (e.g., carboxylic acid group, carboxamide or carboxamidine group), and may also include one or more heteroatoms other than nitrogen. Furthermore, it is contemplated that numerous halogenated and/or amino group-containing benzimidazoles are commercially available. However, where a particular benzimidazole is not commercially available, it is contemplated that such a heterocyclic base can be prepared from a commercially available precursor without expenditure of undue experimentation following protocols well lαiown in the art. Coupling of a benzimidazole heterocyclic base to a desired sugar will follow procedures as described, for example, in Handbook of Nucleoside Synthesis by Helmut Norbruggen and Carmen Ruli- Pohlenz (John Wiley & Sons; ISBN: 0471093831).

Contemplated first reagents generally include all reagents that can replace the chlorine atom in the heterocyclic base, and especially in an aromatic nucleophilic substitution reaction. Thus, suitable first reagents typically include various nucleophiles, and especially contemplated nucleophiles will have a general structure of R-NH₂, RR'N_H, or RSH, wherein R, and R' are independently alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, and a substituted alkynyl, an aryl and a substituted aryl.

Similarly, contemplated second reagents for Scheme 3 include all reagents that can replace the bromine (e.g., via Heck or Stille reaction). Consequently, particularly contemplated reagents for Scheme 3 include all reagents that can form a covalent bond with a carbon atom in a Heck reaction (e.g., R-C≡CH). However, alternative reagents also include reagents suitable for a Suzuki (e.g., arylB(OH)₂) or Stille (e.g., arylSnBu₃) reaction. Reaction conditions for all of such reactions are well known in the art (see e.g., Can. J. Chem. (2000), Vol. 78 (7): 957-962, or Tetrahedron (2001) Vol., 57(14): 2787-2789). Thus, especially preferred reagents include R- C≡CH, wherein R is alkyl, alkenyl, alkynyl, aryl, and alkaryl, all of which may further be substituted, and ArSnBu3, wherein R is aryl and aralkyl, both of which may further be substituted.

With respect to the second reagent in Scheme 4, it is generally contemplated that all reagents that can form a covalent bond with the nitrogen atom of the NH2 group are suitable. However, particularly suitable reagents include electrophilic reagents (e.g., carbonyl group containing compounds and especially ketones and aldehydes) that will form the corresponding imine. Thus, particularly preferred second reagents for Scheme 4 will have the general formula RCOR' or RCHO, wherein R and R' are independently alkyl, alkenyl, alkynyl, aryl, and alkaryl, all of which may further be substituted. There are numerous such reagents known in the art and commercially available, and it should be recognized that all of such suitable reagents are contemplated for use herein.

It is generally contemplated that almost all contemplated first and second reagents for Schemes 3 and 4 are commercially available. However, where a particular reagent is not commercially available, it should be recognized that such reagents can be prepared from commercially available precursors without expenditure of undue experimentation (see e.g.,

Advanced Organic Chemistry: Structure and Mechanisms (Part A) by Francis A. Carey, Richard J. Sundberg; Plenum Pub Corp; ISBN: 0306462435; or Advanced Organic Chemistry : Reactions and Synthesis (Part B) by Francis Carey, Richard J. Sundberg; Plenum Pub Corp; ISBN: 0306434571, or Compendium of Organic Synthetic Methods, Volume 9, by Michael B. Smith, John Wiley & Sons; ISBN: 0471145793).

Therefore, contemplated nucleoside libraries may comprise a plurality of nucleosides according to Formula 5 A or Formula 5B, wherein a first compound of the plurality of compounds has a first set of substituents A, Ri and R₂ wherein a second compound of the plurality of compounds has a second set of substituents A, R] and R₂

Formula 5 A Formula 5B wherein A is a protected or unprotected sugar (e.g., a ribofuranose, a substituted ribofuranose, a carbocyclic ring system, and an arabinose, wherein the sugar is in a D- configuration or in an L-configuration) covalently bound to a solid phase, wherein Ri and R₂ are independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an allcynyl, and a substituted alkynyl, an aryl and a substituted aryl, and wherein not all of the substituents A, Ri andR in the first set are the same as the substituents A, Ri andR₂ in the second set.

