Carbocyclic nucleoside analogs

Abstract

The present invention relates to novel hydrolysis-stable carbocyclic 5-aza-2-deoxycytidine and carbocyclic 5-azacytidine compounds and prodrugs thereof as hypomethylating agents.

Description

[0001] The present invention relates to novel hydrolytically stable carbocyclic nucleoside analogs of 5-aza-2'-deoxycytidine and 5-azacytidine and their use as hypomethylating agents.

[0002] 5-aza-2'-deoxycytidine (decitabine, AzadC) and 5-azacytidine (Vidasa, AzaC) are nucleoside analogs that act as antimetabolites by manipulating epigenetic information [1-5]. Epigenetic information in DNA is associated with the formation of 5-methyl-2'-deoxycytidine (mdC) from 2'-deoxycytidine (dC) with the help of DNA methyltransferase (DNMT) and S-adenosylmethionine (SAM) as methylation cofactors [6-7, 4]. Methylation of dC to mdC in the promoter region is generally associated with transcriptional silencing of genes [8-9]. AzadC is a prodrug that is converted into the corresponding active triphosphate in the cell and subsequently incorporated into the genome during cell division. AzaC is also a prodrug that is 2'-deoxygenated and subsequently converted into the corresponding 5'-triphosphate for incorporation into DNA. AzaC can also be converted into 5'-triphosphate and incorporated into RNA without prior 2'-deoxygenation. The mode of action of Aza(d)C involves the reaction of its electrophilic C6 position with thiol nucleophiles in the active site of DNMTs, e.g. Figure 1 a [10-11]. This produces a covalent intermediate that is methylated by the SAM cofactor and held tightly. Due to the N atom at position 5 of the triazine heterocycle, the final β-elimination reaction that would normally release m(d)C from the DNMT enzyme is no longer possible. The result is the formation of a covalent DNA-DNMT or RNA-DNMT crosslink.

[0003] As a result of the administration of Aza(d)C, a substantial decrease in mdC levels (i.e., hypomethylation effect) was observed, which resulted in the reactivation of silenced tumor suppressor genes in cancer cells [1], which allowed the redifferentiation of cancer cells back to normal proliferating cells or caused cell death. Aza(d)C is currently used as a drug for the treatment of myelodysplastic syndrome (MDS) [2] and acute myeloid leukemia (AML) [4]. Clinically, it is administered in several cycles, each consisting of one week of treatment and three weeks of rest.

[0004] A problem associated with Aza(d)C is that it Figure 1 b) Hydrolysis by the pathway shown in Figure 4. This hydrolysis impairs the activity of Aza(d)C, especially during long treatment times.

[0005] The present inventors have overcome this problem by providing Aza(d)C analogs that can demethylate (and thus react with S-nucleophiles) while resisting hydrolysis (reaction with O-nucleophiles). Surprisingly, they found that replacing the oxygen of the deoxyribose with a CH2 group has a large long-range effect on the reactivity of the heterocycle. These analogs, particularly the carbocyclic form of Aza(d)C, still inhibit methylation by DNA methyltransferases but are hydrolytically stable, e.g. Figure 1 As shown in c.

[0006] The first aspect of the present invention relates to compounds of formula I:

[0007]

[0008] Where R 1 is H, a free or protected, modified or unmodified phosphate group or a hydroxyl protective group,

[0009] R 2 is H, a free or protected, modified or unmodified phosphate group or a hydroxyl protective group,

[0010] R 3 H, F, CH3, CH2F, CHF2, CF3, OH or OR 6 , where R 6 is a hydroxyl protective group,

[0011] R 4 and R 5 Each is H or forms an amino-protecting group,

[0012] or a salt thereof.

[0013] In the compounds of formula I, R 1 It may be selected from H, a free or protected, modified or unmodified phosphate group, such as a monophosphate, diphosphate or triphosphate group, or a hydroxyl protecting group.

[0014] The term "phosphoric acid group" includes unmodified phosphate groups (e.g., unmodified monophosphates, unmodified diphosphates, or unmodified triphosphates) or modified phosphate groups (e.g., modified monophosphates, modified diphosphates, or modified triphosphates), wherein one or more oxygen atoms are replaced by carbon, sulfur, and / or nitrogen atoms. Examples of modified phosphate groups are phosphonates, aminophosphorates, or thiophosphate groups. The term "phosphoric acid group" also includes free phosphate groups and protected phosphate groups, such as phosphates (including monoesters and diesters), and amidates (including monoamidates, diamidates, and amides). A detailed review of protected phosphates and phosphonates can be found in

[28] and

[29] , the contents of which are incorporated herein by reference. Therefore, in pharmaceutical applications, R 1 The phosphate, diphosphate or triphosphate group may be protected, so that upon cleavage in the cell the phosphate, diphosphate or triphosphate group is released (prodrug concept).

[0015] Hydroxyl protecting groups are known in the art, particularly in nucleoside chemistry, and include, but are not limited to, acyl (such as acetyl or benzoyl), benzyl, trityl (such as trityl, methoxytrityl, or dimethoxytrityl), silyl (such as trimethylsilyl, tert-butyldimethylsilyl, or triisopropylsilyl), or alkyl or substituted alkyl (such as methyl, ethoxyethyl, or β-methoxyethoxymethyl).

[0016] R 2 It can be selected from H, unmodified or modified, free or protected phosphate groups as described above, such as monophosphate, diphosphate or triphosphate groups, or hydroxyl protecting groups as described above. 2 The phosphate, diphosphate or triphosphate group may be protected to release the phosphate, diphosphate or triphosphate group upon cleavage in the cell (prodrug concept).

[0017] In certain embodiments, R 3 In this case, the five-membered carbocyclic ring in Formula I can be considered as deoxyribose, wherein the oxygen atom at the 1' position of the ring has been replaced by a CH2 group. In another embodiment, R 3 Can be OH or OR 6 , where R 6 is a hydroxyl protecting group as described above. In these embodiments, the five-membered carbon ring in Formula I can be considered as ribose, wherein the oxygen atom at the 1' position of the ring has been replaced by a CH2 group. 3It may also be CH3, CH2F, CHF2, CF3 or F. In these embodiments, the five-membered carbocyclic ring in Formula I may be considered a ribose analog in which the oxygen atom at the 1' position of the ring has been replaced by a CH2 group.

[0018] R 4 and R 5 Each can be H, thus providing a primary amino group NH2 or they can form an amino protective group, for example one or two H atoms are replaced by a protective group. Amino protective groups are known in the art, particularly in the field of nucleoside chemistry, and include but are not limited to acyl (such as acetyl or benzoyl), benzyl, benzyloxycarbonyl, tosyl, 9-fluorenylmethoxycarbonyl or tert-butyloxycarbonyl.

[0019] In certain embodiments, R 1 For H.

[0020] In certain embodiments, R 2 is H or an unmodified or modified phosphate group, such as a monophosphate, diphosphate or triphosphate group, including a free or protected monophosphate, diphosphate or triphosphate group.

[0021] In certain embodiments, R 3 is H or OH.

[0022] In certain specific embodiments, R 1 , R 2 , R 3 , R 4 and R 5 Each is H, or R 1 , R 2 , R 4 and R 5 Each is H and R 3 OH or OR 6 In a particularly specific embodiment, R 1 , R 2 , R 3 , R 4 and R 5 is H. This compound is a carbocyclic analog of AzadC (decitabine) which, as described above, has surprisingly high hydrolytic stability while maintaining inhibitory activity against methyltransferases, particularly DNA methyltransferases. In another particularly specific embodiment, R 1 , R 2 , R 4 and R 5 H and R 3 The compound is a carbocyclic analog of AzaC (Vidasa).

[0023] In another specific embodiment, the present invention also relates to AzadC and prodrugs of AzaC, particularly wherein R 1 and / or R 2 Compounds other than H.

[0024] The present invention further relates to the salt of the compound of formula I, particularly pharmaceutically acceptable salt.Compounds of the present invention can form acid and / or base salts by the presence of amino and / or phosphate / salt groups or groups similar thereto.The salt of acid addition can be formed by inorganic acid and organic acid, and includes but is not limited to acetate, benzoate, hydrobromide, hydrochloride, citrate, lactate, phosphate, toluenesulfonate and trifluoroacetate.The salt of base addition can be formed in inorganic base and organic base, includes but is not limited to ammonium salt, sodium salt, potassium salt, calcium salt, magnesium salt or the salt derived from primary amine, secondary amine and tertiary amine.

[0025] The compounds of the present invention act as antimetabolites by reducing the level of the epigenetic modification 5-methyl-2'-deoxycytidine (mdC). They can be incorporated into the genome and / or RNA of proliferating cells and inhibit DNA and / or RNA methyltransferases, resulting in a reduction of mdC in DNA and mC in RNA. The loss of mdC in DNA (transcriptional silencers in promoters) can lead to the reactivation of genes including tumor suppressor genes, thereby inducing an anti-hyperproliferative effect.

[0026] Therefore, a further aspect of the present invention relates to a compound of formula I as described above, or a pharmaceutically acceptable salt thereof, for use in medicine, such as in veterinary or human medicine.

[0027] In certain embodiments, the compounds of the invention are used to treat hyperproliferative diseases, including malignant or non-malignant hyperproliferative diseases. The term "hyperproliferative disease" relates to diseases caused by, associated with and / or accompanied by dysfunctional increased proliferation of cells, tissues and / or organs in an organism.

[0028] Therefore, the present invention also relates to a method for treating a hyperproliferative disease, comprising administering a therapeutically effective dose of a compound of formula I or a pharmaceutically acceptable salt thereof to a subject, particularly a human subject, in need thereof.

