Carbocyclic nucleoside analogue

Abstract

To provide a novel hydrolytically stable carbocyclic 5-aza-2'-deoxycytidine.SOLUTION: The present invention provides carbocyclic 5-aza-2'-deoxycytidine as shown below, wherein R1 and R2 are each independently H, a free or protected, modified or unmodified phosphate group, or a hydroxyl protecting group; R3 is H, F, CH3, CH2F, CHF2, CF3, OH, or OR6 (R6 is a hydroxyl protecting group); and R4 and R5 are each independently H or the like.SELECTED DRAWING: None

Description

[Technical field]

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

[0002] 5-Aza-2'-deoxycytidine (decitabine, AzadC) and 5-aza-cytidine (vidaza, AzaC) are nucleoside analogs that act as antimetabolites by manipulating epigenetic information [1-5]. Epigenetic information in DNA involves the formation of 5-methyl-2'-deoxycytidine (mdC) from 2'-deoxycytidine (dC) with the aid of DNA methyltransferases (DNMTs) and S-adenosylmethionine (SAM) as a methylation cofactor [6-7, 4]. Methylation of dC to mdC in promoter regions is typically associated with transcriptional silencing of genes [8-9]. AzadC is a prodrug that is converted intracellularly to the corresponding active triphosphate and subsequently incorporated into the genome during cell division. AzaC is also a prodrug, which is 2'-deoxygenated and then converted to the corresponding 5'-triphosphate for incorporation into DNA. AzaC can also be converted to the 5'-triphosphate without prior 2'-deoxygenation for incorporation into RNA. The mechanism of action of Aza(d)C involves the reaction of its electrophilic C6 position with the DNMT active site thiol nucleophile [10-11], as shown in Figure 1a. This produces a covalent intermediate that is methylated by the SAM cofactor (hold in close proximity). Due to the N atom at the 5-position of the triazine heterocycle, the final β-elimination reaction (which normally releases m(d)C from the DNMT enzyme) is no longer possible. As a result, a covalent DNA-DNMT or RNA-DNMT crosslink is formed.

[0003] As a result of the administration of Aza(d)C, a large decrease in mdC levels, i.e., a hypomethylation effect, is observed, which leads to the reactivation of silenced tumor suppressor genes in cancer cells [1], redifferentiating cancer cells into normal proliferative cells or causing cell death. Aza(d)C is currently used as a drug for the treatment of myelodysplastic syndromes (MDS) [2] and acute myeloid leukemia (AML) [4]. Clinically, it is administered in several cycles, each cycle including one week of treatment and three weeks of rest. Summary of the Invention [Problem to be solved by the invention]

[0004] A problem associated with Aza(d)C is that it undergoes hydrolysis in aqueous solution, following the pathway shown in Figure 1b, which impairs the activity of Aza(d)C, especially over long periods of treatment. [Means for solving the problem]

[0005] The inventors have solved this problem by providing analogs of Aza(d)C that can be demethylated (and thus react with S-nucleophiles) while blocking hydrolysis (reaction with O-nucleophiles). Surprisingly, the inventors found that replacing the deoxyribose oxygen with a CH2 group has a profound remote effect on the reactivity of the heterocycle. These analogs, in particular the carbocyclic version of Aza(d)C, inhibit methylation by DNA methyltransferases, yet are hydrolytically stable, as shown in Figure 1c.

[0006] The present invention is further illustrated in more detail by the following figures and examples. [Brief description of the drawings]

[0007] [Figure 1]5-Aza-2'-deoxycytidine (decitabine, AzadC) is shown with its mechanism of action. a) The active site thiol reacts with the C6 position of AzadC. b) The hydrolytic degradation pathway goes in hand with the reaction of the C6 position of AzadC with a water molecule (O-reactivity), resulting in the final base loss and formation of an abasic site. c) The carbocyclic version of cAzadC 1 is shown in the box. [Diagram 2] Crystal structure of carbocyclic 5-aza-2'-deoxycytidine (cAzadC1) showing the molecule in the observed C6'-endo conformation (a) and C2'-endo conformation (2T1) (b). [Diagram 3] HPLC-based stability measurements showed (a) severe hydrolytic degradation of 5-aza-2'-deoxycytidine (AzadC) solutions at different pH values, while (b) the carbocyclic compound cAzadC 1 was stable at all three pH values. The inset table in (a) shows the chromatograms for AzadC between t1 = 10 min and t2 = 20 min. The AzadC signal is shown in red. [Figure 4] Figure 1 shows quantification data of DNA modification of mouse embryonic stem cells (mESCs) treated with carbocyclic 5-aza-2'-deoxycytidine (cAzadC1) obtained by UHPLC-MS2. For each condition, three biological replicates were measured in technical triplicates. For each technical replicate, 0.5 μg DNA was digested. Bars represent the mean and error bars represent the standard deviation. LLOQ indicates the lower limit of quantification. [Diagram 5] HPLC study of neutral solutions of decitabine 1 (left) versus the carbacyclic compound cAzaC20 (right). 100 mM solutions of compounds were maintained in pure water and tested at t=0 h, 1.5 h, 3 h, 4.5 h, 6 h, 24 h, and 6 days. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] A first aspect of the present invention relates to a compound of formula I: JPEG2025037980000001.jpg68166 Here, R 1 is H, a free or protected modified or unmodified phosphate group, or a hydroxyl protecting group; R 2 is H, a free or protected modified or unmodified phosphate group, or a hydroxyl protecting group; R 3 is H, F, CH3, CH2F, CHF2, CF3, OH or OR 6 where R 6 is a hydroxyl protecting group, and R 4 and R 5 is H or forms an amino protecting group; or a salt thereof.

[0009] In the compounds of formula I, R 1 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.

[0010] The term "phosphate group" includes unmodified phosphate groups, such as unmodified monophosphate, diphosphate or triphosphate groups, or modified phosphate groups, such as modified monophosphate, diphosphate or triphosphate groups, in which one or more oxygen atoms are replaced by carbon, sulfur and / or nitrogen atoms. Examples of modified phosphate groups are phosphonate, phosphoramidate or phosphorothioate groups. The term "phosphate group" also includes free phosphate groups and protected phosphate groups, such as phosphate esters, including monoesters and diesters, and amidates, including monoamidates, diamidates and amidate esters. Detailed reviews of protected phosphate and phosphonate groups can be found in

[28] and

[29] , the contents of which are incorporated herein by reference. Thus, in pharmaceutical applications, R 1may be a protected phosphate, diphosphate or triphosphate group that liberates the phosphate, diphosphate or triphosphate group upon cleavage within the cell (prodrug concept).