Where it is particularly desirable that the sugar portion of contemplated nucleosides is a ribofuranose, contemplated nucleoside libraries may comprise a plurality of nucleosides according to Formula 5C or Formula 5D, wherein a first compound of the plurality of compounds has a first set of substituents Ri and R₂ wherein a second compound of the plurality of compounds has a second set of substituents Ri and R₂

Formula 5C Formula 5D

wherein Ri and R₂ are independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, and a substituted alkynyl, an aryl and a substituted aryl, wherein • is a solid phase, and PG is a hydroxyl protecting group, and wherein not all of the substituents Ri andR₂ in the first set are the same as the substituents Ri and R₂ in the second set.

In still further contemplated aspects, contemplated nucleoside libraries will comprise a plurality of nucleosides according to Formula 5E, wherein a first compound of the plurality of compounds has a first set of substituents Ri and R₂ wherein a second compound of the plurality of compounds has a second set of substituents Ri and R₂

Formula 5E

wherein A is a protected or unprotected sugar (e.g., a ribofuranose, a substituted ribofuranose, a carbocyclic ring system, and an arabinose, wherein the sugar is in a D- configuration or in an L-configuration) covalently bound to a solid phase, wherein Ri and R₂ are independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, and a substituted alkynyl, an aryl and a substituted aryl, and wherein not all of the substituents A, Ri andR₂ in the first set are the same as the substituents A, Ri andR₂ in the second set.

Where it is preferred that such libraries have a ribofuranose as a sugar portion, suitable libraries will comprise a plurality of nucleosides according to Formula 5F, wherein a first compound of the plurality of compounds has a first set of substituents Ri and R₂ wherein a second compound of the plurality of compounds has a second set of substituents Ri and R₂

Formula 5 F

wherein Ri and R₂ are independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, and a substituted alkynyl, an aryl and a substituted aryl, wherein • is a solid phase, and PG is a hydroxyl protecting group, and wherein not all of the substituents Ri andR₂ in the first set are the same as the substituents Ri and R₂ in the second set. Thus, contemplated compounds may have a structure according to Formula 5G, 5H, or

51

Formula 5G Formula 5H Formula 51

wherein Ri and R₂ are independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, and a substituted alkynyl, an aryl and a substituted aryl.

Uses of contemplated libraries and compounds -

It is generally contemplated that all libraries will comprise one or more nucleosides that have numerous biological activities, and especially contemplated biological activities include in vitro and in vivo inhibition of DNA and/or RNA polymerases, reverse transcriptases, and ligases. Therefore, contemplated nucleosides will exhibit particular usefulness as in vitro and/or in vivo antiviral agents, antineoplastic agents, or immunomodulatory agents. Still further, it is contemplated that nucleosides according to the inventive subject matter may be incorporated into oligo- or polynucleotides, which will then exhibit altered hybridization characteristics with single or double stranded DNA in vitro and in vivo.

Particularly contemplated antiviral activities include at least partial reduction of viral titers of respiratory syncytial virus (RSV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex type 1 and 2, herpes genitalis, herpes keratitis, herpes encephalitis, herpes zoster, human immunodeficiency virus (HIV), influenza A virus, Hanta virus (hemorrhagic fever), human papilloma virus (HPV), and measles virus. Especially contemplated immunomodulatory activity includes at least partial reduction of clinical symptoms and signs in arthritis, psoriasis, inflammatory bowel disease, juvenile diabetes, lupus, multiple sclerosis, gout and gouty arthritis, rheumatoid arthritis, rejection of transplantation, giant cell arteritis, allergy and asthma, but also modulation of some portion of a mammal's immune system, and especially modulation of cytokine profiles of Type 1 and Type 2. Where modulation of Type 1 and Type 2 cytokines occurs, it is contemplated that the modulation may include suppression of both Type 1 and Type 2, suppression of Type 1 and stimulation of Type 2, or suppression of Type 2 and stimulation of Type 1.