[0029] In specific embodiments, the compounds of the invention are used to treat cancer. In certain embodiments, the compounds of the invention are used to treat leukemias, such as acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), including previously treated or untreated primary or secondary MDS, such as refractory anemia, refractory anemia with ringed sideroblasts, refractory anemia with excess blasts, refractory anemia with excess blasts in transformation, and chronic myelomonocytic leukemia, as well as intermediate-1, intermediate-2, and high-risk MDS.

[0030] In other embodiments, the compounds of the invention are useful for treating renal insufficiency based on the reported results with decitabine

[26] . In certain embodiments, the compounds of the invention are useful for treating atherosclerosis based on the observation that decitabine prevents the formation of atherosclerotic lesions and reduces the production of inflammatory cytokines by macrophages

[27] .

[0031] The compound of formula I or its pharmaceutically acceptable salt can be administered to a subject in need thereof as a pharmaceutical composition comprising an active agent and a pharmaceutically acceptable carrier. The composition can be administered in any suitable manner, such as orally, parenterally, by inhalation and / or by dermal administration. The carrier can be any suitable pharmaceutical carrier. The compound of formula I is administered in a therapeutically effective dose, which can be determined by a skilled practitioner based on the subject and the condition to be treated.

[0032] In pharmaceutical applications, the composition may be in the form of a solid dosage form (eg, tablet, capsule, etc.) or a liquid dosage form (eg, solution, emulsion, suspension, etc.).

[0033] The compounds of formula I may be administered in one or several treatment cycles, wherein each treatment cycle may comprise at least 1, 2, 3, 4, 5, 6, 7 or 10 days.

[0034] The compounds of formula I can be administered as a monotherapy or in combination with another drug, particularly one suitable for treating a hyperproliferative disease as described above. In certain embodiments, the compounds of formula I can be administered together with an anticancer drug other than formula I as a combination therapy.

[0035] In addition, the compound of formula I can also be used for non-medical applications, such as generally in basic research, diagnosis and / or drug screening fields. Therefore, in certain embodiments, the present invention relates to the compound of formula I for inhibiting the methylation of nucleic acids including DNA and RNA, particularly for inhibiting the methylation of DNA, more particularly for inhibiting the in vitro use of the methylation of genomic DNA. Therefore, the compound is suitable for genome analysis in cells, such as eukaryotic cells (such as animal cells including mammals and human cells), particularly for epigenetic analysis.

[0036] The general scheme for preparing compounds of formula I is described in the following scheme. The synthesis starts with the aminocyclopentane derivative 2 protected by tert-butyloxycarbonyl (Boc), which has been previously used for the synthesis of DNA damage analogs [12-15]. Compound 2 is first benzyl protected to 3, Boc deprotected to 4, and then reacted with carbonimidazole 5, which is prepared in two steps from isomethylurea 6 (after reacting with potassium hydroxide to generate free base 7 and 7 with carbonyldiimidazole). This provides carbamoyl urea-cyclopentane nucleoside analog 8. It is then cyclized to triazine base 9 with triethyl orthoformate. Reaction of 9 with NH3 in methanol and deprotection of the benzyl group with BCl3 in dichloromethane provides the final compound cAzadC 1 as a free nucleoside.

[0037]

[0038] Compound cAzadC 1 was recrystallized from hot methanol to give colorless needle-shaped crystals, which allowed the analysis of Figure 2 The crystal structure depicted in Figure 2 is interesting because cAzadC 1 was observed to exist in two different cyclopentane conformations in the crystals. One of these conformations is typical of 2'-deoxynucleosides in DNA, suggesting that the cAzadC 1 nucleoside can adopt the correct DNA-type conformation and has the potential to be phosphorylated and integrated into the genome.

[0039] Another general scheme for preparing compounds of Formula I is described below, showing the synthesis of a carbocyclic version of azacitidine (Vidaza) cAzaC,20:

[0040]

[0041] A general scheme for preparing prodrugs of compounds of Formula I is described below, showing the synthesis of the 5'-phosphoramidite prodrug 24 of cAzaC,20:

[0042]

[0043] In addition, the present invention will be explained in more detail through the following drawings and examples.

[0044] Figure Legend

[0045] Figure 1 : Description of 5-aza-2'-deoxycytidine (decitabine, AzadC) and its mode of action. a) The active site thiol reacts with the C6 position of AzadC. b) The hydrolytic degradation pathway is closely related to the reaction of water molecules with the C6 position of AzadC (O reactivity). This leads to the final loss of the base and the formation of the abasic site. c) The carbocyclic version of cAzadC 1 is depicted in the box.

[0046] Figure 2: Crystal structure of carbocyclic 5-aza-2'-deoxycytidine (cAzadC 1) showing the molecule in the observed C6'-endo conformation (a) and C2'-endo conformation (2T1) (b).

[0047] Figure 3 : HPLC-based stability measurements showing that (a) 5-aza-2'-deoxycytidine (AzadC) solutions are severely hydrolytically decomposed at different pH values, while (b) the carbocyclic compound cAzadC1 is stable at all three pH values. The inset in (a) shows the chromatogram of AzadC between t1 = 10 min and t2 = 20 min. The signal of AzadC is depicted in red.

[0048] Figure 4 : Describes quantitative data of DNA modification of carbocyclic 5-aza-2'-deoxycytidine treated (cAzadC 1) mouse embryonic stem cells (mESCs) obtained by UHPLC-MS2. For each condition, three biological replicates were measured in three technical replicates. For each technical replicate, 0.5 μg of DNA was digested. The bar graph represents the mean and the error bars represent the standard deviation. LLOQ represents the lower limit of quantification.

[0049] Figure 5 : HPLC study of neutral solutions of decitabine 1 (left) vs. carbocyclic compound cAzaC 20 (right). 100 mM compound solutions were stored in pure water and tested at t=0h, 1.5h, 3h, 4.5h, 6h, 24h, 6d. Example

[0050] Example 1: Synthesis of cAzadC (1)

[0051] Tert-butyl-N-[(1R,3S,4R)-3-hydroxy-4-(hydroxymethyl)cyclopentyl]carbamate (2):

[0052]

[0053] C 11 H 21 NO4

[0054] 231.29g / mol

[0055] Compound 2 was synthesized from tert-butyl-N-((1S,2R,4R,5R)-4-((tert-butyldimethylsilyloxy)methyl)-6-oxa-bicyclo-[3.1.0]-hex-2-yl)-carbamate as described previously [1].

[0056] Tert-butyl-N-[(1R,3S,4R)-3-benzyloxy-4-(benzyloxymethyl)cyclopentyl]carbamate (3):

[0057]

[0058] C 25 H 23 NO4

[0059] 411.54 g / mol

[0060] Boc protected 2 (3.107 g, 13.43 mmol, 1.0 eq) was dissolved in dry DMF (75 mL). The solution was cooled to 0 °C and NaH (60% suspension in mineral oil, 1.182 g, 29.55 mmol, 2.2 eq) was added in three portions. After 15 min at 0 °C, benzyl bromide (4.00 mL, 33.58 mmol, 2.5 eq) was added dropwise. The reaction mixture was stirred at 0 °C for 1.5 h and stirred at room temperature for an additional 2 h. The reaction mixture was diluted with CH2Cl2 (30 mL) and quenched with saturated sodium bicarbonate (30 mL). The mixture was extracted with CH2Cl2 (500 mL). The organic phase was washed three times with saturated sodium bicarbonate (1×400 mL, 2×100 mL) and dried over Na2SO4. The solvent was removed in vacuo. The crude product was purified by column chromatography on silica gel with a step gradient of iHex / EtOAc (9:1→7:1→4:1) to give the product 3 as a colorless solid (3.39 g, 8.24 mmol, 61%).

[0061] R f =0.67 (iHex / EtOAc=2:1)

[0062] Mp=74-76℃

[0063] HRMS[ESI+]:[C 25 H 33 NO4Na] + ,[M+Na + ] + Calculated value: 434.2292, found value: 434.2299.

[0064] 1H-NMR (599MHz, CDCl3): δ / ppm=7.39-7.26(m,10H,Ar-H),4.76(s,1H,NH),4.53-4.37(m,4H,C7-H and C8-H),4.18-4.08(m,1H,C1-H),3.96-3.87(m,1H,C3-H),3.53-3.41(m,2H,C5-H),2.36-2.25(m,2H,C6-H a ,C4-H),2.11-2.05(m,1H,C2-H a ),1.77-1.68(m,1H,C2-H b ),1.43(s,9H,C 2’ -H), 1.29-1.20(m,1H,C6-H b ).

[0065] 13 C-NMR (151MHz, CDCl3): δ / ppm=155.5(C 1’ ),138.60,138.23,128.36,128.28,127.59,127.56,127.42(C-Ar),81.0(C3),79.2(C 2’ ),73.3(C8),71.9(C5),71.3(C7),50.4(C1),44.9(C4),39.6(C2),35.0(C6),28.6(C 2’ ).

[0066] FTIR(ATR): 2973(w),2929(w),2858(w),1692(m),1496(m),1453(m),1390(w),1364 (m),1274(m),1247(m),1166(s),1091(s),1067(s),1027(m),1012(m).

[0067] (1R,3S,4R)-1-amino-3-benzyloxy-4-(benzyloxymethyl)cyclopentane (4):

[0068]

[0069] C 20 H 25 NO4

[0070] 311,42g / mol

[0071] A solution of Boc-protected amine 3 (3.351 g, 8.14 mmol, 1.0 equiv) and trifluoroacetic acid (12 mL, 30% (v / v)) in dry CH2Cl2 (28 mL) was stirred at room temperature for 1 h. After removal of the solvent in vacuo, the residue was resuspended in saturated Na2CO3 (100 mL) solution and stirred for 10 min. The mixture was extracted with EtOAc (3×150 mL) and the combined organic phases were dried over Na2SO4. Removal of the solvent in vacuo gave the free amine 4 as a viscous brown oil, which was used without further purification due to its instability to O2.