[0011] Hydroxyl protecting groups are known in the art, particularly in the field of nucleoside chemistry, and include, but are not limited to, acyl groups such as acetyl or benzoyl, benzyl groups, trityl groups such as trityl, methoxytrityl or dimethoxytrityl, silyl groups such as trimethylsilyl, t-butyl-dimethylsilyl or tri-i-propysilyl, or alkyl- or substituted alkyl groups such as methyl, ethoxyethyl or β-methoxyethoxymethyl.

[0012] R 2 may be selected from H, an unmodified or modified free or protected phosphate group, e.g., a monophosphate, diphosphate or triphosphate group, or a hydroxyl protecting group as described above. Thus, in pharmaceutical applications, R 2 may be a protected phosphate, diphosphate or triphosphate group that liberates the phosphate, diphosphate or triphosphate group upon cleavage within the cell (prodrug concept).

[0013] In certain embodiments, R 3 may be H. In such a case, the five-membered carbocyclic ring in formula I may be considered as deoxyribose in which the oxygen atom at the 1' position of the ring is replaced by a CH group. 3 is OH or OR 6 where R 6 is a hydroxyl protecting group as described above. In these embodiments, the five-membered carbocyclic ring in formula I can be considered as a ribose in which the oxygen atom at the 1' position of the ring is replaced by a CH2 group. 3 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 is replaced by a CH2 group.

[0014] R 4 and R 5 may each be H, thus providing a primary amino group NH2, or may form an amino-protecting group, e.g., where one or both of the H atoms are replaced by a protecting group. Amino-protecting groups are known in the art, particularly in the field of nucleoside chemistry, and include, but are not limited to, acyl groups such as acetyl or benzoyl, benzyl, carbobenzyloxy, tosyl, 9-fluorenylmethyloxycarbonyl, or t-butyloxycarbonyl.

[0015] In certain embodiments, R 1 is H.

[0016] 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.

[0017] In certain embodiments, R 3 is H or OH.

[0018] In certain embodiments, R 1 , R 2 , R 3 , R 4 and R 5 are H, or R 1 , R 2 , R 4 , and R 5 are H and R 3 is OH or OR 6 In particular embodiments, R 1 , R 2 , R 3 , R 4 and R 5is H. This compound is a carbocyclic analogue of AzadC (decitabine), which surprisingly has high hydrolytic stability while retaining inhibitory activity against methyltransferases, particularly DNA methyltransferases (as shown above). More particularly, in certain embodiments, R 1 , R 2 , R 4 and R 5 is H and R 3 is OH. This compound is a carbocyclic analogue of AzaC (Vidaza).

[0019] In a more particular embodiment, the present invention also relates to prodrugs of AzadC and AzaC, in particular 1 and / or R 2 is different from H.

[0020] The present invention also includes salts of the compounds of formula I, particularly pharma- ceutically acceptable salts. The compounds of the present invention are capable of forming acid and / or base salts due to the presence of amino and / or phosphate groups or groups similar thereto. Acid addition salts can be formed with inorganic and organic acids, including but not limited to acetate, benzoate, hydrobromide, hydrochloride, citrate, lactate, phosphate, tosylate and trifluoroacetate. Base addition salts can be formed with inorganic and organic bases, including but not limited to ammonium, sodium, potassium, calcium, magnesium salts or salts derived from primary, secondary and tertiary amines.

[0021] The compounds of the present invention act as antimetabolites by reducing the level of epigenetic modification 5-methyl-2'-deoxycytidine (mdC). It can be incorporated into the genome and / or RNA of proliferating cells, inhibiting DNA- and / or RNA-methyltransferase, resulting in the reduction of mdC in DNA and mC in RNA. The loss of mdC in DNA (which is a transcriptional silencer in promoters) leads to the reactivation of genes, including tumor suppressor genes, thereby inducing anti-hyperproliferative effects.

[0022] Thus, a further aspect of the invention relates to a compound of formula I as defined above or a pharma- ceutically acceptable salt thereof for use in medicine, such as veterinary medicine or human medicine.

[0023] In a particular embodiment, the compounds of the invention are used for the treatment of hyperproliferative disorders, including malignant and non-malignant hyperproliferative disorders. The term "hyperproliferative disorder" relates to disorders caused by, associated with, and / or involving dysfunctional increased proliferation of cells, tissues and / or organs in an organism.

[0024] Thus, the present invention also relates to a method of treating a hyperproliferative disorder, comprising the step of administering to a subject, particularly a human subject, in need thereof a therapeutically effective dose of a compound of formula I, or a pharma- ceutically acceptable salt thereof.

[0025] In certain embodiments, the compounds of the present invention are used for the treatment of cancer.In certain embodiments, the compounds of the present invention are used for the treatment of leukemia, 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 increased blasts, refractory anemia with increased blasts in transformation and chronic myelomonocytic leukemia, as well as intermediate 1, intermediate 2 and high-risk MDS.

[0026] In a further embodiment, the compounds of the invention may be used for the treatment of renal failure, based on the results reported for decitabine.

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

[27]

[0027] 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 pharmacologically acceptable carrier.The composition can be administered in any suitable manner, for example, orally, parenterally, by inhalation and / or dermal application.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 disorder to be treated.

[0028] For pharmaceutical use, the compositions may be in the form of a solid dosage form, such as a tablet, capsule, etc., or a liquid dosage form, such as a solution, emulsion, suspension, etc.

[0029] The compound of formula I may be administered in one or several treatment cycles, each treatment cycle may comprise at least 1, 2, 3, 4, 5, 6, 7, or 10 days.

[0030] The compounds of formula I may be administered as monotherapy or in combination with additional drugs, particularly drugs suitable for the treatment of the hyperproliferative disorders mentioned above. In certain embodiments, the compounds of formula I may be administered as combination therapy with anti-cancer drugs different from formula I.