Where contemplated nucleosides are administered in a pharmacological composition, it is contemplated that suitable nucleosides can be formulated in admixture with a pharmaceutically acceptable carrier. For example, contemplated nucleosides can be administered orally as pharmacologically acceptable salts, or intravenously in physiological saline solution (e.g., buffered to a pH of about 7.2 to 7.5). Conventional buffers such as phosphates, bicarbonates or citrates can be used for this purpose. Of course, one of ordinary skill in the art may modify the formulations within the teachings of the specification to provide numerous formulations for a particular route of administration. In particular, contemplated nucleosides may be modified to render them more soluble in water or another vehicle, which for example, may be easily accomplished by minor modifications (salt formulation, esterification, etc.) that are well within the ordinary skill in the art. It is also well within the ordinary skill of the art to modify the route of administration and dosage regimen of a particular compound in order to manage the pharmacokinetics of the present compounds for maximum beneficial effect in a patient.

In certain pharmaceutical dosage forms, prodrug forms of contemplated nucleosides may be formed for various purposes, including reduction of toxicity, increasing the organ- or target cell specificity, etc. Among various prodrug forms, acylated (acetylated or other) derivatives, pyridine esters and various salt forms of the present compounds are preferred. One of ordinary skill in the art will recognize how to readily modify the present compounds to pro-drug forms to facilitate delivery of active compounds to a target site within the host organism or patient. One of ordinary skill in the art will also take advantage of favorable pharmacokinetic parameters of the pro-drug forms, where applicable, in delivering the present compounds to a targeted site within the host organism or patient to maximize the intended effect of the compound.

Still further, it should also be recognized that contemplated compounds may be metabolized in a cell or extracellular compartment, and that such metabolites may exhibit the same or different pharmacological effect. For example, contemplated compounds may be phosphorylated and thus be more active as nucleoside analogs. On the other hand, reduction or glycosylation may affect bioavailability of contemplated compounds. Consequently, contemplated compounds will not only include those as described in the synthetic sections above, but also include metabolites thereof.

In addition, contemplated compounds may be administered alone or in combination with other agents for the treatment of various diseases or conditions. Combination therapies according to the present invention comprise the administration of at least one compound of the present invention or a functional derivative thereof and at least one other pharmaceutically active ingredient. The active ingredient(s) and pharmaceutically active agents may be administered separately or together and when administered separately this may occur simultaneously or separately in any order. The amounts of the active ingredient(s) and pharmaceutically active agent(s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect.

Examples

It is generally contemplated that, unless stated otherwise, the general reaction conditions for reactions shown in Schemes 1-4 are well known in the art. However, to provide additional guidance to a practitioner, the below exemplary reagents are provided for nucleophilic reactions, Heck, Suzuki, and/or Stille reaction used in the reactions of Schemes 1-4 above.