[0072] Analytical data of purified 4:

[0073] HRMS[ESI + ]:[C 23 H 29 N4O4Na] + ,[M+Na + ] + Calculated value: 312.1958, found value: 312.1955.

[0074] 1 H-NMR (400MHz, CDCl3): δ / ppm=7.38-7.20(m,10H,C-Ar),4.57-4.36(m,4H,C7,C8),3.95-3.85(m, 1H,C1),3.62-3.50(m,1H,C3),3.50-3.39(m,2H,C5),2.41-2.28(m,1H,C4),2.28-2.13(m,1H,C6-H a ),2.13-1.96(m,3H,NH2,C2-H a ),1.65-1.50(m,1H,C2-H b ),1.19-1,.08(m,1H,C6-H a ),

[0075] 13 C-NMR (101MHz, CDCl3): δ / ppm=138.8,138.4,128.5,128.5,127.79,127.75,127.7,127.6(C-Ar) ,81.82(C3),73.23(C7),72.58(C5),71.10(C8),51.40(C1),45.63(C4),42.07(C2),38.08(C6).

[0076] 2-Methyl-1-(1-imidazolylcarbonyl)isourea (5):

[0077]

[0078] C6H8N4O4

[0079] 168.16 g / mol

[0080] To a solution of O-methylisourea hydrochloride 6 (5.00 g, 45.65 mmol, 1.0 equiv) in Et2O:H2O (39:1) was added KOH powder (51.22 g, 912.96 mmol, 20.0 equiv) in portions at -15°C with constant stirring. After 30 min, the mixture was filtered and the residue was washed with ice-cold Et2O (3×50 mL). The combined organic phases were concentrated in vacuo to 20 mL. Cooling to -20°C resulted in precipitation. Vacuum filtration under N2 flow gave O-methylisourea 7 as a colorless wax (1.759 g, 23.74 mmol, 52%). A solution of 7 (1.24 g, 16.74 mmol, 1.0 equiv) and carbonyldiimidazole (2.90 g, 17.91 mmol, 1.07 equiv) in dry THF (35 mL) was stirred at room temperature for 3 h. After 1 h, a colorless precipitate was observed. The reaction mixture was concentrated in vacuo to 3 mL and filtered. The residue was washed with ice-cold THF (2×3 mL) and the combined organic phases were lyophilized to give a colorless solid 5 (1.946 g, 11.57 mmol, 69%).

[0081] 1 H-NMR (400MHz, CDCl3): δ / ppm = 8.60 (br s, 1H, NH), 8.35 (s, 1H, Ar-H), 7.58 (s, 1H, Ar-H), 7.04 (s, 1H, Ar-H), 6.04 (br s, 1H, NH), 3.96 (s, 3H, C2-H).

[0082] 13 C-NMR (101MHz, CDCl3): δ / ppm=165.7(C1), 157.3(C3), 137.5(Ar), 130.0(Ar), 117.2(Ar), 55.3(C2).

[0083] 1-[(1'R,3'S,4'R)-3'-Benzyloxy-4'-(benzyloxymethyl)cyclopentyl]methyl isobiuret (8):

[0084]

[0085] C 23 H 29 N4O4

[0086] 411.50g / mol

[0087] The crude product 4 was dissolved in dry acetonitrile (75 mL) and carbonylimidazole 5 (1.506 g, 8.96 mmol, 1.1 eq.) was added. The mixture was refluxed for 2 h. The solvent was removed in vacuo. The crude product was purified by column chromatography on silica gel with a stepwise gradient of iHex / EtoAc (2:1→1:1) to give methyl isobiuret 8 (2.613 g, 6.35 mmol, 78%) as a yellow oil.

[0088] R f =0.51 (DCM / MeOH=19:1)

[0089] HRMS[ESI + ]:[C 23 H 29 N4O4Na] + ,[M+Na + ] + Calculated value: 434.2050, found value: 434.2049.

[0090] 1 H-NMR (599MHz, CDCl3): δ / ppm=7.35-7.16(m,10H,Ar-H),5.42(d, 3 J 1’,N1 =7.8Hz,1H,N1-H),4.50-4.34(m,4H,C 7’ -H a,b ,C 8’ -H a,b ),4.33-4.16(m,1H,C 1’ -H), 3.93-3.81(m,1H,C 3’ -H),3.62(s,3H,C6-H),3.43-3.31(m,2H,C 5’ -H), 2.34-2.18(m,2H,C 6’ -H a ,C 4’ -H),2.06(ddd, 2 J 2’a,2’b =13,0Hz, 3 J 2’a,1’ / 3’ =6,9Hz, 3 J 2’a,1’ / 3’ =4,6Hz,1H,C 2’ -H a ),1.73(ddd, 2 J 2’a,2b’ =13.0Hz, 3 J 2’b,1’ / 3’ =7.0Hz, 3 J2’b,1’ / 4’ =7.0Hz,1H,C 2’ -H b ),1.28-1.11(m,1H,C 6’ -H b ).

[0091] 13 C-NMR (151MHz, CDCl3): δ / ppm=163.8(C2),162.3(C4),138.9(C Ar ),138.5(C Ar ),128.51(C Ar ),128.41(C Ar ),127.76(C Ar ),127.74(C Ar ),127.70(C Ar ),127.56(C Ar ),81.1(C 3’ ),73.3(C 7’ ),71.9(C 5’ ),71.2(C 8’ ),53.8(C6),49.5(C 1’ ),45.0(C 4’ ),39.5(C 2’ ),35.0(C 6’ ).

[0092] 4-Ethoxy-1-[(1'R,3'S,4'R)-3'-benzyloxy-4'-(benzyloxymethyl)cyclopentyl]-1H-[1,3,5]-triazine-2-one (9a) and 4-methoxy-1-[(1'R,3'S,4'R)-3'-benzyloxy-4'-(benzyloxymethyl)cyclopentyl]-1H-[1,3,5]-triazine-2-one (9b):

[0093]

[0094] Trifluoroacetic acid (75 μL) was added to a solution of 8 (2.613 g, 6.35 mmol, 1.0 eq) in triethyl orthoformate (60 mL). The mixture was refluxed for 3 h. The solvent was removed in vacuo. The residue was co-evaporated once with MeOH and the crude product was purified by column chromatography on silica gel with a stepwise gradient of iHex / EtOAc (2:1→1:1→1:2) to give 9a (1.774 g, 4.07 mmol, 64%, colorless solid) and 9b (268.0 mg, 0.64 mmol, 10%, colorless oil).

[0095] 9a:

[0096] R f =0.71(iHex / EtOAc,1:3)

[0097] HRMS[ESI + ]:[C 25 H 30 N3O4H] + ,[M+H + ] + Calculated value: 436.2232, found value: 436.2231.

[0098] 1 H-NMR (400MHz, CDCl3): δ / ppm=8.23(s,1H,C6-H),7.38-7.24(m,10H,Ar-H),5.07-4.89(m,1H,C 1’ -H), 4.56-4.39(m,6H,C 7’ -H a,b ,C 8’ -H a,b ,C7-H a,b ),4.07-3.08(m,1H,C 3’ -H), 3.61-3.46(m,2H,C 5’ -H a,b ),2.52-2.36(m,2H,C 6’ -H a ,C 4’ -H),2.28(ddd,1H, 2 J 2’a,2’b =13.3Hz, 3 J=7.6Hz, 3 J=2.7Hz,C 2’ -H a ),2.11(ddd,1H, 2 J 2’a,2’b =13.3Hz, 3 J=10.0Hz, 3 J=6.3Hz,C 2’ -H b ),1.84-1.68(m,1H,C 6’ -H b ),1.40(t,3H, 3 J 8,7 =7.1Hz,C8-H),

[0099] 13C-NMR (101MHz, CDCl3): δ / ppm=169.3(C4),158.4(C6),155.0(C2),138.2(C Ar ),138.1(C Ar ),128.6(C Ar ),128.6(C Ar ),127.93(C Ar ),127.85(C Ar ),127.82(C Ar ),80.4(C 3’ ),73.4(C 7’ / 8’ ),71.26(C 7’ / 8’ ),71.25(C 5’ ),65.1(C7),56.8(C 1’ ),44.9(C 4’ ),36.9(C 2’ ),32.6(C 6’ ),14.2(C8).

[0100] 9b:

[0101] R f =0.63 (iHex / EtOAc, 1:3)

[0102] HRMS[ESI + ]:[C 24 H 28 N3O4] + ,[M+H + ] + Calculated value: 422.2074, measured value: 422.2075.

[0103] 1 H-NMR (599MHz, CDCl3): δ / ppm==8.23(s,1H,C6-H),7.38-7.25(m,10H,Ar-H),5.04-4.93(m,1H,C 1’ -H), 4.57-4.43(m,6H,C 7’ -H a,b ,C 8’ -H a,b ),4.09-3.98(m,4H,C 3’ -H,C7-H),3.60-3.50(m,2H,C 5’ -H a,b ),2.50-2.39(m,2H,C 6’ -H a ,C 4’-H),2.28(ddd,1H, 2 J 2’a,2’b =13.1Hz, 3 J=7.4Hz, 3 J=2.7Hz,C 2’ -H a ),2.11(ddd,1H, 2 J 2’a,2’b =13.1Hz, 3 J=10.1Hz, 3 J=6.3Hz,C 2’ -H b ),1.80-1.68(m,1H,C 6’ -H b ).

[0104] 13 C-NMR (101MHz, CDCl3): δ / ppm=169.9(C4),158.5(C6),155.0(C2),138.3(C Ar ),138.0(C Ar ),128.63(C Ar ),128.57(C Ar ),127.93(C Ar ),127.84(C Ar ),127.82(C Ar ),80.4(C 3’ ),73.5(C 7’ / 8’ ),71.3(C 7’ / 8’ ),56.9(C 1’ ),56.0(C7),44.9(C 4’ ),36.9(C 2’ ),32.6(C 6’ ).

