Abstract:
This Divisional application of patent application Ser. No. 10/768,996, entitled “Novel Oligonucleotides And Related Compounds” discloses a class of chemical compounds which have been demonstrated to possess cancer fighting properties. The parent application disclosed oligonucleotides for selectively killing cancerous cells over noncancerous cells by incorporating and covalently linking antimetabolite prodrugs via CpG moieties, for the anitmetabolite Gemcitabine and other compounds with known cancer fighting properties. This application discloses modifications of Gemcitabine for incorporation into CpG oligonucleotides for improved biochemical and biological properties.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application for patent is a Divisional application of patent application Ser. No. 10/768,996, entitled “Novel Oligonucleotides and Related Compounds” filed on Jan. 30, 2004, by Suresh C. Srivastava, Satya P. Bajpai and Kwok-Hung Sit. This Divisional application is being filed as a result of a restriction requirement in the parent application. This application contains no new matter and claims priority from its parent, application Ser. No. 10/768,996, the entire contents of which application are incorporated herein by reference. 
     
    
     FIELD OF THE INVENTION 
       [0002]    This application relates to a class of chemical compounds which have been demonstrated to possess cancer fighting properties. It was determined during the prosecution of the parent application that the originally filed claims  38 - 44  which were directed to chemical modification of the antimetabolite Gemcitabine belonged in class 536, subclass 4.1. This Divisional application pertains to those claims in class 536, subclass 4.1. 
       BACKGROUND OF THE INVENTION 
       [0003]    In the parent application it was disclosed that the antimetabolite prodrug of Gemcitabine possesses strong anti cancer properties provided it is covalently linked to one of the nucleotides of the oligonucletoides having CpG motifs wherein the antimetabolite prodrug is linked by a 3′-3′ linkage, a 5′-5′ linkage, a 3′-5′ linkage, or a 5′-3′ linkage. This invention is sometimes referred in the present application as the “prior” invention. 
         [0004]    A large number of prior art references were given in the parent application as filed and during its prosecution to specify the background for the parent application. The present application recapitulates the segments relevant to the present application. 
         [0005]    The studies of Dr. Kwok-Hung Sit and colleagues (Yee-Jiun Kok, Myint Swe, and Kwok-Hung Sit, Biochemical and Biophysical Communications, 294 934-939, 2002), suggest a relationship between the cell death and immunostimulatory activity. With the effective ODN-CpG binding, there is strong inhibition of CpG DNA fragmentation, resulting in site specific resistance to cleavage, and thereby prevent necrosis and apoptosis. CpG oligonucleotides referred in the previous publication, however, independently seem to enhance the immunostimulatory activity. On the other hand inhibition of megabase fragmentation of the highly conserved GCn*GC motifs by complementary ODN&#39;s will help to protect complementary CpG DNA from degradation. Our approach as disclosed in the prior application for ODN design and incorporation of potent anticancer drug is a novel approach and presents enormous future potential in molecular medicine. The incorporated anticancer drug can be cleaved by one of the several endolytic cleavage mechanisms. This should result in hydrolysis of a phosphodiester bond, esterase hydrolysis of the ester linkages outlined in the details of claims or amidate hydrolysis will liberate the anticancer drug. While the CpG ODN will act as complementary DNA-ODN conjugate for the stability from degradation, it also seems that with proper design selection of the CpGn*CpG ODN, immunostimulatory properties of the ODN could be available within the cell. 
         [0006]    The hydrolysis of Gemcitabine (or another prodrug) which is attached via an ester linkage to a CpG oligo, is envisaged to be easily hydrolyzed by intracellular esterases (Ghosh, M and Mitra, A. K., Pharm. Res., 8, 771-775, 1009). 
         [0007]    It was observed that the sugar and bases of one or more nucleotides that make up the oligonucleotide may have one or more substitutions and examples of one or more nucleotides having a 2′-o-alkyl, 2′-n-alkyl, or 2′-halogen modifications on the sugar. Preferably the alkyl is a C1-C6 alkyl were described. 
         [0008]    It was observed that the sugar and bases of one or more nucleotides that make up the oligonucleotides may include one or more protecting groups for stability during oligonucleotide synthesis or in vivo conditions. 
         [0009]    It was further noted that oligonucleotides can be obtained from existing nucleic acid sources (e.g. 
         [0010]    genomic or cDNA), but are preferably synthetic, and have a defined sequence (e.g. produced by oligonucleotide synthesis). 
         [0011]    The delivery vehicles were discussed in detail. It was noted that an “oligonucleotide delivery complex” is an oligonucleotide associated with (e.g. ionically or covalently bound to; or encapsulated within) a targeting means (e.g. a molecule that results in a higher affinity binding to a target cell, such as that of a B-cell or natural killer (NK) cell, and/or increased cellular uptake by target cells). Examples of oligonucleotide delivery complexes include oligonucleotides associated with: a sterol (e.g. cholesterol), a lipid (e.g. cationic lipid, virosome or liposome), or a target cell specific binding agent (e.g. a ligand recognized by a target cell specific receptor). Preferred complexes must be sufficiently stable in vivo to prevent significant uncoupling prior to internalization by the target cell. However, the complex should be cleavable or otherwise accessible under appropriate conditions within the cell so that the oligonucleotide is functional. (Gursel, J. Immunol. 167: 3324, 2001.) 