Exemplary Amino Building blocks (R-NH₂ or RNHR) used for the libraries

l-(Benzyl)benzylamine, 2-phenyl-n-propylamine, m-trifluorobenzylamine, 2,2- diphenylethylamine, cyclobutylamine, methylcyclohexylamine, 2-methylpropylamine, allylcyclopentanylamine, N-methyl-4-piperidinylmethylamine, 4-hydroxypiperidine, 3- hydroxypiperidine, 1-benzylpiperazine, p-methoxybenzylamine, N,N- bis(isopropyl)aminoethylamine, 2-ethylhexylamine, 5-methyl-2-furanosylmethylamine, N,N- dimethylaminopropylamine, 3 -(N,N-dimethylamino)-2,2-dimethylpropylamine, 2- methylbutylamine, o-ethoxybenzylamine, 3-(2-methyl-N-piρeridinylpropylamine, l-(2- aminoethyl)pyrrolidine, 2-morpholinylethylamine, N4-hydroxyethylpiperazine, N- methylethylenediamine, 3-morpholinylpropylamine, pyridinyl-2-ethylamine, butylamine, hexylamine, methylamine, 2-hydroxyethylamine, N,N-dimethylethylenediamine, 3- methoxypropylamine, 2-mefhoxylethylamine, ethylamine, 2-isopropylamine, methylefhylamine, 2-methylthioethylamine, di-n-butylamine, dimethylamine, allylamine, cyclopantylamine, 2-( - methyl-pyrrolidin-2-yl)ethylamine, tetrahydrofuranosyl-2-methylamine, piperidine, N-benzyl-4- aminopiperidine, aminomethylcyclopropane, cyclopropylamine, 3-methylpiperizine, 4-piperidin- 1-ylpiperidine, cyclohexylamine, piperazine, 4-pyridin-2-ylpiperazine, 1-methylpiperazine, N-(2- methoxyphenyl)piperazine, N-pyrimidin-2-ylpiperazine, cycloheptylamine, p- rifluorobenzylamine, benzylamine, 3-imidazol-l-ylpropylamine, exo-2-aminonorborane, N- phenylethylenediamine, 1-methylbenzylamine, 3,4-(l,3-dioxolanyl)benzylamine, pyridin-2- ylmethylamine, pyridin-3-ylmethylamine, pyridin-4-ylmethylamine, thiophen-2-ylmethylamine, 3,3-dimethylbutylamine, o-methoxybenzylamine, l-(3-aminopropyl)pyrrolidin-2-one, N- methylethylenediamine, m-methylbenzylamine, 3-methylbutylamine, 2-methylbutylamine, heptylamine, 3-butoxypropyamine, 3-isopropoxypropylamine, 2-morpholin-4-ylpropylamine, 1 ,N 1 -diethylethylenediamime, 2-ethylthioethylamine, 4-(2-aminoethyl)phenol, furfurylamine, 4-aminomethylpiperidine, 2-(2-aminoethyl)pyridine, 2-phenoxyethylamine, 2- aminoethylthiophene, p-methoxybenzylamine, 2-(N,N-dimethylamino)ethylamine, 1- amino-2-propanol, 5-methylfurfurylamine, 3-(dimethylamino)propylamine, o- methoxybenzylamine, l-(3-aminopropyl)-2-pipecoline, hydrazine, 4-hydroxypiperidine, ethylenediamine, 1 ,4-diaminobutane, N-methylpropylamine, trans- 1,4-diaminocyclohexane, 2,2,2-trifluoroethylamine, 3-chloropropylamine, 3-ethoxypropylamine, aminoacetaldehyde dimethyl acetal, 3-amino-l,2-propanediol, l,3-diamino-2-hydroxypropane, 1-aminopyrrolidine, 2-(2-aminoethyl)-l-methylpyrrolidine, 3-methylpiperidine, 2-piperidine methanol, 3 -piperidine methanol, 1-aminohomopiperidine, homopiperazine, 4-aminomorpholine, 3-bromobenzylamine, piperonylamine, 1,2,3,4-tetrahydroisoquinoline, L-proline methyl ester, l-(2-pyridyl)piperazine, 4-(2-aminoethyl)morpholine, l-(2-aminoefhyl)piperidine, 3-aminopropipnitrile, 3- (aminomethyl)pyridine, 2-(aminomethyl)pyridine, thiomorpholine, l,4-dioxa-8-azaspiro(4,5)- decane, 2-hydroxylethylamine, l-(2-aminoethyl)pyrrolidine, aminomethylcyclohexane, 2- hydroxymethylpyrrolidine, 3-amino-l,2-propanediol acetone ketal, N-(2- hydroxyethyl)piperazine, N-phenylethylenediamine, 4-amino-2,2,6,6-tetramethylpiperidine, N- (4-nitrophenyl)ethylenediamine, 1 ,2-diphenylethylamine, 1 -(N,N-dimethylamino)-2- propylamine, 2-phenylpropylamine, 2-methylcyclopropylamine, 2-methylaziridine, aminomethylcyclopropane, l-aminomethyl-2-methylcyclopropane, butten-3-ylamine, 3-methyl- buten-2-ylamine, 3-methyl-buten-3-ylamine, 4-aminomethyl-l-cyclohexene, 3-phenylallylamine, 2,2-dimethylethylenediamine, 3-ethylhexylamine, 3-(N,N-dimethylamino)-2,2- dimethylpropylamine, 2-methyl-N-aminopropylpiperidine, as well as other related aliphatic and aromatic primary and secondary amines that are good nucleophiles to react with leaving groups on the scaffolds. Exemplary Building Blocks For Heck Reaction