[0031] Furthermore, the compounds of formula I are also useful in non-medical applications, such as in the fields of basic research, diagnostics and / or drug screening in general.Accordingly, in a particular embodiment, the present invention relates to the in vitro use of the compounds of formula I for inhibiting methylation of nucleic acids, including DNA and RNA, in particular for inhibiting methylation of DNA, more particularly for inhibiting methylation of genomic DNA.The compounds are therefore useful for genomic analysis, in particular epigenetic analysis, in cells, such as eukaryotic cells, for example animal cells, including mammalian and human cells.

[0032] A general scheme for the preparation of compounds of formula I is shown in the following scheme. The synthesis begins with t-butyloxycarbonyl (Boc) protected aminocyclopentane derivative 2, previously used to synthesize DNA damage analogs [12-15]. Compound 2 was first benzyl protected to 3 and Boc deprotected to 4, which was then reacted with carbimidazole 5 (prepared in two steps from isomethylurea 6 after generation of the free base 7 with potassium hydroxide and reaction of 7 with carbonyldiimidazole). This provided the carbamoylurea-cyclopentane nucleoside analog 8. Subsequent cyclization to the triazine base 9 was achieved with triethyl orthoformate. Reaction of 9 with NH3 in methanol and deprotection of the benzyl group with BCl3 in dichloromethane provided the final compound cAzadC 1 as the free nucleoside.

[0033] JPEG2025037980000002.jpg87166

[0034] Recrystallization of compound cAzadC1 from hot methanol afforded colorless needles, allowing the elucidation of the crystal structure shown in Figure 2. It was interesting to observe that cAzadC1 exists in two different cyclopentane conformations in the crystals. One of these conformations is typical of 2'-deoxynucleosides in DNA, indicating that cAzadC1 nucleosides can adopt the correct DNA-type conformation and have the potential to be phosphorylated and incorporated into the genome.

[0035] Another general scheme for the preparation of compounds of formula I is shown below, illustrating the synthesis of a carbacyclic version of azacitidine (Vidaza) cAzaC (20):

[0036] JPEG2025037980000003.jpg132166

[0037] A general scheme for the preparation of prodrugs of compounds of formula I is shown below, illustrating the synthesis of the 5′ phosphoroamidate prodrug 24 of cAzaC (20):

[0038] JPEG2025037980000004.jpg109166 EXAMPLES

[0039] Example 1 - Synthetic Procedure for cAzadC (1) Tert.-Butyl-N-[(1R,3S,4R)-3-hydroxy-4-(hydroxymethyl)cyclopentyl]-carbamate (2) JPEG2025037980000005.jpg68166

[0040] Compound 2 was synthesized from tert-butyl-N-((1S,2R,4R,5R)-4-((tert-butyl-dimethylsilyloxy)methyl)-6-oxa-bicyclo-[3.1.0]-hex-2-yl)-carbamate as previously described [1].

[0041] Tert.-Butyl-N-[(1R,3S,4R)-3-benzyloxy-4-(benzyloxymethyl)-cyclopentyl]-carbamate (3) JPEG2025037980000006.jpg71166

[0042] 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% dispersion 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 at room temperature for an additional 2 h. The reaction mixture was diluted with CHCl (30 mL) and the reaction was quenched with saturated NaHCO (30 mL). The mixture was extracted with CHCl (500 mL). The organic phase was washed three times with saturated NaHCO (1 × 400 mL, 2 × 100 mL) and dried over NaSO. The solvent was removed in vacuum. The crude product was purified by column chromatography on silica gel with a stepwise 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%).

[0043] R f =0.67(iHex / EtOAc=2:1) Mp=74-76°C HRMS[ESI+]:[C 25 H 33 NO4Na] + , [M+Na + ] + Calculated value: 434.2292, Measured value: 434.2299 1 H-NMR(599 MHz, 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-Hb ). 13 C-NMR(151 MHz, 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’ ). FTIR(ATR):ν / cm -1 =3339(w),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).

[0044] (1R,3S,4R)-1-Amino-3-benzyloxy-4-(benzyloxymethyl)cyclopentane (4) JPEG2025037980000007.jpg72166

[0045] A solution of Boc-protected amine 3 (3.351 g, 8.14 mmol, 1.0 eq.) and trifluoroacetic acid (12 mL, 30% (v / v)) in dry CHCl (28 mL) was stirred at room temperature for 1 h. After the solvent was removed in vacuo, the residue was resuspended in saturated NaCO (100 mL) and stirred for 10 min. The mixture was extracted with EtOAc (3×150 mL) and the combined organic phases were dried over NaSO. The solvent was removed in vacuo to give the free amine 4 as a viscous brown oil, which was used without further purification due to its instability towards O.

[0046] Analytical data for purified 4: HRMS[ESI + ]:[C 23 H 29 N4O4Na] + , [M+Na + ] +Calculated value: 312.1958, Measured value: 312.1955 1 H-NMR(400 MHz, 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 )、 13 C-NMR(101 MHz, 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).

[0047] 2-Methyl-1-(1-imidazolylcarbonyl)-isourea (5) JPEG2025037980000008.jpg60166

[0048] To a solution of O-methylisourea hydrochloride 6 (5.00 g, 45.65 mmol, 1.0 eq.) in Et2O:H2O (39:1) at -15 °C, KOH powder (51.22 g, 912.96 mmol, 20.0 eq.) was added portion wise under constant stirring. After 30 min, the mixture was filtered and the residue was washed with ice-cold Et2O (3 x 50 mL). The combined organic phase was concentrated in vacuo to 20 mL. Precipitation occurred upon cooling to -20 °C. Vacuum filtration under a stream of N2 afforded 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 eq.) and carbonyldiimidazole (2.90 g, 17.91 mmol, 1.07 eq.) 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%).

[0049] 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). 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).

[0050] 1-[(1'R,3'S,4'R)-3'-benzyloxy-4'-(benzyloxymethyl)-cyclopentyl]-methylisobiuret (8) JPEG2025037980000009.jpg77166

[0051] 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 as a yellow oil (2.613 g, 6.35 mmol, 78%).

[0052] R f =0.51 (DCM / MeOH=19:1) HRMS[ESI + ]:[C 23 H 29 N4O4Na] + , [M+Na + ] + Calculated value: 434.2050, Measured value: 434.2049 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, 3J 2’b,1’ / 3’ =7.0Hz, 3 J 2’b,1’ / 4’ =7.0Hz,1H,C 2’ -H b ), 1.28-1.11(m,1H,C 6’ -H b ). 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’ ).