         [0012]    “Pharmaceutically acceptable carriers” that were shown to be useful in the prior invention are conventional; see for example, Remington&#39;s Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975), which is incorporated herein by reference. 
         [0013]    It was noted that in general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, such as sodium acetate or sorbitan monolaurate. 
         [0014]    It was also discussed that the oligonucleotides of the invention can be formulated for intratracheal administration or for inhalation. Such compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Other pharmacological excipients are known in the art. 
         [0015]    A prefered, pharmaceutically acceptable carrier is lipofectin. 
         [0016]    For therapeutic or prophylactic treatment, oligonucleotides are administered in accordance with the prior invention. Oligonucleotides may be formulated in a pharmaceutical composition, which may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like, in addition to the oligonucleotide. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like in addition to oligonucleotides. Conventional chemotherapeutic agents may also be included. 
         [0017]    In one embodiment, the oligonucleotides of the invention are included in a delivery complex. The delivery complex can include the oligonucleotide of the invention and a targeting means. Any suitable targeting means can be used. For example, the oligonucleotide of the invention can be associated with (e.g., ionically or covalently bound to, or encapsulated within) a targeting means (e.g., a molecule that results in higher affinity binding to a target cell, such as a B cell). A variety of coupling or cross-linking agents can be used to form the delivery complex, such as protein A, carbodiamide, and N-succinimidyl-3-(2-pyridyl-dithio) propionate (SPDP). The complex is sufficiently stable in vivo to prevent significant uncoupling prior to delivery to the target cell. In one embodiment, the delivery complex is cleavable such that the oligodeoxynucleotide is released in a functional form at the target cells. 
         [0018]    Dosing is dependent on severity and responsiveness of the condition to be treated. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be calculated based on in vitro and in vivo animal studies. Thus, in the context of this invention, by “therapeutically effective amount” is meant the amount of the compound required to have a therapeutic effect on the treated mammal. This amount, which will be apparent to the skilled artisan, will depend upon the type of mammal, the age and weight of the mammal, the type of disease to be treated, perhaps even the gender of the mammal, and other factors which are routinely taken into consideration when treating a mammal with a disease. A therapeutic effect is assessed in the mammal by measuring the effect of the compound on the disease state in the animal. For example, in mammals being treated for cancer, therapeutic effects are assessed by measuring the rate of growth or the size of the tumor, or by measuring the production of compounds such as cytokines, which production is an indication of the progress or regression of the tumor. 
         [0019]    Some of the fundamental principles of prodrug approach were discussed in the previous application. 
         [0020]    A “prodrug” is a moiety of the oligonucleotide that can be hydrolyzed to form a purine or pyrimidine antimetabolite. In some embodiments, the antimetabolite is selected from the group consisting of 2′-deoxy, 2′,2′-difluorcytidine, 2′-deoxy-3′-thiacytidine, 3′-azido-3′-deoxythymidine, 2′,3′-dideoxycytidine, 2′,3′-didehydro-3′-deoxythymidine, 2′,3′-dideoxyinosine, 5-fluoro-2′-deoxy uridine, 2-fluoro-9-b-D-arabinofuranosyladenine, 1-B-D-arabinofuranosylcytosine, 5-azacytidine, 5-aza-2′-deoxycytidine, 6-mercaptopurineribo side, 2-chlorodeoxyadeno sine, and pentostatin. To obtain the antimetabolite, the linkage(s) between the prodrug and other nucleotide(s) of the oligonucleotide is hydrolyzed by natural or non-naturally occurring enzymes to obtain an antimetabolite nucleoside. In additional embodiments, hydroxyl-protecting groups (e.g., dimethoxytritryl, monomethoxytrityl, trimethoxytrityl, 9-fluorenyl carbonyl, tetrahydropyranyl, benzoyl, phenoxyacetyl, acetyl, propyl, butyryl, isobutyryl, or other higher homologs) also need to be removed by a deblocking agent in order to obtain the antimetabolite. In some embodiments, amine-protecting groups (e.g., benzoyl, acetyl, propyl, butyryl, isobutryl, phenoxy acetyl, substituted phenoxy acetyl, 9-fluorenyl carbonyl, also need to be removed by hydrolysis to obtain the antimetabolite. Antimetabolites kill both cancer and non-cancerous cells at about the same rate. The prior invention was based on the finding that oligonucleotides that include at least two CpG moieties and at least one prodrug of an antimetabolite preferentially kill cancerous cells. The oligonucleotide may have one or more ribonucleotides and/or one or more deoxyribonucleotides. 
         [0021]    It was noted in the previous application that the oligonucleotides of the invention may also have modifications that are not normally found in nature or that are found in nature in small quantities. A number of embodiments were provided for the invention where oligonucleotides having at least one nucleotide having a 2′-o-alkyl modification on the sugar of at least one nucleotide. Examples of alkyls include methyl, ethyl, propyl, ethenyl, and higher homologs. Preferably, the homolog has no more than 6 carbon atoms. Most preferably, the alkyl is methyl. In another embodiment, the invention provides an oligonucleotide having at least one 2′-N-alkyl modification. In yet another embodiment, the invention provides an oligonucleotide having at least one 2′-halogen modification. 