4-n-pentylphenylacetylene, 1-ethynyl-l-cyclohexanol, 1-ethynylcyclohexene, N,N- dimethylpropenoamide, 2-ethynylpyridine, 5 -phenyl- 1-pentyne, 4-(tert-butyl)phenylacetylene, phenylacetylene, 3 -dibutylamino- 1-propyne, phenyl propargyl ether, 5 -chloro- 1-pentyne, 3- diethylamino- 1-propyne, 4-phenyl-l-butyne, 1-heptyne, l-dimethylamino-2-propyne, 1-pentyne, 2-methyl-l-hexene, (triethylsilyl)acetylene, 3 -phenyl- 1-propyne, methyl propargyl ether, 3- cyclopentyl- 1-propyne, 1-ethynylcyclohexene, 3-butyn-l-ol, styrene, vinylcyclohexane, 2- (tributylstannyl)furan, 2-(tributylstannyl)thiophene, tetraphenyltin, 3 -cyclohexyl- 1-propyne, 4- methoxyphenylacetylene, 4-(trifluoromethyl)phenyleneacetylene, 4-fluorophenylacetylene, 4- pentayn- 1 -ol, 4-methylphenylacetylene, 1 -ethynylcyclopentanol, 3 -methyl- 1 -propyne, 5 -cyano- 1 - pentyne, cyclohexylethyne, 1-ethynylcyclohexene, 5-cyano- 1-pentyne, l-dimethylamino-2- propyne, N-methyl-N-propargylbenzylamine, 2-methyl-l-buten-3-yne, cyclopentylethyne, 4- nitrophenylacetylene, phenyl propargylsulfide, 4-methyl- 1-pentyne, propargyl ethylsulfide, 2- prop-2-ynyloxybenzothiazole, 4-ethoxy-l-prop-2-ynyl-l,5-dihydro-2H-pyrrol-2-one, 6-methyl-5- (2-propynyl)-2-thioxo-2,3-dihydro-4(lH)-pyrimidinone and related end-alkenes and alkynes.

Exemplary Building Blocks For Stille Reaction

Tetraethyltin, 2-(tributylstannyl)pyridine, tributylstannyl-4-t-butylbenzene, ethynyltri-n- butyltin, vinyltri-n-butyltin, allyltri-n-butyltin, phenylethynyltri-n-butyltin, phenyltri-n-butyltin, (2-methoxy-2-cyclohexen-l-yl)tributyltin, 5,6-dihydro-2-(tributylstannyl)-4H-pyran, tri-n- butyl(2-furanyl)tin, tri-n-butyl(2-thienyl)tin, tributyl(phenylethenyl)tin, 4-fluoro-(tri-n- butylstannyl)benzene, 5-fluoro-2-methoxy(tri-n-butylstannyl)benzene, 1 -methyl-2- (tributylstannyl)-lH-pyrrole, 5-methyl-2-tributylstannylthiophene, 2-tributylstannylthiazole, 2- trybutylstannylpyrrazine, tributyl[3-(trifluoromethyl)phenyl]stannane and other related organic tin reagents.