[0053] 4-Ethoxy-1-[(1'R,3'S,4'R)-3'-benzyloxy-4'-(benzyloxymethyl)-cyclopentyl]-1H-[1,3,5]-triazin-2-one (9a) and 4-Methoxy-1-[(1'R,3'S,4'R)-3'-benzyloxy-4'-(benzyloxymethyl)-cyclopentyl]-1H-[1,3,5]-triazin-2-one (9b) JPEG2025037980000010.jpg80166

[0054] 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 vacuum. 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).

[0055] 9a: R f =0.71 (iHex / EtOAc, 1:3) HRMS [ESI + : [C 25 H 30 N3O4H] + 、[M + H + + Calculated value: 436.2232, Measured value: 436.2231. 1 1H-NMR (400 MHz, 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, 3 Hz, 3 J = 7, 6 Hz, 3 J = 2, 7 Hz, C 2’ -H a ), 2.11 (ddd, 1H, 2 J 2’a,2’b = 13.3 Hz, 3 J = 10.0 Hz, 3 J = 6.3 Hz, C 2’ -H b ), 1.84 - 1.68 (m, 1H, C 6’ -H b ), 1.40 (t, 3H, 3 J 8,7 = 7, 1 Hz, C8-H), 13 13C-NMR (101 MHz, 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).

[0056] 9b: R f = 0.63 (iHex / EtOAc, 1:3) HRMS [ESI + : [C 24 H 28 N3O4] + 、[M + H + + Calculated value: 422.2074, Measured value: 422.2075. 1 1H-NMR (599 MHz, 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, 1 Hz, 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 ). 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’ ).

[0057] 4-Amino-1-[(1'R,3'S,4'R)-3'-benzyloxy-4'-(benzyloxymethyl)-cyclopentyl]-1H-[1,3,5]triazin-2-one (10) JPEG2025037980000011.jpg78166

[0058] Ethoxytriazine 9a (1.630 g, 3.74 mmol, 1.0 eq.) was dissolved in methanolic NH3 (7N in MeOH, 60 mL) and stirred at room temperature for 3 h. The mixture was diluted with HO (340 mL) to give a colorless precipitate, which was extracted with EtOAc (3 x 200 mL). The combined organic phases were dried and the solvent was removed in vacuo to give benzyl protected cAzadC 10 (1.478 g, 3.63 mmol, 97%) as a colorless foam. The synthesis of 10 was also successful following the same procedure starting with methoxytriazine 9b.

[0059] R f = 0.50 (CH2Cl2 / MeOH, 9:1) HRMS [ESI + : [C 23 H 27 N4O3] + , [M + H + + Calculated value: 407.2078, Measured value: 407.2081. 1 H-NMR (599 MHz, 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 )。 13 C-NMR (151 MHz, 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’ )。

[0060] ​ 4-Amino-1-[(1'R,3'S,4'R)-3'-hydroxy-4'-(hydroxymethyl)-cyclopentyl]-1H-[1,3,5]triazin-2-one (cAzadC,1) JPEG2025037980000012.jpg70166

[0061] A solution of benzyl protected 10 (1.478 g, 3.63 mmol, 1.0 eq) in CHCl (100 mL) was cooled to −78° C. and BCl (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 adding 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 CHCl / MeOH (9:1→7:3) to give cAzadC (1, 0.740 g, 3.27 mmol, 90%). Recrystallization from hot MeOH afforded 57% cAzadC (1,465.3 mg, 2.06 mmol) as colorless, needle-like single crystals.

[0062] R f =0.1 (CH2Cl2 / MeOH, 9:1) Mp.:198-200°C HRMS[ESI + ]:[C9H 15 N4O3] + , [M+H + ] + Calculated value: 227,1138, Actual value: 227,1139. 1 H-NMR(400MHz,D2O):δ / ppm=8.29(s,1H,C6-H),4.90-4.62(m,1H,C 1’ -H, overlapping with DO 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). 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’ ). FTIR(ATR):ν / cm -1 =3194(m),1653(m),1540(s),1437(s),1278(s),1036(s).

[0063] Example 2 - X-ray crystal structure analysis of cAzadC Single crystals of cAzadC were analyzed by X-ray crystallography. Table 1 shows the solved structures of cAzadC1 in two different cyclopentane conformations in the crystals. One conformer adopts the C6'-endo (P = 88.2°, max = 47.8°) conformation (Fig. 2a), and the second exists as the C2'-endo-C3'-exo (South, P = 150.8°, max = 45.4°) conformer (Fig. 2b). The latter conformation is typical for 2'-deoxynucleosides in DNA.

[0064] Further figures were generated using the program Mercury 3.5.1. (Cambridge Crystallographic Data Center), the data of which have been deposited under CCDC 1910952.

[0065] JPEG2025037980000013.jpg184166

[0066] Example 3 - Hydrolytic stability test We next studied the hydrolytic stability of cAzadC1 in a head-to-head comparison against formulated AzadC.

[0067] Both nucleosides were dissolved at 100 mM concentration in aqueous KH2PO4 buffer (100 mM, pH=7.4, 5.5 and 8.5). The solution was analyzed immediately by HPLC (t=0 h) and subsequent HPLC analyses were performed every 1.5 h or at the indicated time points. An EC 250 / 4 NUCLEOSIL 120-3 C18 (Macherey-Nagel) chromatography column was used as stationary phase. The mobile phase consisted of water (buffer A) and acetonitrile (buffer B) at a flow rate of 0.5 mL / min as follows: 0→25 min, 0→5% buffer B; 25 min→28 min, 5%→80%; 28 min→38 min, 80%; 38 min→43 min 80%→0%; 43 min→45 min, 0%.

[0068] Since one treatment cycle for AzadC lasts for 4 weeks, we decided to measure the stability at half-cycle (14 days) time points. The NMR spectra were measured after keeping the solutions at room temperature.

[0069] Since tumor cells often present an acidic microenvironment

[16] , stability at slightly acidic pH is of particular interest. As is evident from the data shown in Figure 3, formulated AzadC was highly degraded within these 14 days. Importantly, at pH=5.5 and pH=8.5, intact AzadC was barely detectable any more. At physiological pH (7.4), AzadC was still present after 14 days, but the level of degradation was significant.

[0070] In contrast to these results, surprisingly, no decomposition was observed for cAzadC1 at all pH values ​​tested, including pH = 5.5. This led to the surprising finding that the simple exchange of O for CH2 produces a strong remote disarming effect that appears to change the properties of the triazine ring such that it ceases to react with water.