         [0022]    In one embodiment it was noted that the prodrug can be attached to the oligonucleotide by a 3′-3′ linkage. In another embodiment, the prodrug was shown to attach to the oligonucleotide by a 5′-5′ linkage. These types of chemical modifications are known to the skilled person and are described, for example, in M. Koga et al., J. Org. Chem. 56:3757, 1991, EP 0 464 638, and EP 0 593 901, U.S. Pat. No. 5,750,669, each of which is incorporated herein by reference. The synthesis of an oligonucleotide having a 3′-3′ linkage at the 3′ end can be achieved by attaching the 5′ end of a nucleoside attached to a solid support; this nucleoside then will allow growth of an oligonucleotide from the 3′ end. Similarly, the synthesis of an oligonucleotide having a nucleotide linked by a 5′-5′ linkage at the 5′ end of the oligonucleotide can be achieved by using a nucleotide having a support attached at the 3′ end that will allow growth of the oligo from the 5′-end. 
         [0023]    It was also discussed that the prodrug may also be linked by a 3′-5′ linkage or a 5′-3′ linkage, both of which are well known to the skilled artisan. Attachment of a prodrug to the oligo via a linker, will cause liberation of the prodrug at the cancer cell sites by hydrolytic enzymes and will have the effect of the prodrug as well as the CGn*CG oligonucleotide. An example of a point of cleavage between a prodrug and the oligonucleotide is an ester linkage. The aliphatic esters are chosen for this purpose, since they are stable, yet can be easily hydrolyzed inside cells by intracellular esterases. Aliphatic phosphate esters, alkyl substituted phosphate esters, and amidates are also part of this discovery, since they are also hydrolyzed by intracellular enzymes. Aliphatic amide linkages are chosen at the other side of linkage. The amide linkage is generally required in order to attach the prodrug and oligonucleotide. Thus, one end bears a carboxylic or activated ester of carboxylic acid, and the other end has a free amino function, to effect the joining of two moieties. This approach is extensively used in oligonucleotide labeling with various chromophores and ligands (See, P. S. Nelson, M. Kent and Sylvester Muthini, Nucleic Acids Research, Vol. 20, No. 23: 6253-6259, 1992; Misiura, K., Durrant, I., Evans, M. R., and Gait, M., Nucleic Acids Research, Vol., 18: 4345-4354, 1990; Zendegui, J. G., Vasquez, K. M., Tinsley, J. H., Kessler, D. J. and Hogan, M. E., Nucleic Acids Research, Vol. 20: 307-314, 1992. Each of these is incorporated herein by reference.). 
         [0024]    Exemplary oligonucleotides within the scope of the invention were depicted in Formulas I-XI, reproduced below. Each of these includes at least two CpG motifs. These oligonucleotides do not limit the scope of the invention. In some embodiments, the prodrug replaces one of the nucleotides of one of the CpG motifs. In other embodiments, the antimetabolite prodrug of Gemcitabine does not replace one of the nucleotides of the CpG motifs. 
         [0025]    Each of these formulas shows oligonucleotides that include the prodrug of the antimetabolite 2′-deoxy, 2′,2′-difluorocytidine which has been chemically modified so that the antimetabolite may offer increased biological potency and enhanced structure activity relationship, improved diseased recognition. Oligonucleotides with prodrugs for other antimetabolites may be used within the scope of the invention may be used in the same way and position as shown for 2′-deoxy, 2′,2′-difluorocytidine or in other positions. Intermediates useful in the synthesis of these oligonucleotides are described below as well. Oligonucleotides of the invention may further include one or more protecting groups as defined herein. 
         [0026]    For Formulas I through XI below, the subsequent symbols used have the following meaning: C represents cytosine or a modified cytosine including: a 5-alkyl cytosine such as 5-methyl cytosine, a 5-alkenyl homolog, a 5-alkynyl homolog, or a 5-halogen analog. The nucleotide with the C base is the prodrug for 2′-deoxy, 2′,2′-difluorocytidine. In another embodiment, the nucleotide with the C base is an analog of 2′-deoxy, 2′,2′-difluorocytidine. 
         [0027]    The phosphodiester bond is selected from a natural phosphodiester, alkoxy phosphotriester containing a lower alkoxy containing 1 to 6 carbon atoms such as OCH 3 , OC 2 H 5 , n-C 3  H 7 , iso-C 3 H 7 , a substituted lower alkoxy, such as OCH 3 , OC 2 H 5 , phosphorothioate, a straight or branched C-1 to C-6 alkyl, and phosphoramidate. Other internucleotide linkages may be used within the context of the prior invention. 
         [0028]    In the formula&#39;s it was noted that B, B′ and B″ were the same or different natural or modified bases. Natural bases include adenine, cytosine, guanine, thymine, inosine, and uridine. Modified bases include 5-methylcytosine, 5-azacytosine, 5-halogen substituted (F, Cl, Br, I) uracil or cytosine, and 5-alkyl substituted uracil or cytosine, such as, C-5 propyne uracil and C-5 propyne cytosine. Purine modifications can include 7-deazaadenine, 7-deazaguanine, 7-iodo-7-deaza adenine, 7-iodo-7-deazaguanine, 7-propyne-7-deazaadenine, 7-propyne-7-deazaguanine. Other bases are well known to the skilled artisan. The repeating portion of the oligonucleotide may have the same or different bases, and the term B′ should not be construed to imply that the same nucleotide is repeated n number of times, but rather that there are n nucleotides each of which has a base that is within the definition of B′. 
         [0029]    The number n was noted to be between 2 to 50. 
         [0030]    S stands for fluorine, chlorine, a sulfur derivative (e.g., S-alkyl), or a nitrogen with alkyl groups (e.g., N—R′, R″, in which R′ and R″ are the same or different alkyl groups with up to 8 carbon atoms). 
         [0000]    
       