Exemplary Building Blocks For Suzuki Reaction

Phenylboronic acid, 4-tolylboronic acid, 2-thiopheneboronic acid, thiophene-3-boronic acid, furan-2-boronic acid, cyclopentylboronic acid, 4-methylfuran-2-boronic acid, 3- hydroxyphenyl)boronic acid, 5-methylfuran-2-boronic acid, 3-cyanophenylboronic acid, 4- cyanophenylboronic acid, (5-formyl-3-furanyl)boronic acid, furan-3-boronic acid and other related organic boronic acids. Thus, specific embodiments and applications of substituted cytidine libraries and compounds have been disclosed. It should be apparent, however, to those slcilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the spirit of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced.

Claims

What is claimed is:

1. A nucleoside library comprising a plurality of nucleosides according to Formula 1 A, wherein a first compound of the plurality of compounds has a first set of substituents A, Ri, R₂, and R₃ wherein a second compound of the plurality of compounds has a second set of substituents A, Ri R₂ and R₃

Formula A

wherein A is a protected or unprotected sugar bound to a solid phase;

Ri, R₂, and R₃ are independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an allcynyl, a substituted alkynyl, NH₂, NHNH₂, NHR', NHOH, NHOR', NHNHCONH₂, NHNHCONHNH₂, SR', CN, C(NH)NH₂, guanidine, hydroxyguanidine, mercaptoguanidine, COR', OR', CR', OH, and SH;

wherein R' is selected from the group consisting of hydrogen, an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, a substituted alkynyl, an aryl, a substituted aryl; and

wherein not all of the substituents A, R_l3 R₂, and R₃ in the first set are the same as the substituents A, R_1; R₂, and R₃ in the second set.

The nucleoside library of claim 1 wherein the sugar is selected from the group consisting of a ribofuranose, a substituted ribofuranose, a carbocyclic ring system, and an arabinose, wherein the sugar is in a D-configuration or in an L-configuration. A compound according to Formula IB

Formula IB

wherein A is a sugar, R_l5 R₂, and R₃ are independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, a substituted allcynyl, NH₂, NHNH₂, NHR', NHOH, NHOR', NHNHCONH₂, SR', CN, SH, NHNHCONHNH₂, C(NH)NH₂, guanidine, hydroxyguanidine, mercaptoguanidine, COR', OR', CR', and OH; and

wherein R' is selected from the group consisting of hydrogen, an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, a substituted alkynyl, an aryl, a substituted aryl.

4. The compound of claim 3 wherein the sugar is selected from the group consisting of a ribofuranose, a substituted ribofuranose, a carbocyclic ring system, and an arabinose, wherein the sugar is in a D-configuration or in an L-configuration.

5. A nucleoside library comprising a plurality of nucleosides according to Formula 3 A, wherein a first compound of the plurality of compounds has a first set of substituents A, Ri andR₂ wherein a second compound of the plurality of compounds has a second set of substituents A, Ri and R₂

Formula 3A wherein A is a protected or unprotected sugar that is covalently bound to a solid phase;

wherein Ri and R₂ are independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an allcynyl, and a substituted alkynyl, an aryl and a substituted aryl; and

wherein not all of the substituents A, R_l5 R₂, and R₃ in the first set are the same as the substituents A, R_ls R₂, and R₃ in the second set.

6. The nucleoside library of claim 5 wherein the sugar is selected from the group consisting of a ribofuranose, a substituted ribofuranose, a carbocyclic ring system, and an arabinose, wherein the sugar is in a D-configuration or in an L-configuration.