[0071] Example 4 - Biological function test Cell culture of mESCs upon cAzadC treatment Feeder-independent wt J1 (strain 129S4 / SvJae)

[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 subcultured, where appropriate, in inhibitor-free DMEM supplemented with FBS and LIF as above. For drug treatment, cells were shifted to a primed state by removing 2i from the medium. Cells were incubated for 2 days in DMEM supplemented with FBS and LIF in 6-well plates (VWR). After splitting, 2 × 10 5 Cells were transferred to 6-well culture plates and supplemented with either 1 μM (in 0.01% DMSO) or 5 μM cAzadC (in 0.05% DMSO) for 72 h. After removing the medium and washing the cells with DPBS, RLT was incubated for 72 h as previously described

[18] . + The solution was dissolved directly using buffer.

[0072] gDNA isolation, complete enzymatic digestion and UHPLC-MS 2 gDNA was isolated as previously described

[18] . gDNA was directly subjected to complete enzymatic digestion and analyzed by UHPLC-MS as previously described

[19] . 2An external calibration curve was constructed by serially diluting pure cAzadC [20 pmol, 10 pmol, 5 pmol, 2.5 pmol, 1.25 pmol, 0.625 pmol, 0.3125 pmol] and measuring in technical triplicates before each measurement. Linear regression was performed by OriginPro 2016G.

[0073] result We tested whether the release effect shown in Example 3 affects biological function. For this purpose, we used mouse embryonic stem cells (mESCs) primed in serum / Lif as a model system, since mdC levels increase from naïve to primed conditions

[17] . We added cAzadC1 at two different concentrations (1 μM and 5 μM) to mESCs primed for 48 h and further expanded the cells in the presence of cAzadC1 under primed conditions for another 72 h. After 72 h, cells were harvested, DNA was isolated, and DNA was digested to nucleoside levels using a protocol we described

[18] .

[0074] The levels of mdC were finally analyzed by isotope dilution UHPLC-MS. 2 To this end, isotopically labeled nucleoside standards were spiked-in for precise 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 quantified. In mESCs, the absolute levels of hmdC are more than 10-fold lower than mdC levels [20, 22]. As a result, there should be enough mdC present to keep hmdC levels constant even after a substantial reduction in mdC. Thus, the question of whether and how much hmdC levels are affected can inform how epigenetic reprogramming is orchestrated.

[0075] In parallel with the quantification of mdC and hmdC, we also quantified to what extent cAzadC1 itself is incorporated into the genome of mESCs. Detection of AzadC in the genome of treated cells is only possible after treatment of DNA with NaBH4. Application of NaBH4 reduces the C(5)=C(6) double bond and stabilizes the compound in such a way that its quantification becomes possible [23, 19]. Pleasingly, we found that the stabilization of cAzadC1 allows its quantification without this pretreatment. We also found that the enzymatic digestion protocol used allows digestion of genomic DNA (gDNA) even in the presence of high amounts of cAzadC1. Taken together, the UHPLC-MS with external calibration curves allows the detection of AzadC1 in the genome of treated cells. 2 Quantification of cAzadC1 by ELISA was possible in parallel with quantification of standard and epigenetic criteria.

[0076] At a concentration of 1 μM cAzadC, 5 × 10 -4 cAzadC / dN levels of cAzadC1 were detected (Fig. 4a), which means that nearly 3 million cAzadC nucleotides were integrated into the genome. At higher concentrations (5 μM cAzadC1), the levels increased threefold to 1.7 × 10 -3 cAzadC / dN, resulting in more than 8 million cAzadC integrations per genome. -3 Compared to the incorporation of AzadC reaching AzadC / dN

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

[0077] Importantly, after 72 h exposure of mESCs to 1 M cAzadC in the medium, a nearly 30% decrease in mdC values ​​was detected (Figure 4b). At a concentration of 5 M in the medium, mdC levels were further reduced to approximately 50% of the original value. A 50% reduction is also observed for AzadC; however, here the 50% reduction is already reached earlier (24 h) and at a lower AzadC concentration (1 μM)

[19] .

[0078] The data indicate that the carbocyclic version cAzadC1 simply requires more time to affect mdC levels in the same amount. We attribute this effect to a potentially slower conversion of cAzadC1 to the triphosphate. However, the slower reaction rate of cAzadC1 is not necessarily a disadvantage, given the long treatment times applied in the hospital.

[0079] It is also very interesting to find that hmdC levels were already reduced by about 50% in the 1 μM experiment. At 5 μM, we could not even detect hmdC above background levels with 0.5 μg of genomic DNA. The results show that hmdC levels declined even faster than mdC levels, but hmdC is more than 10 times less abundant in the genome. This result is intriguing. It points to the potential that hmdC may be mainly produced in the mdC maintenance process during cell division. We now consider that compound cAzadC 1 is a perfect tool molecule that allows us to gain further insights into the interplay between methylation of dC to mdC and oxidation of mdC to hmdC. With the novel compound cAzadC 1 in hand, we can elegantly link genomic demethylation with the corresponding cellular effects, without compromising the DNA damage effects. Finally, cAzadC 1 could not only be a valuable tool compound, but also a very next generation epigenetic drug.

[0080] summary It is shown that the replacement of the ring O atom with a CH2 unit stabilizes the formulation, thereby terminating its nucleophilic reaction with water. The novel nucleoside cAzadC1 is accepted by the intracellular phosphorylation enzyme, and the corresponding cAzadC-triphosphate is efficiently incorporated into the genome. cAzadC1 is incorporated into genomes containing millions of nucleotides, reducing mdC levels by up to 70% relative to control levels.

[0081] Example 5 - Synthetic Procedure for cAzaC (20) Tert-Butyl-(1R,4S,5R,6S)-5,6-dihydroxy-3-oxo-2-azabicyclo[2.2.1]heptane-2-carboxylate 12 JPEG2025037980000014.jpg33166 The Boc-protected Vince lactam 11

[30] (11.66 g, 55.7 mmol, 1.0 eq.) was dissolved in 214 mL THF, 92 mL tert-butanol and 31 mL H2O, 22.59 g N-methylmorpholino-N-oxide (167.2 mmol, 3.0 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. After that, 200 mL water and 35.1 g Na2SO3 (278.5 mmol, 5.0 eq.) were added and the mixture was stirred for another hour at room temperature. The reaction mixture was then extracted with EtOAc (3 x 300 mL). The combined organic phase was washed with saturated aqueous NaCl (300 mL) and dried over Na2SO4. After evaporation of the solvent in vacuo, the residue was purified by silica gel column chromatography (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.