                 
         
             
             
         
       
     
         [0031]    In one embodiment (Formula I), the prodrug was shown to attach at the 3′-end of an oligonucleotide via a 3′-5′ linkage. 
         [0032]    In another embodiment, the invention provided the oligonucleotide shown in Formula II. In this case, the prodrug was shown to be attached at the 5′-end of the oligonucleotide via a 5′-5′-Linkage. The oligonucleotide can be synthesized with a prodrug phosphoramidite, in which the phosphoramidite is at the 5′ of the oligonucleotide. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0033]    In another embodiment, the invention provided an oligonucleotide of the following formula: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0034]    The prodrug was shown attached at the 5′-end of the oligonucleotide via a 3′-5′-linkage. In another embodiment, the invention provided an oligonucleotide of the following formula: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0035]    In yet another embodiment the invention provided the oligonucleotide shown in Formula V. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0036]    In this embodiment, the prodrug was envisaged to be at an internal position of the oligonucleotide. It was noted that the synthesis of such an oligonucleotide can be achieved by the use of a phosphoramidite nucleotide of the prodrug using well-known oligonucleotide synthesis techniques. 
         [0037]    The invention further provided an oligonucleotide having the following formula (VI) : 
         [0000]    
       
                 
         
             
             
         
       
     
         [0038]    In this embodiment, the prodrug was shown to be attached to the 3′-end of an oligonucleotide via a lipophilic ester group at the prodrug&#39;s 5′-position. “X, Y=” means that X and Y are independently chosen from the three groups shown. The coupling could be done with an amino linker oligonucleotide. The amino linker oligonucleotides having various spacer lengths are well known in the oligonucleotide chemistry. (See, P. S. Nelson, M. Kent, S. Muthini, Nucleic Acids Research, Vol. 20, No. 23: 6253-6259, 1992; F. Berg, D. Praseuth, A. Zerial, N. Thoung, U. Asseline, T. Le Doan, C. Helene, Nucleic Acids Research, 18: 2901-2908, 1990, which are hereby incorporated by reference.) 
         [0039]    Suitable ester groups include carboxylic ester, methylene and phosphate ester groups. The assembly of oligonucleotides having a lipophilic ester is well known to the skilled artisan. For example, if the prodrug is 2′-deoxy, 2′,2′-difluorocytidine, the oligonucleotide can be synthesized using the 2,2′-difluorocytidineactive ester, an example of which is shown in formula XVIII. The oligonucleotide in this case will have a 3′-amino linker, which is well known in the art. The final oligonucleotide as depicted in Formula VII will be formed from the reaction of 3′-amino linker oligonucleotide and the active ester of formula (XVII). 
         [0040]    In another embodiment, the invention provides an oligonucleotide of the following formula: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0041]    In this oligonucleotide, the prodrug was shown to be attached via a linker, at its 3′-position to the 5′-end of an oligonucleotide. The synthesis of oligonucleotides having a lipophilic group can be accomplished by procedures cited in the literature, e.g., (Nikolai N. Polushin and Jack Cohen, Nucl. Acids Res., 22: 5492-5496, 1995). One such procedure will require the prodrug attached to solid support at the 5′-end, which will subsequently be treated with an amino linker at the 3′-end. The 3′-amino linker prodrug can then be coupled with an oligonucleotide having a carboxylic linker at its 5′-end. The general synthesis of oligonucleotide having a 5′-aliphatic carboxylic group can be derived from commercially available products, such as DMT-Thymidine-succinyl hexamide amidite, which is available from ChemGenes Corporation, Wilmington, Mass., catalog item number CLP-2244. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0042]    In yet another embodiment, the invention provided an oligonucleotide of the following formula (VIII): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0043]    In this embodiment, the prodrug was shown to be attached at the 3′ end of RNA or a 2′-modified RNA oligonucleotide, via a 3′-3′ linkage. The X stands for H, methyl, ethyl, a higher C3-C6 alkyl homolog, a C2-C6 alkenyl, a C2-C6 straight or branched alkynyl, an amino C1-C6 alkyl, an amino C2-C6 alkenyl, cyclopropyl, an allyl, a C1-C6 alkynylalkoxy, or an aminoalkoxy. 