[0105] 4-Amino-1-[(1'R,3'S,4'R)-3'-benzyloxy-4'-(benzyloxymethyl)cyclopentyl]-1H-[1,3,5]triazine-2-one (10):

[0106]

[0107] C 23 H 26 N4O3

[0108] 406.47 g / mol

[0109] Ethoxytriazine 9a (1.630 g, 3.74 mmol, 1.0 equiv) was dissolved in methanolic NH3 (7N in MeOH, 60 mL) and stirred at room temperature for 3 h. The mixture was diluted with H2O (340 mL) to produce a colorless precipitate, which was extracted with EtOAc (3×200 mL). The organic phases were combined, dried and the solvent removed in vacuo to give benzyl protected cAzadC 10 (1.478 g, 3.63 mmol, 97%) as a colorless foam. Starting from methoxytriazine 9b and following the same procedure, 10 was also successfully synthesized.

[0110] R f =0.50 (CH2Cl2 / MeOH, 9:1).

[0111] HRMS[ESI + ]:[C 23 H 27 N4O3] + ,[M+H + ] + Calculated value: 407.2078, found value: 407.2081.

[0112] 1 H-NMR (599MHz, CDCl3): δ / ppm=8.01(s,1H,C6-H),7.35-7.23(m,10H,Ar-H),5.78(s,2H,NH),4.92-4.76(m,4H,C 7’ -H and C 8’ -H),4.59-4.46(m,1H,C1'-H),4.07-3.95(m,1H,C 3’ -H), 3.55-3.45(m,2H,C 5’ -H), 2.44-2.28(m,2H,C 6’ -H a ,C 4’ -H), 2.24-2.16(m,1H,C 2’ -H a ),2.14-2.05(m,1H,C 2’ -H b ),1.78-1.67(m,1H,C 6’ -H b ).

[0113] 13C-NMR (151MHz, CDCl3): δ / ppm=165.7(C4),157.1(C6),154.2(C2),138.3,138.3,128.6,128.5,127.84,127.78(C-Ar),80.4(C 3’ ),73.4(C 8’ ),71.4(C 5’ ),71.2(C 8’ ),56.7(C 1’ ),44.9(C 4’ ),36.8(C 2’ ),32.5(C 6’ ).

[0114] 4-Amino-1-[(1'R,3'S,4'R)-3'-hydroxy-4'-(hydroxymethyl)cyclopentyl]-1H-[1,3,5]triazine-2-one (cAzadC,1):

[0115]

[0116] C9H 14 N4O3

[0117] 226.24 g / mol

[0118] A solution of benzyl protected 10 (1.478 g, 3.63 mmol, 1.0 eq) in CH2Cl2 (100 mL) was cooled to -78 °C and BCl3 (1 M in DCM, 12.7 mL, 12.7 mmol, 3.5 eq) was added dropwise. The mixture was stirred at -78 °C for 1 h and at room temperature for an additional 2 h. The reaction was quenched by the addition of MeOH (85 mL) under constant stirring. The solvent was removed in vacuo. The crude product was purified by column chromatography on silica gel with a stepwise gradient of CH2Cl2 / MeOH (9:1→7:3) to afford cAzadC (1, 0.740 g, 3.27 mmol, 90%). Recrystallization from hot MeOH afforded 57% of cAzadC (1, 465.3 mg, 2.06 mmol) as colorless needle-shaped single crystals.

[0119] R f =0.1 (CH2Cl2 / MeOH, 9:1).

[0120] Mp.:198-200℃.

[0121] HRMS[ESI + ]:[C9H 15 N4O3] + ,[M+H + ]+ Calculated value: 227.1138, found value: 227.1139.

[0122] 1 H-NMR (400MHz, D2O): δ / ppm=8.29(s,1H,C6-H),4.90-4.62(m,1H,C 1’ -H, overlap with D2O signal), 4.29-4.16 (m, 1H, C 3’ -H),3.73(dd, 2 J 5’a,5’b =11.2Hz, 3 J 5’a,4’ =5.7Hz,1H,C 5’a -H),3.62(dd, 2 J 5’a,5’b =11.2Hz, 3 J 5’b,4’ =6.8Hz,1H,C 5’b -H), 2.41-2.29(m,1H,C 6’a -H), 2.28-2.18(m,1H,C 2’a -H), 2.17-2.02(m,2H,C 2’b -H,C 4’b -H), 1.77-1.55(m,1H,C 6’b -H).

[0123] 13 C-NMR (101MHz, CDCl3): δ / ppm=165.5(C4),158.5(C6),156.4(C2),72.2(C 3’ ),62.8(C 5’ ),56.2(C 1’ ),48.11(C 4’ ),38.0(C 2’ ),32.0(C 6’ ).

[0124] FTIR(ATR): 1653(m),1540(s),1437(s),1278

[0125] (s),1036(s).

[0126] Example 2: X-ray crystal structure analysis of cAzadC

[0127] Single crystals of cAzadC were analyzed by X-ray crystallography. Table 1 shows the solved structures of cAzadC 1 in two different cyclopentane conformations in the crystals. One conformer adopts a C6′-endo (P = 88.2°, max = 47.8°) conformation ( Figure 2 a), while the second exists as a C2'-endo-C3'-exo (south, P = 150.8°, max = 45.4°) conformer ( Figure 2 b) The latter conformation is typical for 2'-deoxynucleosides in DNA.

[0128] Additional figures were generated using the Mercury 3.5.1 program (Cambridge Crystallographic Data Center). The data are deposited under CCDC1910952.

[0129] Table 1: Crystallographic data of cAzadC:

[0130]

[0131] Example 3: Testing for hydrolytic stability

[0132] Next, the hydrolytic stability of cAzadC 1 was investigated and directly compared with the drug AzadC.

[0133] Both nucleosides were dissolved in KH2PO4 aqueous buffer (100mM, pH=7.4, 5.5 and 8.5) at a concentration of 100mM. The solution was analyzed by HPLC immediately (t=0h), followed by HPLC analysis every 1.5h or at a specified time point. EC 250 / 4NUCLEOSIL 120-3C18 (Macherey-Nagel) chromatographic column was used as the stationary phase. The mobile phase consisted of water (buffer A) and acetonitrile (buffer B) with a flow rate of 0.5mL / min, as follows: 0→25min, 0→5% buffer B; 25min→28min, 5%→80%; 28min→38min, 80%; 38min→43min, 80%→0%; 43min→45min, 0%.

[0134] Since one treatment cycle of AzadC lasts more than 4 weeks, it was decided to measure stability at time points associated with half a cycle (14 days). NMR spectra were measured after keeping the solutions at room temperature.

[0135] Since tumor cells often provide an acidic microenvironment

[16] , stability at slightly acidic pH is particularly useful. Figure 3As is evident from the data shown in , the drug AzadC is strongly degraded during these 14 days. Importantly, at pH = 5.5 and pH = 8.5, intact AzadC is barely detectable anymore. At physiological pH (7.4), AzadC is still present after 14 days, but is significantly degraded.

[0136] In contrast to these results, it was surprisingly observed that cAzadC 1 did not degrade at all pH values ​​tested, including pH = 5.5. This result surprisingly suggests that a simple O→CH2 exchange induces a strong long-range desorption effect, which appears to change the properties of the triazine ring, thereby stopping the reaction with water.

[0137] Example 4 - Biological Function Test

[0138] mESC cell culture for cAzadC treatment

[0139] Feeder-independent wild-type J1 (129S4 / SvJae strain)

[24] cells were cultured in the presence of serum and LIF as previously described

[25] . They were routinely maintained on gelatinized plates in 2i / L medium. For priming experiments, 2i cultures were passaged in DMEM supplemented with FBS and LIF, as described above but lacking inhibitors, when applicable. For drug treatments, cells were primed by removing 2i from the culture medium. Cells were incubated in 6-well plates (VWR) in DMEM supplemented with FBS and LIF for 2 days. After splitting, 2x10 5 The cells were transferred into 6-well plates and supplemented with 1 μM (in 0.01% DMSO) or 5 μM cAzadC (in 0.05% DMSO) and treated for 72 h. After removing the medium and washing the cells with DPBS, they were directly treated with RLT + Buffer lysis was performed as described in a previous publication

[18] .

[0140] gDNA isolation, total enzyme digestion and UHPLC-MS 2

[0141] gDNA was isolated as previously described

[18] . gDNA was directly subjected to total enzyme digestion and analyzed by UHPLC-MS 2 The analysis was performed as described in a previous publication

[19] . An external calibration curve was generated by serial dilution of pure cAzadC [20 pmol, 10 pmol, 5 pmol, 2.5 pmol, 1.25 pmol, 0.625 pmol, 0.3125 pmol] and measuring in technical triplicate before each measurement. Linear regression was performed with OriginPro 2016G.

[0142] result

[0143] It was investigated whether the relief effect described in Example 3 affects biological functions. For this purpose, mouse embryonic stem cells (mESCs) primed in serum / Lif were used as a model system, since mdC levels increase from the naive to the primed state

[17] . Two different concentrations (1 μM and 5 μM) of cAzadC 1 were added to mESCs primed for 48 h, and the cells were allowed to proliferate further for 72 h under primed conditions in the presence of cAzadC1. After 72 h, the cells were harvested, the DNA isolated and digested to the nucleoside level using the protocol we described

[18] .

[0144] Isotope dilution UHPLC-MS2 was used to precisely quantify the levels of mdC. To this end, isotope-labeled nucleoside standards were spiked for accurate quantification [19,18]. In addition to mdC, the levels of 5-hydroxymethyl-2'-deoxycytidine (hmdC), which is formed from mdC by the action of TET enzymes [20-21], were also quantified. The absolute levels of hmdC in mESCs are more than tenfold lower than those of mdC [20,22]. The result is that even after a large reduction in mdC, there should be enough mdC to keep hmdC levels unchanged. Therefore, the question of whether and to what extent hmdC levels are affected can tell how epigenetic reprogramming is organized.