[0082] 1H-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)。 13 C-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)。 IR(ATR):ν(cm -1 )=3415(w),2979(w),1777(m),1714(m),1457(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)。 R f (CH2Cl2:MeOH 20:1):0.24。

[0083] Tert-Butyl-((1R,2S,3R,4R)-2,3-dihydroxy-4-(hydroxymethyl)-cyclopentyl)-carbamate 13 JPEG2025037980000015.jpg50166

[0084] A solution of bishydroxylated lactam 12 (2.90 g, 11.9 mmol, 1.0 eq.) in 40 mL 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 another 1 h. Afterwards, 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, CHCl:MeOH 5:1 → CHCl:MeOH 4:1) and coevaporation with dry toluene (2 × 150 mL) afforded the product 313 as its acetate salt (2.96 g, 9.6 mmol, 81%) as a brownish oil.

[0085] 1 H-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). 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). R f (CH2Cl2:MeOH 8:1): 0.43.

[0086] Tert-Butyl-((1R,2S,3R,4R)-2,3-bis(benzyloxy)-4-((benzyloxy)methyl)-cyclopentyl)-carbamate 14 JPEG2025037980000016.jpg49166

[0087] A solution of cyclopentane 13 (2.93 g, 9.5 mmol, 1.0 eq.) in 67 mL dry DMF was cooled to 0 °C and NaH (1.57 g, 39.3 mmol, 4.1 eq., 60% in mineral oil) was added in three portions. After the mixture was stirred for 15 min at 0 °C, 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 an additional 3.18 mL of BnBr (26.8 mmol, 2.8 eq.) were added and the mixture was warmed to 80 °C for 3.5 h. Afterwards, the mixture was cooled to room temperature and poured into 300 mL of saturated aqueous NaHCO3 and extracted with CHCl2 (3 × 300 mL). The combined organic phases 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.

[0088] 1 H-NMR (400MHz, CDCl3): δ / ppm=7.45-7.09(m,15H,3xOCH2Ph),4.95(s,1H,NH),4.76-4.25(m,6H,3xOCH2Ph),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). 1313C-NMR (101 MHz, 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). HRMS (ESI + ): Calculated value for C 32 H 40 NO5 + [M + H] + 518.2901; Measured value: 518.2911. IR (ATR): ν(cm -1 ) = 3331 (br w), 3029 (w), 2974 (w), 2867 (w), 1705 (s), 1495 (m), 1453 (m), 1390 (w), 1364 (s), 1245 (m), 1165 (s), 1092 (s), 1071 (s), 1027 (m), 732 (s), 695 (s). R f (iHex:EtOAc 4:1): 0.69.

[0089] (1R,2S,3R,4R)-2,3-Bis(benzyloxy)-4-((benzyloxy)methyl)-cyclopentan-1-amine 15 JPEG2025037980000017.jpg37166

[0090] Under Ar atmosphere, 1.02 g of Boc-protected cyclopentane 14 (1.97 mmol, 1.0 eq.) was dissolved in 7.0 mL of dry CHCl, 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 aqueous NaHCO 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 phase was further extracted with EtOAc (2×60 mL). The combined organic phases were dried over NaSO and concentrated to dryness in vacuo to give the deprotected amine 15 as a yellow oil (882 mg, 1.97 mmol, quant.), which was used in the next step without further purification due to its instability to oxidation.

[0091] 1 H-NMR (400MHz, CDCl3): δ / ppm=7.39-7.26(m,15H,3xOCH2Ph),4.63-4.33(m,6H,3xOCH2Ph),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). 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). HRMS(ESI +): The calculated value is C 27 H 32 No.3 + [M+H] + 418.2377;Actual value: 418.2378.

[0092] 1-[(1'R,2'S,3'R,4'R)-2',3'-Bis(benzyloxy)-4'-((benzyloxy)methyl)-cyclopentyl]-methylisobiuret 16 JPEG2025037980000018.jpg49166

[0093] Under Ar atmosphere, 882 mg of cyclopentylamine 15 (1.87 mmol, 1.97 mmol, 1.0 eq.) was dissolved in 18.20 mL of dry MeCN and 431 mg of 2-methyl-1-(1-imidazoylcarbonyl)-isourea [1] (2.56 mmol, 1.3 eq.) was added. 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 as a colorless oil (886 mg, 1.17 mmol, 87%).

[0094] 1 H-NMR (400MHz, CDCl3): δ / ppm=7.43-7.22(m,15H,3xOCH2Ph),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). 1313C-NMR (101 MHz, CDCl3): δ / ppm = 207.11 (4-C), 162.27 (2-C), 138.74 (Ar-C), 138.71 (Ar-C), 138.45 (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-Ph), 71.20 (OCH2-Ph), 70.60 (5’-C), 52.56 (1’-C), 41.68 (4’-C), 31.07 (6-C), 30.57 (6’-C). HRMS (ESI + ): Calculated for C 30 H 36 N3O5 + [M + H] + 518.2650; Found: 518.2652. 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). R f (iHex:EtOAc 2:1): 0.37.

[0095] 4-Methoxy-1-[(1'R,2'S,3'R,4'R)-2',3'-bis(benzyloxy)-4'-((benzyloxy)methyl)-cyclopentyl]-1H-[1,3,5]-triazin-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]-triazin-2-one (18) JPEG2025037980000019.jpg46166

[0096] Methyl isobiuret 16 (837 mg, 1.62 mmol, 1.0 eq.) was dissolved in 15.30 mL of triethyl orthoformate and 20 μL of 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 coevaporated with dry MeOH 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 give 373 mg of ethoxytriazine 18 (0.69 mmol, 43%) as a colorless wax and 389 mg of methoxytriazine 17 (0.73 mmol, 46%) as a colorless solid.