         [0044]    In yet another embodiment, the invention provided oligonucleotides of the following formula (IX): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0045]    Where X was defined as used for Formula VII. Such oligonucleotides have a prodrug attached via a 5′-5′linkage at the 5′ end of RNA or a 2′-modified RNA. The attachment of a prod rug via its 5′-end can be achieved using a prodrug phosphoramidite, for example, the prodrug that is depicted in formula XX. The X is used as defined in reference to Formula VIII. 
         [0046]    In yet another embodiment the invention provided an oligonucleotide of the following formula (X): 
         [0000]    
       
                 
         
             
             
         
       
     
         [0047]    In this embodiment, the prodrug is attached at its 3′ end to the 3′-end of RNA or a 2′-modified RNA oligonucleotide. The attachment of the prodrug at its 3′-end can be achieved using the prodrug bound to a solid support at its 5′-end, as depicted in formula XV for the prodrug of 2′-deoxy,2′,2′-difluorocytidine. 
         [0048]    In yet another embodiment, the invention provides an oligonucleotide of the following formula: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0049]    Here, the prodrug is attached at its 3′ end to the 5′-end of RNA or a 2′-modified RNA. 
         [0050]    Oligonucleotides can be synthesized de novo using any of a number of procedures well known in the art. For example, the 0-cyanoethyl phosphoramidite method (S. L. Beaucage and M. H. Caruthers, Tet. Let. 22:1859, 1981; U.S. Pat. Nos. 4,415,732 and 4,458,066, (Caruthers), and U.S. Pat. No. Re 34,069, (Koster)) the nucleoside H-phosphonate method (Garegg et al., Tet. Let. 27: 4051-4054, 1986; Froehler et al., Nucl. Acid. Res 14: 5399-5407, 1986; Garegg eg al., Tet. Let. 27: 4055-4058, 1986; Gaffney et al., Tet. Let. 29: 2619-2622, 1988) can be used to synthesize oligonucleotides of the invention. Each of the above references is hereby incorporated by reference. These chemistries can be performed by a variety of automated oligonucleotide synthesizers available in the market. The oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including Applied Biosystems (Foster City, Calif.). It is also well known to use similar techniques to prepare other oligonucleotides such as phosphorothioates or alkylated derivatives. It is also well known to use similar techniques and commercially available modified phosphoramidites and solid supports, such as polystyrene, various silica gels in beads or powder forms, and controlled-pore glass (CPG) products to synthesize naturally-occurring and modified oligonucleotides. An example of use of a solid support to synthesize oligonucleotides is provided in U.S. Pat. No. 6,646,118 which is incorporated herein by reference. 
       SUMMARY OF THE INVENTION 
       [0051]    The present application pertains to chemical compounds that are modifications of Gemcitabine which provide improved biochemical and biological effectiveness of the cancer fighting property of Gemcitabine. 
         [0052]    Similar to the prior invention in one embodiment, the oligonucleotide of the invention has at least one nucleotide having a ribose sugar moiety. In another embodiment, the oligonucleotide of the invention has at least one nucleotide having a 2′-deoxyribose sugar moiety. In yet another embodiment, the oligonucleotide has at least one 2′-o-alkyl nucleotide, 2′-n-alkyl nucleotide, or 2′-O-halogen nucleotide, wherein the alkyl has between about 1 and about 6 carbon atoms. Nucleotides of the oligonucleotides are connected by covalent internucleoside linkages. Examples of covalent internucleoside linkages include phosphodiester linkages, C1-C6 alkoxy phosphotriester linkages, phosphorothioate linkages and phosphoramidate linkages. In some embodiments, the prodrug is attached to at least one of the multiple nucleotides by a linker of the following formula: 2 
         [0053]    Linker; 
         [0000]    
       