[0145] In parallel with the quantification of mdC and hmdC, the expansion of cAzadC 1 itself incorporated into the mESC genome was also quantified. AzadC could only be detected in the genome of treated cells after treatment of the DNA with NaBH4. The application of NaBH4 reduces the C(5)=C(6) double bond, thus stabilizing the compound and making its quantification possible [23,19]. Interestingly, we noticed that the stability of cAzadC 1 allowed its quantification without such pretreatment. It was also noted that the enzymatic digestion protocol applied allowed the digestion of genomic DNA (gDNA) even in the presence of large amounts of cAzadC 1. In summary, the quantification of cAzadC 1 by UHPLC-MS using an external calibration curve was performed. 2 Quantification of cAzadC1 can be performed in parallel with quantification of canonical and epigenetic bases.

[0146] At 1 μM cAzadC 1 concentration, cAzadC 1 levels of 5x10 per dN were detected. -4 cAzadC( Figure 4 a). This corresponds to nearly 3 million cAzadC nucleotides integrated into the genome. At a higher concentration of 5 μM cAzadC 1, levels increased 3-fold to 1.7x10 per dN. -3cAzadC, thus the integrated cAzadC per genome exceeds 8 million. Compared to the incorporation of AzadC, which reached 1.2x10 per dN when 1 μM was applied, -3 AzadC

[19] , cAzadC 1 reached levels approximately one-third of that. The data clearly show that the carbocyclic version of AzadC (cAzadC 1) is incorporated and that it ultimately reaches comparable levels in the genome at a concentration of 5 μM.

[0147] Importantly, after exposure of mESCs to 1 M cAzadC in culture for 72 h, a decrease of nearly 30% in mdC values ​​was detected ( Figure 4 b). At a concentration of 5 M in the culture medium, mdC levels even dropped to about 50% of the original value. A reduction of 50% was also observed for AzadC. However, here, the 50% reduction was reached much faster (24 h) and already at a lower AzadC concentration (1 μM)

[19] .

[0148] The data show that the carbocyclic version of cAzadC 1 simply takes more time to affect mdC levels by the same amount. We believe that this effect is caused by a potentially slower conversion of cAzadC 1 to the triphosphate. However, given the longer treatment durations for clinical applications, the slower kinetics of cAzadC 1 are not necessarily a disadvantage.

[0149] It is very interesting to find that hmdC levels were already reduced to about 50% in the 1 μM experiment. At 5 μM, hmdC could not even be detected above background levels using 0.5 μg of genomic DNA. The results show that hmdC levels dropped even faster than mdC levels, although hmdC is more than ten times less abundant in the genome. This result is very interesting. This suggests that hmdC might be mainly produced during mdC maintenance during cell division. The compound cAzadC 1 is seen here as a perfect tool molecule that now allows to further understand the interplay between methylation of dC to mdC and oxidation of mdC to hmdC. With the new compound cAzadC 1, it is possible to clearly correlate the demethylation of the genome with the corresponding cellular effects without compromising the DNA damaging effects. Finally, cAzadC 1 might not only be a valuable tool compound but also the next generation of epigenetic drugs.

[0150] Summarize

[0151] We show that replacing the O atoms within the ring with CH2 units stabilizes the drug, thereby stopping its nucleophilic reaction with water. The new nucleoside cAzadC 1 is accepted by phosphorylases in the cell and the corresponding cAzadC-triphosphate is efficiently incorporated into the genome. cAzadC 1 is incorporated into the genome at millions of nucleotides and it causes a reduction in mdC levels to 70% relative to control levels.

[0152] Example 5: Synthesis of cAzaC (20)

[0153] Tert-butyl-(1R,4S,5R,6S)-5,6-dihydroxy-3-oxo-2-azabicyclo[2.2.1]heptane-2-carboxylate 12:

[0154]

[0155] Boc protected Vince lactam 11

[30] (11.66 g, 55.7 mmol, 1.0 eq) was dissolved in 214 mL THF, 92 mL tert-butyl alcohol and 31 mL H2O, 22.59 g N-methylmorpholine-N-oxide (167.2 mmol, 3 eq) and 1.03 g K2OsO4 dihydrate (2.79 mmol, 5 mol%) were added and the mixture was stirred at room temperature for 16 h. Subsequently, 200 mL water and 35.1 g Na2SO3 (278.5 mmol, 5.0 eq) were added and the mixture was stirred at room temperature for an additional 1 h. Subsequently, the reaction mixture was extracted with EtOAc (3×300 mL). The combined organic layers were washed with saturated NaCl aqueous solution (300 mL) and dried over Na2SO4. After evaporation of the solvent in vacuo, the residue was purified by column chromatography on silica gel (CH2Cl2:MeOH 40:1→CH2Cl2:MeOH 30:1) to give 2.91 g of the exo-bishydroxylated product 12 (12.0 mmol, 22%) as a yellow foam.

[0156] 1 H-NMR (400MHz, CDCl3): δ / ppm=4.35-4.32(m,1H,1-H),4.23(d,J=6.0Hz,1H,5-H),4.08(d,J=6.1Hz,1H,6-H),3.89-3.80(br s,-OH),3.78-3.68(br s,-OH)2.82-2.76(m,1H,4-H),2.09(dt,J=10.9,1.5Hz,1H,7-H),1.97(m,1H,7-H),1.51(s,9H,3′-H).

[0157] 13C-NMR (101MHz, CDCl3): δ / ppm=173.30 (3-C), 149.20 (1′-C), 83.82 (2′-C), 70 .57(6-C),68.14(5-C),62.29(1-C),53.74(4-C),31.99(7-C),28.16(3′-C).

[0158] IR(ATR):ν(cm -1 )=3415(w),2979(w),1777(m),1714(m),1457

[0159] (w),1394(w),1368(m),1350(m),1326(m),1297(m),1256(m),1144(s),1081(s) ,1043(w),1031(w),1016(m),947(s),902(w),838(w),776(m),733(s),702(m).

[0160] R f (CH2Cl2:MeOH 20:1):0.24.

[0161] Tert-butyl-(1R,2S,3R,4R)-2,3-dihydroxy-4-(hydroxymethyl)-cyclopentyl)-carbamate 13:

[0162]

[0163] A solution of bishydroxylated lactam 12 (2.90 g, 11.9 mmol, 1.0 eq) in 40 mL of dry MeOH was cooled to 0 °C and NaBH4 (2.25 g, 59.61 mmol, 5.0 eq) was added in 5 portions over 15 min. The mixture was further stirred at 0 °C for 45 min, warmed to room temperature and stirred at room temperature for an additional 1 h. Subsequently, 100 mL of acetic acid (10% v / v in MeOH) and silica gel were added and the mixture was concentrated to dryness in vacuo. Purification by silica gel column chromatography (dry loading, CH2Cl2:MeOH 5:1 → CH2Cl2:MeOH 4:1) and co-evaporation with dry toluene (2×150 mL) gave the product 13 as its acetate (2.96 g, 9.6 mmol, 81%) as a slightly brown oil.

[0164] 1H-NMR (400MHz, DMSO-d6): δ / ppm=6.70(d,J=7.9Hz,1H,NH), 3.65-3.49(m,2H,1-H,3-H), 3.52(dd,J=6.3,5.2Hz,1H,2-H), 3.37(dd,J=10.5,5.8Hz,1 H,5-H),3.30(dd,J=10.5,6.2Hz,1H,5-H),1.98(dt,J=13.1,8.4Hz,1H,6- H),1.86-1.77(m,4H,4-H,OAc),1.37(s,9H,3′-H),1.04-0.90(m,1H,6-H).

[0165] 13 C-NMR (101MHz, DMSO-d6): δ / ppm=173.28 (OAc-C1), 155.23 (C-1′), 77.37 (2′-C), 75.93 (2-C), 7 2.07(1-C),63.02(5-C),55.07(3-C),44.94(4-C),30.61(6-C),28.30(3′-C),22.49(OAc-C2).

[0166] R f (CH2Cl2:MeOH 8:1):0.43.

[0167] Tert-butyl-((1R,2S,3R,4R)-2,3-bis(benzyloxy)-4-((benzyloxy)methyl)-cyclopentyl)-carbamate 14:

[0168]

[0169] A solution of cyclopentane 13 (2.93 g, 9.5 mmol, 1.0 eq) in 67 mL of dry DMF was cooled to 0 °C and NaH (1.57 g, 39.3 mmol, 4.1 eq, 60% solution in mineral oil) was added in 3 portions. After the mixture was stirred at 0 °C for 15 min, 5.31 mL of BnBr (44.6 mmol, 4.7 eq) was slowly added and the reaction mixture was stirred at 0 °C for 2.5 h and at room temperature for 3 h. Then, 220 mg of TBAI (0.6 mmol, 6 mol%) and another 3.18 mL of BnBr (26.8 mmol, 2.8 eq) were added and the mixture was heated to 80 °C for 3.5 h. Subsequently, the mixture was cooled to room temperature, poured into 300 mL of saturated aqueous NaHCO3 and extracted with CH2Cl2 (3×300 mL). The combined organic layers were washed with saturated aqueous NaHCO3 (300 mL), saturated aqueous NaCl (300 mL), dried over anhydrous Na2SO4 and concentrated in vacuo at 50° C. The residue was purified by column chromatography (iHex:EtOAc 10:1→iHex:EtOAc 8:1→iHex:EtOAc 7:1→iHex:EtOAc 6:1→iHex:EtOAc 4:1) to give the tribenzyl protected product 14 (2.47 g, 4.8 mmol, 51%) as a colorless solid wax.