[0097] Methoxytriazine 17: 1 H-NMR (400MHz, CDCl3): δ / ppm=7.96(s,1H,6-H),7.38-7.17(m,15H,3xOCH2Ph),4.70-4 .40(m,6H,1'-H,2.5xOCH2Ph),4.36-4.24(m,2H,2'-H,0.5xOCH2Ph),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). 1313C-NMR (101 MHz, CDCl3): δ / ppm = 169.87 (4-C), 159.59 (6-C), 154.67 (2-C), 138.16 (Ar-C), 138.14 (Ar-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). HRMS (ESI + ): Calculated for C 31 H 34 N3O5 + [M+H] + 528.2493; Found: 528.2498. 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). R f (iHex:EtOAc 2:1): 0.27.

[0098] Ethoxytriazine 18: 11H-NMR (400 MHz, CDCl3): δ / ppm = 7.97 (s, 1H, 6-H), 7.39 - 7.15 (m, 15H, 3 x OCH2Ph), 4.66 - 4.21 (m, 10H, 1’-H, 2’-H, 7-H, 3 x 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, 4.7 Hz, 1H, 6’-H), 1.88 - 1.74 (m, 1H, 6’-H), 1.25 (q, J = 6.9 Hz, 3H, 8-H). 13 13C-NMR (101 MHz, CDCl3): δ / ppm = 169.31 (4-C), 159.53 (6-C), 154.74 (2-C), 138.18 (Ar-C), 138.16 (Ar-C), 137.64 (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). HRMS (ESI + ): Calculated value for C 32 H 36 N3O5 + [M + H] + 542.2650; Found: 542.2656. 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), 7348s), 697 (s). R f (iHex:EtOAc 2:1): 0.30.

[0099] 4-Amino-1-[(1'R,2'S,3'R,4'R)-2',3'-bis(benzyloxy)-4'-((benzyloxy)methyl)-cyclopentyl]-1H-[1,3,5]-triazin-2-one 19 JPEG2025037980000020.jpg43166

[0100] 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 MeOH and stirred at room temperature for 3 h. Afterwards, 125 mL of H2O was added to the reaction mixture, which was extracted with EtOAc (3 x 200 mL). The combined organic phases were dried over Na2SO4 and evaporated to dryness in vacuo to give 593 mg of the aminated product 19 (1.16 mmol, 86%) as a colorless foam, which was used in the next step without further purification.

[0101] 1 H-NMR (400MHz, CDCl3): δ / ppm=7.80(s,1H,6-H),7.26(m,15H,3xOCH2Ph),5.70(s ,1H,NH),4.63-4.42(m,6H,1'-H,2.5xOCH2Ph),4.39-4.28(m,2H,2'-H,0.5xOCH2P h),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) 1313C-NMR (101 MHz, CDCl3): δ / ppm = 165.84 (4-C), 158.30 (6-C), 154.11 (2-C), 138.27 (Ar-C), 138.24 (Ar-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), 128.05 (Ar-C), 127.84 (Ar-C), 127.82 (Ar-C), 127.78 (Ar-C), 78.99 (2’-C), 77.39 (3’-C), 73.30 (OCH2-Ph), 72.18 (OCH2-Ph), 71.39 (OCH2-Ph), 71.3 (5’-C), 63.53 (1’-C), 41.21 (4’-C), 26.79 (6’-C). HRMS (ESI + ): Calculated for C 30 H 33 N4O4 + [M + H] + 513.2496; Found: 513.2496. IR (ATR): ν(cm -1 ) = 3331 (br w), 3182 (br w), 3060 (w), 3028 (w), 2925 (w), 2857 (w), 1633 (s), 1506 (m), 1495 (m), 1470 (m), 1452 (s), 1361 (w), 1262 (w), 1092 (s), 1070 (s), 1027 (m), 912 (w), 799 (m), 734 (s), 696 (s). R f (CH2Cl2:MeOH 15:1): 0.42.

[0102] 4-Amino-1-[(1'R,2'S,3'R,4'R)-2',3'-bishydroxy-4'-(hydroxymethyl)-cyclopentyl]-1H-[1,3,5]-triazin-2-one 20 JPEG2025037980000021.jpg42166

[0103] A solution of benzyl protected 5-aza-carbaC 19 (576 mg, 1.12 mmol, 1.0 eq.) was dissolved in 30.8 mL of dry CHCl under Ar atmosphere and cooled to −78 °C. Then, 5.88 mL of BCl (1 M in CHCl, 5.88 mmol, 5.25 eq.) was added dropwise and the mixture was stirred at −78 °C for 1 h and at room temperature for 2 h. After that, 40 mL of dry MeOH was added and the mixture was stirred for another 20 min at room temperature and concentrated to dryness in vacuo. The residue was purified by column chromatography on silica gel (dry loading, CHCl:MeOH 8:1 → CHCl:MeOH 5:1 → CHCl:MeOH 4:1 → CHCl:MeOH 7:3) to give 228 mg of deprotected compound 20 (0.94 mmol, 84%) as a colorless solid.

[0104] For cell biology applications, 34.5 mg of the compound was further purified by reverse-phase HPLC (0%–3% MeCN in HO, 45 min, flow rate 5 mL / min) to give 25.3 mg of HPLC-purified 20 (73%).

[0105] 1 H-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). 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). HRMS(ESI + ): Calculated value is C9H 15 N4O4 + [M+H] +243.1088;Actual value: 243.1086. HRMS(ESI - ): Calculated value is C9H 13 N4O4 - [MH] - 241.0942;Actual value: 241.0941. R f (CH2Cl2:MeOH 7:3): 0.29.

[0106] In HPLC stability studies, the carbacyclic compound cAzaC 20 was shown to be more stable than azacitidine (decitabine) (Figure 5).

[0107] Example 6 Synthetic procedure for cAzaC prodrug 24 To improve intracellular delivery, we also prepared the 5′-phosphoroamidate prodrug 24 of cAzaC 20.

[0108] 4-Amino-1-((3aS,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydro-4H-cyclopenta-[d][1,3]dioxol-4-yl)-1,3,5-triazin-2(1H)-one 21 JPEG2025037980000022.jpg54166

[0109] In a flame-dried flask, 5-aza-carbaC 20 (64 mg, 264 μmol, 1.0 eq.) 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 eq.) were added and the mixture was stirred at room temperature for 18 h. To the resulting solution, 150 μL of NEt3 (1.08 mmol) was added and the mixture was stirred at room temperature for another 10 min. The mixture was then concentrated to dryness and the resulting residue was purified by column chromatography on silica gel (CH2Cl2:MeOH 10:1, containing 1% NEt3) to give 81 mg of isopropylidene-acetal protected compound 21 (182 μmol, 69%, 1.6 eq. NEt3 added) as a colorless solid.