                 
         
             
             
         
       
     
         [0054]    The invention further provides a pharmaceutical composition that includes a therapeutically effective amount of any of the oligonucleotides disclosed herein. In some embodiments, the pharmaceutically acceptable carrier is lipofectin. 
         [0055]    The invention further provides a compound having purity in excess of 98% by HPLC, and having the following formula; 
         [0000]    
       
                 
         
             
             
         
       
     
         [0056]    Wherein R is selected from the group consisting of H, a C1-C6 alkyl, a halogen, a C2-C6 alkenyl, and a C2-C6 alkynyl; 
         [0057]    x is an amine-protecting group that is stable in oligonucleotide synthesis conditions; and 
         [0058]    y, and z are each selected from H, a hydroxyl-protecting group that is stable in oligonucleotide synthesis conditions and a group that can be attached to a solid support. In some embodiments, the compound group that is attachable to a solid support has the formula C(O)-M-C(O)—NH, where M is selected from the group consisting of succinyl, oxalyl, and hydroquinolynyl, and wherein the Spacer is a C1-C20 alkyl, ethyloxyglycol, or a combination of alkyl and ethyleneglycoxy, and the Spacer is attached to the solid support. 
         [0059]    Inter alia, the present application also discloses a compound represented by the formula 
         [0000]    
       
                 
         
             
             
         
       
     
         [0060]    Wherein R is selected from the group consisting of H, a C1-C6 alkyl, a halogen, a C2-C6 alkenyl, and a C2-C6 alkynyl; 
         [0061]    x is an amine-protecting group that is stable in oligonucleotide synthesis conditions; 
         [0062]    z is a hydroxyl-protecting group that is stable in oligonucleotide synthesis conditions; and, in some embodiments, the compound group that is attachable to solid support has the formula C(O)-M-C(O)—NH, where M is selected from the group consisting of succinyl, oxalyl, and hydroquinolynyl, and wherein the Spacer is a C1-C20 alkyl, ethyloxyglycol, or a combination of alkyl and ethyleneglycoxy, and the Spacer is attached to the solid support. 
         [0063]    Other modifications of the compounds and the requirements of the purity levels of the disclosed compounds are discussed further in the detailed description below. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0064]    Similar to the invention in the parent application the present invention relates to CpG oligonucleotides having at least one modified Gemcitabine, a nucleoside antimetabolite which can be classified as a “prodrug”. Various modifications were envisaged and disclosed for the Gemcitabine nucleoside in order to modulate biochemical and biological properties and to develop ideal therapeutic candidates. The present invention addresses the chemical modifications which are considered germane to this approach. The detailed descriptions of representative structures are illustrated diagrammatically in the Background section in Formulas I-XI. 
         [0065]    Various embodiments are briefly summarized in the following paragraphs. 
         [0066]    In one embodiment, the oligonucleotide of the invention has at least one nucleotide having a 2′-deoxyribose sugar moiety. In yet another embodiment, the oligonucleotide has at least one 2′-o-alkyl nucleotide, 2′-n-alkyl nucleotide, or 2′-halogen nucleotide, wherein the alkyl has between 1 and 6 carbon atoms. Nucleosides of the oligonucleotides are connected by covalent internucleotide linkages. Examples of covalent internucleotide linkages include phosphodiester linkages, C1-C6 alkoxy phosphotriester linkages, phosphorothioate linkages and phosphoramidate linkages. In some embodiments, the prodrug is attached to at least one of the multiple nucleotides by a linker described by the following class of formulas. 
         [0067]    Description of Linkers: 
         [0068]    A linker may have a formula illustrated below in formula (XII); 
         [0000]    
       