7. The nucleoside library of claim 5 comprising a plurality of nucleosides according to Formula 4 A, wherein a first compound of the plurality of compounds has a first set of substituents R_l3 R₂, and R wherein a second compound of the plurality of compounds has a second set of substituents R_1; R₂ and R

Formula 4A

wherein Ri and R2 are independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an allcynyl, and a substituted allcynyl, an aryl and a substituted aryl;

wherein R₃ is selected from the group consisting of hydrogen, OH, OCH₃, SH, SCH₃, O- allcyl, O-alkenyl, O-alkynyl; and wherein • is a solid phase, and wherein not all of the substituents A, R_l5 R₂, and R in the first set are the same as the substituents A, R_ls R₂, and R₃ in the second set.

i. A compound according to Formula 3B

Formula 3B

wherein A is a protected or unprotected sugar; and

wherein Ri and R₂ are independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an allcynyl, and a substituted allcynyl, an aryl and a substituted aryl.

9. The compound of claim 8 wherein the sugar is selected from the group consisting of a ribofuranose, a substituted ribofuranose, a carbocyclic ring system, and an arabinose, wherein the sugar is in a D-configuration or in an L-configuration.

10. The compound of claim 8 having a structure according to Formula 4B

Formula 4B wherein Ri and R2 are independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, and a substituted allcynyl, an aryl and a substituted aryl; and

^ wherein R₃ is selected from the group consisting of hydrogen, OH, OCH₃, SH, SCH₃, O- allcyl, O-alkenyl, O-alkynyl.

11. A nucleoside library comprising a plurality of nucleosides according to Formula 5 A or Formula 5B, wherein a first compound of the plurality of compounds has a first set of substituents A, Ri and R2 wherein a second compound of the plurality of compounds has a second set of substituents A, Ri and ₂

Formula 5 A Formula 5B

wherein A is a protected or unprotected sugar covalently bound to a solid phase;

wherein not all of the substituents A, Ri and R₂ in the first set are the same as the substituents A, R_\ and R₂ in the second set.

12. The nucleoside library of claim 11 wherein the sugar is selected from the group consisting of a ribofuranose, a substituted ribofuranose, a carbocyclic ring system, and an arabinose, wherein the sugar is in a D-configuration or in an L-configuration.

13. The nucleoside library of claim 11 comprising a plurality of nucleosides according to Formula 5C or Formula 5D, wherein a first compound of the plurality of compounds has a first set of substituents Ri and R₂ wherein a second compound of the plurality of compounds has a second set of substituents Ri and R₂

Formula 5C Formula 5D

wherein Ri and R₂ are independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an allcynyl, and a substituted allcynyl, an aryl and a substituted aryl;

wherein • is a solid phase, and PG is a hydroxyl protecting group; and

wherein not all of the substituents Ri andR₂ in the first set are the same as the substituents Ri and R₂ in the second set.

14. A nucleoside library comprising a plurality of nucleosides according to Formula 5E, wherein a first compound of the plurality of compounds has a first set of substituents Ri and R₂ wherein a second compound of the plurality of compounds has a second set of substituents _\ and R₂

Formula 5E

wherein Ri and R₂ are independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, and a substituted alkynyl, an aryl and a substituted aryl; and wherein not all of the substituents A, Ri andR₂ in the first set are the same as the substituents A, Ri and R in the second set.

15. The nucleoside library of claim 14 wherein the sugar is selected from the group consisting of a ribofuranose, a substituted ribofuranose, a carbocyclic ring system, and an arabinose, wherein the sugar is in a D-configuration or in an L-configuration.

16. The nucleoside library of claim 14 comprising a plurality of nucleosides according to Formula 5F, wherein a first compound of the plurality of compounds has a first set of substituents Ri and R wherein a second compound of the plurality of compounds has a second set of substituents Ri and R₂

Formula 5F

wherein Ri and R₂ are independently selected from the group consisting of an alkyl, a substituted alkyl, an alkenyl, a substituted alkenyl, an alkynyl, and a substituted allcynyl, an aryl and a substituted aryl;

wherein • is a solid phase, and PG is a hydroxyl protecting group; and

wherein not all of the substituents Ri and R₂ in the first set are the same as the substituents Ri andR₂ in the second set.