[0110] 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). 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). HRMS(ESI + ):Definition C 12 H 19 N4O4 + 283.1401[M+H] + ;Direction:283.1397. 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). R f (CH2Cl2:MeOH 6:1):0.36.

[0111] 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]dioxol-4-yl)methoxy)(phenoxy)phosphoryl)-L-alaninato 22 JPEG2025037980000023.jpg56166

[0112] The protected 5-aza-carbaC 21 (69 mg, 155 μmol, 1.0 eq., as 1.6 eq., NEt3 added) 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 eq.) was added and the mixture was warmed directly to 0 °C and stirred at this temperature for 30 min. After that, the mixture was cooled again to -78 °C and 538 μL of a solution of phosphoroxyaryl chloridate

[31] 23 (1.0 M in THF, 538 μmol, 3.5 eq.) was added dropwise. The reaction mixture was allowed to warm slowly to room temperature and stirred for 17 h. The resulting bright yellow solution was quenched by the addition of H2O (20 mL) and the aqueous phase was extracted with EtOAc (3 × 25 mL). The combined organic phases were washed with saturated aqueous NaCl (25 mL), dried over anhydrous 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%, 5.0 eq. NEt3 added) as a mixture of both diastereoisomers on P as a colorless solid.

[0113] 11H-NMR (800 MHz, CDCl3): δ / ppm = 8.05 (2xs, 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). 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). 31 31P-NMR (162 MHz, CDCl3): δ / ppm = 2.46, 2.36. HRMS (ESI + ): Calculated value for C 24 H 35 N5O8P + 552.2218 [M + H] + ; Measured value: 552.2211. HRMS(ESI - ): The calculated value is C 24 H 33 N5O8P - 550.2072[MH] - ;Actual value: 550.2074. 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). R f (CH2Cl2:MeOH 20:1):0.25.

[0114] Isopropyl((((1R,2R,3S,4R)-4-(4-amino-2-oxo-1,3,5-triazin-1(2H)-yl)-2,3-dihydroxy-cyclopentyl)methoxy)(phenoxy)phosphoryl)-L-alaninato 24 JPEG2025037980000024.jpg48166

[0115] To the isopropylidene acetal protected compound 22 (38 mg, 69 μmol, 1.0 eq.), 1.0 mL of 80% TFA (in ddH2O) was added, 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 of MeCN, 500 μL of DMSO, and 4.0 mL of ddH2O (containing 0.1% TFA), and the cloudy solution was filtered and purified by reverse phase HPLC (5% MeCN in ddH2O cont. 0.1% TFA to 60% MeCN in ddH2O cont. 0.1% TFA, 45 min, flow rate 5 mL / min) to give 10.4 mg of the target compound 24 (20 μmol, 29%) as a colorless solid.

[0116] 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). 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). 31 P-NMR (162MHz, D3CCN): δ / ppm=3.08,2.92. HRMS(ESI + ): Calculation value, C 21 H 31 N5O8P + 512.1905[M+H] + ; Test value: 512.1901. HRMS(ESI - ): Calculation value, C 21 H 29 N5O8P - 510.1759[MH] -;Actual value: 510.1757.

[0117] JPEG2025037980000025.jpg227166JPEG2025037980000026.jpg233166JPEG2025037980000027.jpg60166

Claims

1. Compounds of Formula I: Here, R １ is H, a free or protected modified or unmodified phosphate group, or a hydroxyl protecting group; R ２ is H, a free or protected modified or unmodified phosphate group, or a hydroxyl protecting group; R ３ is H, F, CH ３ , C.H. ２ F, C.H.F. ２ , C.F. ３ , OH or OR ６ where R ６ is a hydroxyl protecting group, R ４ and R ５ are each H or form an amino protecting group; Or its salt.

2. 2. The compound of claim 1 , wherein each phosphate group is independently: below: (i) free or protected unmodified phosphate, wherein the phosphate group is a monophosphate, diphosphate or triphosphate group; and (ii) a free or protected modified phosphate, such as a monophosphate, diphosphate or triphosphate group, wherein the modified phosphate group is selected from a phosphonate, phosphoramidate or phosphorothioate group; Selected from: compound.

3. R １ The compound of claim 1 or 2, wherein is H.

4. R ２ The compound of any one of claims 1 to 3, wherein is H.

5. R ３ The compound of any one of claims 1 to 4, wherein is H or OH.

6. R ４ and R ５ The compound of any one of claims 1 to 5, wherein is H.

7. R １ , R ２ , R ３ , R ４ and R ５ are each H, or R １ , R ２ , R ４ and R ５ are H and R ３ is OH or OR ６ That is, 7. A compound according to any one of claims 1 to 6.

8. R １ , R ２ , R ３ , R ４ and R ５ are H, 8. A compound according to any one of claims 1 to 7.

9. R １ , R ２ , R ４ and R ５ are H and R ３ is OH; 8. A compound according to any one of claims 1 to 7.

10. 10. A compound according to any one of claims 1 to 9, or a pharma- ceutically acceptable salt thereof, for use in medicine.

11. 10. A compound of any one of claims 1 to 9, or a pharma- ceutically acceptable salt thereof, for the treatment of a hyperproliferative disorder.

12. 10. A compound of any one of claims 1 to 9, or a pharma- ceutically acceptable salt thereof, for the treatment of cancer.

13. 10. A compound of any one of claims 1 to 9, or a pharma- ceutically acceptable salt thereof, for the treatment of acute myeloid leukemia (AML), or myelodysplastic syndrome (MDS).

14. 10. A compound according to any one of claims 1 to 9, or a pharma- ceutically acceptable salt thereof, for the treatment of atherosclerosis or renal failure.

15. 10. In vitro use of a compound according to any one of claims 1 to 9 for inhibiting methylation of nucleic acids, in particular DNA.

16. 16. Use according to claim 15 for epigenetic analysis.

17. 1. A method for the treatment of a hyperproliferative disorder, comprising:

10. The method of claim 1, further comprising administering to a subject, particularly a human subject, a therapeutically effective dose of a compound according to any one of claims 1 to 9 or a pharma- ceutically acceptable salt thereof. method.

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