                 
         
             
             
         
       
     
         [0069]    Aliphatic amide linkages are chosen at the other side of linkage of the modified nucleoside antimetabolite, while one end is attached to oligonucleotide chain. The amide linkage is generally required in order to attach the prodrug and oligonucleotide. Thus, one end bears a carboxylic or activated ester of carboxylic acid, and the other end has a free amino function, to effect the joining of two moieties. 
         [0070]    The coupling could be done with an amino linker oligonucleotide. The amino linker oligonucleotides having various spacer lengths are well known in the oligonucleotide chemistry. 
         [0071]    Suitable ester groups include carboxylic ester and phosphate ester groups. 
         [0072]    For instance, the modified antimetabolite prodrug is attached to the 3′-end of an oligonucleotide via a lipophilic ester group at the prodrug&#39;s 5′-position. “X, Y═” in Formula XII means that X and Y are independently chosen from the three groups shown. 
         [0073]    The modified antimetabolite prodrug may also be linked by a 3′-5′ linkage or a 5′-3′ linkage. 
         [0074]    The general synthesis of oligonucleotide having a 5′-aliphatic carboxylic group can be derived from commercially available products, such as DMT-thymidine-succinyl hexamide amidite, which is available from ChemGenes Corporation, Wilmington, Mass., catalog item number CLP-2244. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0075]    The invention also provides a pharmaceutical composition that includes a therapeutically effective amount of any of the oligonucleotides disclosed. In some embodiments, the pharmaceutically acceptable carrier is lipofectin. 
         [0076]    The invention provides generic compounds having purity in excess of 97% by HPLC, and having the following formulas XIV and XV; and further provides the compounds having purity in excess of 98% by HPLC. 
         [0077]    Further embodiments relate to 5′-OH protected derivatives of 2′-deoxy, 2′,2′-difluorocytidine or of the 4-amine protected 2′-deoxy, 2′,2′-difluorocytidine. These are useful in intermediates for the preparation of 2′-deoxy, 2′,2′-difluorocytidine attached oligonucleotides. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0078]    Wherein, R is selected from the group consisting of H, a C1-C6 alkyl, a halogen, a C2-C6 alkenyl, and a C2-C6 alkynyl; 
         [0079]    x is an amine-protecting group that is stable in oligonucleotide synthesis conditions; and y, and z are each selected from the group consisting of (H, a hydroxyl-protecting group that is stable in oligonucleotide synthesis conditions, phosphoramidites, and a group that can be attached to a solid support). 
         [0080]    In some cases y is hydroxyl-protecting group such as DMT (dimethoxytritryl), MMT (monomethoxytrityl), TMT (trimethoxytrityl), FMOC (9-fluorenyl carbonyl chloride), tetrahydropyranyl, benzoyl, phenoxyacetyl, acetyl, propyryl, butyryl, isobutyryl, or other higher homologs. 
         [0081]    The amine-protecting group x for the exocyclic amine as an amide bond could further comprise one or more of the following: a lower (i.e., C1-C6) alkanoyl group containing a straight or a branched chain alkyl group as defined above, an aryl or substituted aryl having a C1-C6 alkyl or halogen as a substituent on the aryl ring, a phenoxy acetyl or appropriately protected phenoxy acetyl for fast deprotection, a trifluroacetyl or FMOC group, an imine derivative such as formamidine or dimethylformamidine. Such protecting groups are required to offer mild and convenient deprotection conditions after the synthesis of the oligonucleotides derived from the compounds of the present invention, and can be cleaved with a suitable reagent to generate free NH2 groups at the end of the oligonucleotide synthesis. 
         [0082]    In some embodiments, the group of spacer compounds used for attachment to a solid support has the general formula O—C(═O)-M-C(═O)—NH, where M can be selected from the group consisting of succinyl, oxalyl, and hydroquinolynyl, a C1-C6 alkyl, ethyloxyglycol, or a combination of alkyl and ethyleneglycoxy. 
         [0083]    The invention further discloses an active ester having the formula: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    wherein R, M, x, y and z are as defined above for the active esters as shown by the formula XVII, having a purity level approximately 95% and above by HPLC. 
