Oligonucleotide Analogues Incorporating 5-Aza-Cytosine Therein

Abstract

Oligonucleotide analogues are provided that incorporate 5-aza-cytosine in the oligonucleotide sequence, e.g., in the form of 5-aza-2′-deoxycytidine (decitabine) or 5-aza-cytidine. In particular, oligonucleotide analogues rich in decitabine-deoxyguanosine islets (DpG and GpD) are provided to target the CpG islets in the human genome, especially in the promoter regions of genes susceptible to aberrant hypermethylation. Such analogues can be used for modulation of DNA methylation, such as effective inhibition of methylation of cytosine at the C-5 position. Methods for synthesizing these oligonucleotide analogues and for modulating nucleic acid methylation are provided. Also provided are phosphoramidite building blocks for synthesizing the oligonucleotide analogues, methods for synthesizing, formulating and administering these compounds or compositions to treat conditions, such as cancer and hematological disorders.

1 .- 98 . (canceled) 
     
     
         99 . A method of preparing a dinucleotide analogue incorporating 5-aza-cytosine as a base residue, comprising:
 reacting a compound with a 3′- or 5′-protected nucleoside attached to a solid support to produce a protected dinucleotide analogue attached to the solid support; and   deprotecting the protected dinucleotide analogue to produce the dinucleotide analogue,   wherein the compound has the formula:   
       
         
           
           
               
               
           
         
         wherein R 1  is selected from the group consisting of phenoxyacetamide (Pac-NH), O-methyl (OMe), S-methyl (SMe), S-ethyl (SEt), dimethylamine (NMe 2 ), and 9-fluorenylmethyl carbamate (Fmoc-NH); R 2  is selected from the group consisting of dimethoxytritoxyl (DMTr-O), hydroxyl (OH), O-methyl (OMe), ethylene glycol, tetraethylene glycol, hexaethylene glycol, and hexaethylene glycol phosphate; R 3  is selected from the group consisting of cyanoethyl diisopropyl phosphoramidite, O-methyl (OMe), ethylene glycol, tetraethylene glycol, and solid support; and R 4  is selected from the group consisting of hydrogen, hydroxyl (OH), O-methyl (OMe), ethylene glycol, tetraethylene glycol, hexaethylene glycol, and hexaethylene glycol phosphate. 
       
     
     
         100 . The method of  claim 99 , wherein the nucleoside attached to the solid support is a 3′- or 5′-protected guanosine, cytidine, adenosine, thymidine, uridine, inosine, 5-methyl-cytidine, 5-aza-cytidine, 2′-deoxyguanosine, 2′-deoxycytidine, 2′-deoxyadenosine, 2′-deoxythymidine, 2′-deoxyuridine, 2′-deoxyinosine, 5-methyl-2′-deoxycytidine, or 5-aza-2′-deoxycytidine attached to a solid support. 
     
     
         101 . The method of  claim 99 , wherein the solid support is controlled pore glass solid support, or polystyrene solid support. 
     
     
         102 - 115 . (canceled) 
     
     
         116 . The method of  claim 99 , wherein the nucleoside attached to the solid support is 2′-deoxyguanosine. 
     
     
         117 . The method of  claim 99 , wherein R 1  is phenoxyacetamide (Pac-NH). 
     
     
         118 . The method of  claim 99 , wherein R 2  is dimethoxytritoxyl (DMTr-O). 
     
     
         119 . The method of  claim 99 , wherein R 3  is cyanoethyl diisopropyl phosphoramidite. 
     
     
         120 . A method of treating a condition, the method comprising administering to a subject in need thereof a therapeutically effective amount of a dinucleotide analogue or a pharmaceutically acceptable salt thereof, wherein the dinucleotide analogue has a general formula of 5′-DpG-3′ or 5′-GpD-3′, wherein D is decitabine; p is a phospholinker, and G is 2′-deoxyguanosine, wherein the administering comprises injecting the subject with the dinucleotide analogue at a dose of 0.1-1000 mg/m 2  per day over a duration of a treatment cycle. 
     
     
         121 . The method of  claim 120 , wherein upon administration to the subject, the dinucleotide analogue converts to decitabine. 
     
     
         122 . The method of  claim 120 , wherein the administration is subcutaneous. 
     
     
         123 . The method of  claim 120 , wherein the administration is intravenous. 
     
     
         124 . The method of  claim 120 , wherein the duration of the treatment cycle is from three to five days. 
     
     
         125 . The method of  claim 120 , wherein the dose is 2-50 mg/m 2  per day. 
     
     
         126 . The method of  claim 120 , wherein the number of phosphorous atoms in the phospholinker is one, wherein the linker is not a phosphorothioate linker. 
     
     
         127 . The method of  claim 120 , wherein the condition is a hematological condition. 
     
     
         128 . The method of  claim 127 , wherein the hematological condition is acute promyelocytic leukemia. 
     
     
         129 . The method of  claim 127 , wherein the hematological condition is acute myeloid leukemia. 
     
     
         130 . The method of  claim 127 , wherein the hematological condition is chronic myelogenous leukemia. 
     
     
         131 . The method of  claim 120 , wherein the subject is human. 
     
     
         132 . The method of  claim 120 , wherein the pharmaceutically-acceptable salt thereof is a sodium salt.