[0170] 1 H-NMR (400MHz, CDCl3): δ / ppm=7.45-7.09(m,15H,3×OCH2Ph),4.95(s,1H,NH),4.76-4.25(m,6H,3×OCH2Ph),4.15-3.90(m,1H,1-H),3.82(dd,J=6 .7,4.4Hz,1H.3-H),3.79-3.63(m,1H,2-H),3.57-3.34(m,2H,5-H),2.51 -2.24(m,2H,4-H,6-H),1.50-1.34(m,9H,3′-H),1.31-1.19(m,1H,6-H).

[0171] 13C-NMR (101MHz, CDCl3): δ / ppm=155.09 (1′-C), 138.48 (Ar-C), 138.43 (Ar-C), 138.13 (Ar-C), 128. 44(Ar-C),128.30(Ar-C),128.24(Ar-C),128.03(Ar-C),127.88(Ar-C),127.82(Ar-C),127.70(Ar -C),127.54(Ar-C),127.51(Ar-C),81.27(2-C),79.29(3-C),79.13(2′-C),73.30(OCH2-Ph),71. 52(OCH2-Ph),71.02(OCH2-Ph),70.41(5-C),52.85(1-C),41.49(1-C),30.34(6-C),28.46(3′-C).

[0172] HRMS(ESI + ):For C 32 H 40 NO5 + [M+H] + , calculated value: 518.2901; found value: 518.2911.

[0173] IR(ATR):ν(cm -1 )=3331(br w),3029(w),2974(w),2867(w),1705(s),1495(m),1453(m),1390(w),1 364(s),1245(m),1165(s),1092(s),1071(s),1027(m),732(s),695(s).

[0174] R f (iHex:EtOAc 4:1):0.69.

[0175] (1R,2S,3R,4R)-2,3-bis(benzyloxy)-4-(benzyloxy)methyl)-cyclopentane-1-amine 15:

[0176]

[0177] Under Ar atmosphere, 1.02 g of Boc protected cyclopentane 14 (1.97 mmol, 1.0 equiv) was dissolved in 7.0 mL of dry CH2Cl2, 3.0 mL of TFA was added and the mixture was stirred at room temperature for 1 h. The red solution was concentrated to dryness, 20.0 mL of saturated NaHCO3 aqueous solution was added and the mixture was stirred at room temperature for 10 min. Then, 60 mL of EtOAc was added, the phases were separated, and the aqueous layer was further extracted with EtOAc (2×60 mL). The combined organic layers were dried over Na2SO4 and concentrated to dryness in vacuo to give the deprotected amine 15 as a yellow oil (882 mg, 1.97 mmol, quantitative), which was used in the next step without further purification due to its instability to oxidation.

[0178] 1 H-NMR (400MHz, CDCl3): δ / ppm=7.39-7.26(m,15H,3×OCH2Ph), 4.63-4.33(m,6H,3×OCH2Ph), 3.81(dd,J=5.0,3.1Hz,1H,3-H), 3.49(dt,J=9.1,7.8 Hz,1H,1-H),3.41(dd,J=9.2,5.1Hz,1H,5-H),3.34-3.26(m,2H,2-H,5-H ),2.47-2.34(m,1H,4-H),2.24-2.13(m,1H,6-H),1.06-0.94(m,1H,6-H).

[0179] 13 C-NMR (101MHz, CDCl3): δ / ppm=138.66(Ar-C), 138.52(Ar-C), 138.48(Ar-C), 128.72(A r-C),128.52(Ar-C),128.47(Ar-C),128.38(Ar-C),128.31(Ar-C),128.00(Ar-C),127 .74(Ar-C),127.67(Ar-C),127.13(Ar-C),86.10(2-C),78.11(3-C),73.21(OCH2-Ph), 72.27(5-C),71.86(OCH2-Ph),70.89(OCH2-Ph),54.32(1-C),41.49(4-C),32.19(6-C).

[0180] HRMS(ESI + ):For C 27 H 32 NO3 + [M+H] +, calculated value: 418.2377; found value: 418.2378.

[0181] 1-[(1'R,2'S,3'R,4'R)-2',3'-bis(benzyloxy)-4'-((benzyloxy)methyl)-cyclopentyl]-methyl isobiuret 16

[0182]

[0183] Under Ar atmosphere, 882 mg of cyclopentylamine 15 (1.87 mmol, 1.97 mmol, 1.0 equivalent) was dissolved in 18.20 mL of dry MeCN, and 431 mg of 2-methyl-1-(1-imidazolylcarbonyl)-isourea was added. [1] (2.56 mmol, 1.3 eq.). The mixture was refluxed at 92 °C for 2 h, then cooled to room temperature and evaporated to dryness. The residue was purified by column chromatography on silica gel (iHex:EtOAc 3:1 → iHex:EtOAc 5:2 → iHex:EtOAc 2:1 → iHex:EtOAc 1:1) to give methyl isobiuret 16 (886 mg, 1.17 mmol, 87%) as a colorless oil.

[0184] 1 H-NMR (400MHz, CDCl3): δ / ppm=7.43-7.22(m,15H,3×OCH2Ph),5.60(s,1H,1-H),4.80-4.61(m,2H, OCH2Ph),4.55-4.41(m,3H,OCH2Ph),4.32(d,J=11.8Hz,1H,OCH2Ph),4.24(td,J=7.6,3.4Hz,1H,1 ′-H),3.87(dd,J=6.9,4.5Hz,1H,3′-H),3.82(dd,J=4.5,3.1Hz,1H,2′-H),3.61(s,2H,3-H,5-H), 3.57-3.45(m,2H,5′-H),2.54-2.39(m,2H,4′-H,6′-H),2.17(s,3H,6-H),1.39-1.31(m,1H,6′-H).

[0185] 13C-NMR (101MHz, CDCl3): δ / ppm=207.11(4-C),162.27(2-C),138.74(Ar-C),138.71(Ar-C),1 38.45(Ar-C),128.51(Ar-C),128.34(Ar-C),128.28(Ar-C),127.86(Ar-C),127.70(Ar-C), 127.59(Ar-C),127.56(Ar-C),81.65(2′-C),79.79(3′-C),73.35(OCH2-Ph),71.64(OCH2-P h),71.20(OCH2-Ph),70.60(5′-C),52.56(1′-C),41.68(4′-C),31.07(6-C),30.57(6′-C).

[0186] HRMS(ESI + ):For C 30 H 36 N3O5 + [M+H] + , calculated value: 518.2650; found value: 518.2652.

[0187] IR(ATR):ν(cm -1 )=3384(br w),3028(w),2857(w),1694(w),1635(s),1495(s),1453(m),1364(m),1300(w),1197(w),1096(s),1027(m)799(w),736(m),697(s).

[0188] R f (iHex:EtOAc 2:1):0.37.

[0189] 4-Methoxy-1-[(1'R,2'S,3'R,4'R)-2',3'-bis(benzyloxy)-4'-((benzyloxy)methyl)-cyclopentyl]-1H-[1,3,5]-triazine-2-one (17) and 4-ethoxy-1-[(1'R,2'S,3'R,4'R)-2',3'-bis(benzyloxy)-4'-((benzyloxy)methyl)-cyclopentyl]-1H-[1,3,5]-triazine-2-one (18):

[0190]

[0191] Methyl isobiuret 16 (837 mg, 1.62 mmol, 1.0 equivalent) was dissolved in 15.30 mL triethyl orthoformate, and 20 μL TFA was added. The mixture was heated to 90 ° C for 3 h, then cooled to room temperature and concentrated in vacuo at 50 ° C. The residue was co-evaporated with dry methanol, and purified by column chromatography on silica gel (iHex: EtOAc 3: 1 → iHex: EtOAc 5: 2 → iHex: EtOAc 2: 1 → iHex: EtOAc 1: 1 → iHex: EtOAc 1: 2) to obtain 373 mg ethoxy triazine 18 (0.69 mmol, 43%) colorless wax and 389 mg methoxy triazine 17 (0.73 mmol, 46%) colorless solid.

[0192] Methoxytriazine 17:

[0193] 1 H-NMR (400MHz, CDCl3): δ / ppm=7.96 (s, 1H, 6-H), 7.38-7.17 (m, 15H, 3×OCH2Ph), 4.70-4 .40(m,6H,1′-H,2.5×OCH2Ph),4.36-4.24(m,2H,2′-H,0.5×OCH2Ph),3.98(s,3H,7-H),3 .93(dd,J=4.9,3.1Hz,1H,3′-H),3.54-3.40(m,2H,5′-H),2.51(ddt,J=9.8,4.4,2.3Hz, 1H, 4′-H), 2.35 (dt, J=13.3, 9.4Hz, 1H, 6′-H), 1.82 (ddd, J=13.4, 9.0, 6.7Hz, 1H, 6′-H).

[0194] 13 C-NMR (101MHz, CDCl3): δ / ppm=169.87(4-C), 159.59(6-C), 154.67(2-C), 138.16(Ar-C), 138.14(A r-C),137.63(Ar-C),128.63(Ar-C),128.60(Ar-C),128.51(Ar-C),128.24(Ar-C),128.20(Ar-C), 128.17(Ar-C),127.90(Ar-C),127.86(Ar-C),78.78(2′-C),77.27(3′-C),73.40(OCH2-Ph),72.17 (OCH2-Ph),71.41(OCH2-Ph),71.20(5′-C),63.34(1′-C),55.95(7-C),41.14(4′-C),26.75(6′-C).

[0195] HRMS(ESI + ):For C 31 H 34 N3O5 + [M+H] + , calculated value: 528.2493; found value: 528.2498.