         [0084]    The invention additionally provides a phosphoramidite having purity in excess of 97% and 98% by HPLC, as shown by the formula XVIII and XIX. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0085]    Wherein, each of y and z is a hydroxyl-protecting group that is stable in oligonucleotide synthesis conditions; x and R are as defined above, and R′ and R″ are independently selected and are either a C1-C6 alkyl or a C2-C6 cycloalkyl. 
         [0086]    The sugar and bases of one or more nucleotides that make up the oligonucleotide may be a chimera consisting of ribonucleosides, or ribonucleosides in one or more positions. In the case of ribo bases nucleosides may have a 2′-o-alkyl, 2′-n-alkyl, or 2′-halogen modifications on the sugar. Examples of alkyls include methyl, ethyl, propyl, ethenyl, and higher homologs. Preferably, the homolog has no more than 6 carbon atoms. Most preferably, the alkyl is methyl. In such modification the oligonucleotide may have at least one nucleotide having at least one 2′-n-alkyl modification. Similarly, such Oligonucleotide may have at least one nucleoside having at least one 2′-halogen modification. 
         [0087]    Oligonucleotides based on the present invention may include one or more protecting groups for stability during oligonucleotide delivery or in vivo conditions. 
         [0088]    Further, the oligonucleotides can be obtained from existing nucleic acid sources (e.g. genomic or cDNA), but are preferably synthetic, and have a defined sequence (e.g. produced by oligonucleotide synthesis). 
         [0089]    The sugar hydroxyl-protecting groups can be selected from the group (dimethoxytritryl, monomethoxytrityl, trimethoxytrityl, 9-fluorenyl carbonyl, tetrahydropyranyl, benzoyl, phenoxyacetyl, acetyl, propyl, butyryl, isobutyryl, or other higher homologs); these groups also need to be removed by a deblocking agent in order to obtain the oligonucleotides incorporating modified antimetabolites. 
         [0090]    This invention is based on the finding that oligonucleotides that include at least two CpG moieties and at least one prodrug of an antimetabolite or modified antimetabolite preferentially kill cancerous cells. The oligonucleotide may have one or more ribonucleotides and/or one or more deoxyribonucleotides. 
         [0091]    The modified antimetabolite prodrug can be attached to the oligonucleotide by a 3′-3′ linkage, or by a 5′-5′ linkage, or by a 3′-5′ or a 5′-3′ linkage. These chemical modifications are well known in the art. See M. Koga et al., J. Org. Chem. 56:3757, 1991, EP 0 464 638, and EP 0 593 901, U.S. Pat. No. 5,750,669. 
         [0092]    In another embodiment the oligonucleotide of this invention, the prodrug is attached via a linker, at its 3′-position to the 5′-end of an oligonucleotide. The synthesis of oligonucleotides having a lipophilic group can be accomplished by procedures cited in the literature, e.g., (Nikolai N. Polushin and Jack Cohen, Nucl. Acids Res., 22: 5492-5496, 1995). One such procedure will require the prodrug attached to solid support at the 5′-end, which will subsequently be treated with an amino linker at the 3′-end. The 3′-amino linker prodrug can then be coupled with an oligonucleotide having a carboxylic linker at its 5′-end. 
         [0093]    In one embodiment the invention relates to exocyclic amine protected derivatives of 2′-deoxy, 2′,2′-difluorocytidine which are shown in Formula XX, which is an intermediate for the preparation of various 2′-deoxy, 2′,2′-difluorocytidine attached oligonucleotides. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0094]    Yet another embodiment relates to 3′- or 5′-phosphoramidite derivatives of 2′-deoxy-2′,2′-difluorocytidine (formulas XIV and XV), preferably synthesized with the phosphorylating reagent 2-cyanoethyl-n,n-diisopropyl-amino phosphoramidite. Alternately, the phosphorylating reagent methoxy-n,n-diisopropylaminophosphinyl phosphoramidites may be used in the place of the 2-cyanoethyl-n,n-diisopropylamino phosphoramidite. These compounds have purity exceeding 97% and have coupling efficiency of greater than 98% in less than 100 seconds under standard DNA/RNA synthesis coupling conditions. The standard coupling conditions are outlined in the DNA/RNA synthesizer manual of MerMade Instrument. The coupling efficiency was monitored by carrying out oligo synthesis in the instrument model, Expedite 8909, and using the built in per step coupling monitor.