[0196] IR(ATR):ν(cm -1 )=3029(w),2867(w),1698(s),1613(s),1515(m),1464(m),1453(m),1398(m),1337( m),1265(w),1211(w),1096(m),1070(m),1027(m),913(w),802(m),733(s),696(s).

[0197] R f (iHex:EtOAc 2:1):0.27.

[0198] Ethoxytriazine 18:

[0199] 1 H-NMR (400MHz, CDCl3): δ / ppm=7.97(s,1H,6-H),7.39-7.15(m,15H,3×OCH2Ph),4.66-4.21(m,10H,1′-H,2′-H,7-H,3×OCH2Ph),3.96-3.87(m,1H,3′- H),,3.54-3.36(m,2H,5′-H),2.56-2.41(m,1H,4′-H),2.33(ddd,J=14.5, 9.5, 4.7Hz, 1H, 6′-H), 1.88-1.74 (m, 1H, 6′-H), 1.25 (q, J=6.9Hz, 3H, 8-H).

[0200] 13C-NMR (101MHz, CDCl3): δ / ppm=169.31(4-C), 159.53(6-C), 154.74(2-C), 138.18(Ar-C), 138.16(Ar-C), 1 37.64(Ar-C),128.79(Ar-C),128.64(Ar-C),128.61(Ar-C),128.51(Ar-C),128.25(Ar-C),128.20(Ar-C) ,128.19(Ar-C),127.90(Ar-C),127.87(Ar-C),79.11(2′-C),77.36(3′-C),73.46(OCH2-Ph),72.28(OCH2 -Ph),71.55(OCH2-Ph),71.20(5′-C),65.06(7-C),63.33(1′-C),41.15(4′-C),26.81(6′-C),14.24(8-C).

[0201] HRMS(ESI + ):For C 32 H 36 N3O5 + [M+H] + , calculated value: 542.2650; found value: 542.2656.

[0202] IR(ATR):ν(cm -1 )=3064(w),3030(w),2924(w),2962(w),1694(s),1615(s),1514(m),1495(m),1452 (s),1361(m),1326(m),1270(m),1206(w),1096(s),1070(s),1027(m),803(w),734 8s),697(s).

[0203] R f (iHex:EtOAc 2:1):0.30.

[0204] 4-amino-1-[(1'R,2'S,3'R,4'R)-2',3'-bis(benzyloxy)-4'-((benzyloxy)methyl)-cyclopentyl]-1H-[1,3,5]-triazine-2-one 19:

[0205]

[0206] A mixture of ethoxytriazine 18 (358 mg, 0.66 mmol) and methoxytriazine 17 (363 mg, 0.69 mmol) was dissolved in 26.8 mL of 7N NH3 in methanol and stirred at room temperature for 3 h. Subsequently, 125 mL of H2O was added to the reaction mixture, which was then extracted with EtOAc (3×200 mL). The combined organic layers were dried over Na2SO4 and evaporated to dryness in vacuo to give 593 mg of the amination product 19 (1.16 mmol, 86%) as a colorless foam, which was used in the next step without further purification.

[0207] 1 H-NMR (400MHz, CDCl3): δ / ppm=7.80(s,1H,6-H),7.26(m,15H,3×OCH2Ph),5.70(s, 1H,NH),4.63-4.42(m,6H,1′-H,2.5×OCH2Ph),4.39-4.28(m,2H,2′-H,0.5×OCH2Ph ),3.91(dd,J=5.0,3.5Hz,1H,3′-H),3.53-3.42(m,2H,5′-H),2.59-2.35(m,1H,4′ -H), 2.26 (dt, J=13.3, 9.3Hz, 1H, 6′-H), 1.86 (ddd, J=13.3, 9.2, 7.1Hz, 1H, 6′-H).

[0208] 13 C-NMR (101MHz, CDCl3): δ / ppm=165.84(4-C), 158.30(6-C), 154.11(2-C), 138.27(Ar-C), 138.24(A r-C),137.83(Ar-C),128.57(Ar-C),128.56(Ar-C),128.47(Ar-C),128.21(Ar-C),128.12(Ar-C),1 28.05(Ar-C),127.84(Ar-C),127.82(Ar-C),127.78(Ar-C),78.99(2′-C),77.39(3′-C),73.30(OCH 2-Ph), 72.18(OCH2-Ph), 71.39(OCH2-Ph), 71.3(5′-C)7, 63.53(1′-C), 41.21(4′-C), 26.79(6′-C).

[0209] HRMS(ESI + ):For C 30 H 33 N4O4 + [M+H]+ , calculated value: 513.2496; found value: 513.2496.

[0210] IR(ATR):ν(cm -1 )=3331(br w),3182(br w),3060(w),3028(w),2925(w),2857(w),1633(s),1506(m),1495 8m),1470(m),1452 8s),1361(w),1262(w),1092(s),1070(s),1027(m),912(w),799(m),734(s),696(s).

[0211] R f (CH2Cl2:MeOH 15:1):0.42.

[0212] 4-amino-1-[(1'R,2'S,3'R,4'R)-2',3'-dihydroxy-4'-(hydroxymethyl)-cyclopentyl]-1H-[1,3,5]-triazine-2-one 20:

[0213]

[0214] Under Ar atmosphere, a solution of benzyl protected 5-aza-carbaC 19 (576 mg, 1.12 mmol, 1.0 equiv) was dissolved in 30.8 mL dry CH2Cl2 and cooled to -78 °C. Then, 5.88 mL BCl3 (1 M in CH2Cl2, 5.88 mmol, 5.25 equiv) was added dropwise, and the mixture was stirred at -78 °C for 1 h and at room temperature for 2 h. Subsequently, 40 mL dry MeOH was added, and the mixture was stirred at room temperature for another 20 min and concentrated to dryness in vacuo. The residue was purified by column chromatography on silica gel (dry loading, CH2Cl2: MeOH 8:1 → CH2Cl2: MeOH 5:1 → CH2Cl2: MeOH 4:1 → CH2Cl2: MeOH 7:3) to give 228 mg deprotected compound 20 (0.94 mmol, 84%) as a colorless solid.

[0215] For cell biology applications, 34.5 mg of compound was further purified by reverse phase HPLC (0% to 3% MeCN in H2O, 45 min, flow rate 5 mL / min) to afford 25.3 mg of HPLC purified 20 (73%).

[0216] 1H-NMR (400MHz, D2O): δ / ppm=8.32(s,1H,6-H),4.57-4.33(m,2H,1′-H,2′-H),4.04(dd,J=5.3,4.4Hz,1H,3′-H) ,3.76-3.57(m,2H,5′-H),2.29(dt,J=13.0,8.2Hz,1H,6′-H),2.23-2.15(m,1H,4′-H),1.76-1.59(m,1H,6′-H).

[0217] 13 C-NMR (101MHz, D2O): δ / ppm=165.45(4-C), 159.03(6-C), 156.15(2-C), 73.12( 2′-C), 71.77(3′-C), 63.55(1′-C), 62.90(5′-C), 44.42(4′-C), 27.17(6′-C).

[0218] HRMS(ESI + ):For C9H 15 N4O4 + [M+H] + , calculated value: 243.1088; found value: 243.1086.

[0219] HRMS(ESI - ):For C9H 13 N4O4 - [MH] - , calculated value: 241.0942; found value: 241.0941.

[0220] R f (CH2Cl2:MeOH 7:3):0.29.

[0221] In HPLC stability tests, the carbocyclic compound cAzaC 20 was shown to be much more stable than azacitidine (decitabine) ( Figure 5 ).

[0222] Example 6

[0223] Synthetic procedure of cAzaC prodrug 24

[0224] To improve delivery to cells, the 5′-phosphoramidate prodrug 24 of cAzaC 20 was also prepared.

[0225] 4-Amino-1-((3aS,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydro-4H-cyclopenta-[d][1,3]dioxolan-4-yl)-1,3,5-triazine-2(1H)-one 21

[0226]

[0227] In a heated dry flask, 5-aza-carbaC 20 (64 mg, 264 μmol, 1.0 equivalent) was suspended in 6.60 mL of dry acetone. p-Toluenesulfonic acid (5 mg, catalytic amount) and 2,2-dimethoxypropane (49 μL, 396 μmol, 1.5 equivalents) were added, and the mixture was stirred at room temperature for 18 h. 150 μL NEt3 (1.08 mmol) was added to the resulting solution and the mixture was stirred at room temperature for another 10 min. Subsequently, the mixture was concentrated to dryness, and the resulting residue was purified by column chromatography on silica gel (CH2Cl2: MeOH 10: 1, containing 1% NEt3) to obtain 81 mg of isopropylidene-acetal protected compound 21 (182 μmol, 69%, with 1.6 equivalents of NEt3 adduct), as a colorless solid.

[0228] 1 H-NMR (400MHz, D3COD): δ / ppm=8.29(s,1H,6-H),4.94(dd,J=7.0,4.9Hz,1H,2′-H),4.62-4.39(m,2H,1′-H,3′-H),3.73-3.51(m,2H,5′-H),3 .21(q,J=7.3Hz,10H,N(CH2CH3)3),2.32-2.07(m,3H,4′-H,6′-H),1.50(s,3H,1″-H),1.32(t,J=7.3Hz,15H,N(CH2CH3)3),1.29(s,3H,1″-H).

[0229] 13 C-NMR (101MHz, D3COD): δ / ppm=167.87(4-C), 159.88(6-C), 156.66(2-C), 114.31(2″-C), 83.95(2′-C), 82.98(3′-C), 66.2 6(1′-C), 64.17(5′-C), 47.88(N(CH2CH3)3), 47.81(4′-C), 33.72(6′-C), 27.97(1″-C), 25.53(1″-C), 9.23(N(CH2CH3)3).

[0230] HRMS(ESI +):For C 12 H 19 N4O4 + [M+H] + , calculated value: 283.1401; found value: 283.1397.

[0231] IR(ATR):ν(cm -1 )=3346(br w(,2983(w),2658(w),1634(s),1472(s),1210(m),1160(m),1065(m),868(w),800(m),682(w).

[0232] R f (CH2Cl2:MeOH 6:1):0.36.

[0233] Isopropyl ((((3aR,4R,6R,6aS)-6-(4-amino-2-oxo-1,3,5-triazin-1(2H)-yl)-2,2-dimethyltetrahydro-4H-cyclopenta[d][1,3]dioxolan-4-yl)methoxy)(phenoxy)phosphoryl)-L-alaninate 22:

[0234]

[0235] The protected 5-aza-carbaC 21 (69 mg, 155 μmol, 1.0 equiv., as 1.6 equiv. NEt3-adduct) was suspended in 2.40 mL of dry THF and cooled to -78 ° C. Then 855 μL of tBuMgCl solution (1.0 M in THF, 855 μmol, 5.5 equiv.) was added, and the mixture was directly heated to 0 ° C and stirred at this temperature for 30 min. Subsequently, the mixture was cooled to -78 ° C again and 538 μL of a solution of chlorinated phosphorus oxyaryl ester

[31] 23 (1.0 M in THF, 538 μmol, 3.5 equiv.) was added dropwise. The reaction mixture was slowly warmed to room temperature and stirred for 17 h. The resulting clear yellow solution was quenched by the addition of H2O (20 mL), and the aqueous layer was extracted with EtOAc (3×25 mL). The combined organic phases were washed with saturated aqueous NaCl solution (25 mL), dried over Na2SO4 and concentrated to dryness in vacuo. The residue was purified by column chromatography on silica gel (CH2Cl2: MeOH 60:1 → CH2Cl2: MeOH 50:1 → CH2Cl2: MeOH 40:1) to give 83 mg of product 12 (78 μmol, 51%, adduct with 5.0 equivalents of NEt3) as a mixture of two diastereomers on P as a colorless solid.

[0236] 11H-NMR (800 MHz, CDCl3): δ / ppm = 8.05 (2 × s, 1H), 7.32 - 7.27 (m, 2H, OPh), 7.23 - 7.17 (m, 2H, OPh), 7.15 - 7.07 (m, 1H, OPh), 5.04 - 4.87 (m, 2H, 2′-H, 4″′-H), 4.59 - 4.54 (m, 1H, 3′-H), 4.31 - 4.13 (m, 3H, 1′-H, 5′-H), 4.02 - 3.93 (m, 1H, 2″′-H), 3.78 (m, 1H, NH), 3.09 (q, J = 7.4 Hz, 30H, N(CH2CH3)3), 2.53 - 2.17 (m, 3H, 4′-H, 6′-H), 1.49 - 1.47 (m, 3H, 1″-H), 1.39 (t, J = 7.3 Hz, 45H, N(CH2CH3)3), 1.37 - 1.34 (m, 3H, 3″-H), 1.26 - 1.24 (m, 3H, 1″-H), 1.21 - 0.18 (m, 6H, 5″′-H).

[0237] 13 13C-NMR (201 MHz, CDCl3): δ / ppm = 173.10 (1″′-C), 165.39 (4-C), 158.57 (6-C), 153.13 (O-C Ar ), 150.83 (2-C), 129.73 (CH Ar ), 124.99 (CH Ar ), 120.45 (CH Ar ), 113.57 (2″-C), 82.42 (2′-C), 81.17 (3′-C), 69.28 (4″′-C), 67.50 (1′-C), 65.93 (5′-C), 50.50 (2″′-C), 45.91 (N(CH2CH3)3), 44.93 (4′-C), 32.34 (6′-C), 29.79 (5″′-C), 27.73 (1″-C), 25.36 (1″-C), 21.72 (5″′-C), 21.13 (3″′-C), 8.75 (N(CH2CH3)3).

[0238] 31 31P-NMR (162 MHz, CDCl3): δ / ppm = 2.46, 2.36.

[0239] HRMS (ESI + ): For C 24 H 35 N5O8P + [M + H] +, calculated value: 552.2218; found value: 552.2211.

[0240] HRMS(ESI - ): For C 24 H 33 N5O8P - [MH] - , calculated value: 550.2072; found value: 550.2074.

[0241] IR(ATR):ν(cm -1 )=3379(m),2980(s),2946(m),2606(s),2498(s),1640(s),1476(s),1398(m),1 383(m),1212(s),1158(m),1107(w),1070(m),1037(s),929(m),802(w),730(m).

[0242] R f (CH2Cl2:MeOH 20:1):0.25.

[0243] Isopropyl ((((1R,2R,3S,4R)-4-(4-amino-2-oxo-1,3,5-triazin-1(2H)-yl)-2,3-dihydroxycyclopentyl)methoxy)(phenoxy)phosphoryl)-L-alaninate 24:

[0244]

[0245] To the isopropylidene acetal protected compound 22 (38 mg, 69 μmol, 1.0 equiv) was added 1.0 mL of 80% TFA in ddH2O, and the mixture was stirred at 30°C for 20 min. Then, the mixture was poured into 5.0 mL of ddH2O and lyophilized. The resulting residue was dissolved in a mixture of 500 μL MeCN, 500 μL DMSO and 4.0 mL of ddH2O (containing 0.1% TFA), the turbid solution was filtered and purified by reverse phase HPLC (5% MeCN in ddH2O (containing 0.1% TFA) to 60% MeCN in ddH2O (containing 0.1% TFA), 45 min, flow rate 5 mL / min) to obtain 10.4 mg of the target compound 24 (20 μmol, 29%) as a colorless solid.

[0246] 1H-NMR (400MHz, D3CCN): δ / ppm=8.11(s,1H),7.50(s,1H,NH2),7.41-7.29(m,2H,OPh),7.25-7.15 (m,3H,OPh),6.61(s,1H,NH2),4.98-4.88(m,1H,4″-H),4.39-4.23(m,3H,1′-H,2′-H,NH),4.20- 4.07(m,2H,5′-H),3.97(dt,J=5.2,3.8Hz,1H,3′-H),3.93-3.82(m,1H,2″-H),2.33-2.09(m,2H, 4′-H, 6′-H), 1.83-1.67 (m, 1H, 6′-H), 1.30-1.26 (m, 3H, 3″-H), 1.19 (dt, J = 6.2, 3.4Hz, 6H, 5″-H).

[0247] 13 C-NMR (101MHz, D3CCN): δ / ppm=174.00(1″-C), 165.92(4-C), 159.92(6-C), 153.84(2-C), 151.97(OC Ar ),130.62(CH Ar ),125.75(CH Ar ),121.36(CH Ar ),121.31(CH Ar ),74.12(2′-C),72.50(3′-C),69.64(4″-C),68.53(5′-C),64.96(1′-C),51.44 (2″-C), 44.44(4′-C), 28.08(6′-C), 21.89(5″-C), 21.81(5″-C), 20.80(3″-C).

[0248] 31 P-NMR (162MHz, D3CCN): δ / ppm=3.08, 2.92.

[0249] HRMS(ESI + ):For C 21 H 31 N5O8P + [M+H] + , calculated value: 512.1905; found value: 512.1901.

[0250] HRMS(ESI - ):For C 21 H 29 N5O8P- [M-H] - , Calculated value: 510.1759; Found value: 510.1757.

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[27] Dunn, J;Thabet, S;Jo, H (July 2015). "Flow - Dependent Epigenetic DNAMethylation in Endothelial Gene Expression and Atherosclerosis". Arteriosclerosis, Thrombosis, and Vascular Biology. 35(7):1562–9.

[0279]

[28] Hecker, S. J.;Erion, M. D. (2008), "Prodrugs of phosphates andphosphonates". J. Med. Chem 51:2328 - 2345.

[0280] It should be noted that there is an unclear "Ly熔点homa" in the text which might be a typo. If it's a known term in a specific field, it should be corrected before a more accurate translation. Also, the tags -

[0280] are likely some kind of internal identifiers in a specific system and are left unchanged as per the requirement.

[29] Wiemer,J.;Wiemer D.F.(2015),"Prodrugs of phosphonates andphosphates:Crossing the membrane barrier".Top.Curr.Chem.360:115-160.

[0281]

[30] Wildenhof,T.M.,Schiffers,S.,Traube,F.R.,Mayer,P.,Carell,T.,Angew.Chem.Int.Ed.2019,58,12984-12987.

[0282]

[31] Davey,S.M.,Malde,R.,Mykura,R.C.,Baker,A.T.,Taher,T.E.,Le Duff,C.S.,Willco×,B.E.,Mehellou,Y.,J.Med.Chem.2018,61,2111-2117.

Claims

1. Compounds of formula I: Where R 1 is H, a free, modified or unmodified phosphate group, wherein the modified phosphate group is selected from a phosphonate, phosphoramidate or thiophosphate group, and the unmodified phosphate group is selected from a monophosphate, diphosphate or triphosphate group, R 2 For H, R 3 For H, R 4 and R 5 Each is H, or a salt thereof.

2. The compound according to claim 1, wherein R 1 It is a triphosphate group.

3. A compound according to any one of claims 1 to 2, wherein R 1 For H.

4. A pharmaceutical composition comprising as an active agent a compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof and a pharmacologically acceptable carrier.

5. Use of the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof for preparing a medicament for treating a hyperproliferative disease.

6. Use of the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof for preparing a medicament for treating cancer.

7. Use of the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof for preparing a medicament for treating acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS).

8. Use of the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof for preparing a medicament for treating atherosclerosis or renal insufficiency.

9. In vitro use of the compound according to any one of claims 1 to 3 for the preparation of a medicament for inhibiting the methylation of nucleic acids.

10. The use according to claim 9, wherein the nucleic acid is DNA.

11. The use according to claim 9, which is used for epigenetic analysis.

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