CARBOCYCLIC NUCLEOSIDE ANALOGUE
20220332737 · 2022-10-20
Assignee
Inventors
Cpc classification
C12Q1/683
CHEMISTRY; METALLURGY
A61P35/00
HUMAN NECESSITIES
International classification
Abstract
The present invention relates to novel hydrolytically stable carbon-cyclic 5-aza-2-deoxycytidine and carbocyclic 5-aza-cytidine compounds and pro-drugs thereof as hypomethylating agents.
##STR00001##
Claims
1. A compound of Formula I ##STR00027## wherein R.sup.1 is H, a free or protected, modified or unmodified phosphate group or a hydroxyl-protecting group, R.sup.2 is H, a free or protected, modified or unmodified phosphate group, or a hydroxyl-protecting group, R.sup.3 is H, F, CH3, CH2F, CHF2, CF3, OH or OR.sup.6 wherein R.sup.6 is a hydroxyl-protecting group, R.sup.4 and R.sup.5 each are H or form an amino-protecting group, or a salt thereof.
2. The compound of claim 1 wherein each phosphate group is independently selected from: (i) a free or protected unmodified phosphate, wherein the phosphate group is a monophosphate, diphosphate or triphosphate group, and (ii) a free or protected modified phosphate, e.g. monophosphate, diphosphate or triphosphate group, wherein the modified phosphate group is selected from a phosphonate, phosphoramidate or phosphorothioate group.
3. The compound of claim 1 wherein R.sup.1 is H.
4. The compound of claim 1 wherein R.sup.2 is H.
5. The compound of claim 1 wherein R.sup.3 is H or OH.
6. The compound of claim 1 wherein R.sup.4 and R.sup.5 are H.
7. The compound of claim 1 wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 each are H, or wherein R.sup.1, R.sup.2, R.sup.4 and R.sup.5 each are H and R.sup.3 is OH or OR.sup.6.
8. The compound of claim 1 wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4 and R.sup.5 each are H.
9. The compound of claim 1 wherein R.sup.1, R.sup.2, R.sup.4 and R.sup.5 each are H and R.sup.3 is OH.
10. The compound of claim 1 or a pharmaceutically acceptable salt thereof for use in medicine.
11. The compound of claim 1 or a pharmaceutically acceptable salt thereof for the treatment of a hyperproliferative disorder.
12. The compound of claim 1 or a pharmaceutically acceptable salt thereof for the treatment of cancer.
13. The compound of claim 1 or a pharmaceutically acceptable salt thereof for the treatment of acute myeloid leukemia (AML), or a myelodysplastic syndrome (MDS).
14. The compound of claim 1 or a pharmaceutically acceptable salt thereof for the treatment of atherosclerosis or renal insufficiency.
15. An in vitro use of the compound of claim 1 for inhibiting the methylation of nucleic acids, particularly of DNA.
16. The use of claim 15 for an epigenetic analysis.
17. A method for the treatment of a hyperproliferative disorder, comprising administering a therapeutically effective dose of the compound of claim 1 or a pharmaceutically acceptable salt thereof to a subject in need thereof, particularly to a human subject.
Description
FIGURE LEGENDS
[0039]
[0040]
[0041]
[0042]
[0043]
EXAMPLES
Example 1—Synthetic Procedures for cAzadC (1)
Tert.-Butyl-N-[(1R,3S,4R)-3-hydroxy-4-(hydroxymethyl)cyclopentyl]-carbamate (2)
[0044] ##STR00008##
[0045] 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 described previously [1].
Tert.-Butyl-N-[(1R,3S,4R)-3-benzyloxy-4-(benzyloxymethyl)-cyclopentyl]-carbamate (3)
[0046] ##STR00009##
[0047] 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 for 1.5 h at 0° C. and for additional 2 h at rt. The reaction mixture was diluted using CH.sub.2Cl.sub.2 (30 mL) and the reaction was quenched with sat. is NaHCO.sub.3 (30 mL). The mixture was extracted with CH.sub.2Cl.sub.2 (500 mL). The organic phase was washed three times with sat. NaHCO.sub.3 (1×400 mL, 2×100 mL) and dried over Na.sub.2SO.sub.4. The solvent was removed in vacuo. The crude product was purified by column chromatography on silica gel with a stepwise gradient iHex/EtOAc (9:1.fwdarw.7:1.fwdarw.4:1) to afford product 3 as a colorless solid (3.39 g, 8.24 mmol, 61%).
[0048] R.sub.f=0.67 (iHex/EtOAc=2:1)
[0049] Mp=74-76° C.
[0050] HRMS [ESI+]: [C.sub.25H.sub.33NO.sub.4Na].sup.+, [M+Na.sup.+].sup.+ calculated.: 434.2292, found: 434.2299
[0051] .sup.1H-NMR (599 MHz, CDCl.sub.3): 5/ppm=7.39-7.26 (m, 10H, Ar—H), 4.76 (s, 1H, NH), 4.53-4.37 (m, 4H, C.sub.7—H and C.sub.8—H), 4.18-4.08 (m, 1H, C.sub.1—H), 3.96-3.87 (m, 1H, C.sub.3—H), 3.53-3.41 (m, 2H, C.sub.5—H), 2.36-2.25 (m, 2H, C.sub.6—H.sub.a, C.sub.4—H), 2.11-2.05 (m, 1H, C.sub.2—H.sub.a), 1.77-1.68 (m, 1H, C.sub.2—H.sub.b), 1.43 (s, 9H, C.sub.2′—H), 1.29-1.20 (m, 1 H.sub.7 C.sub.6—H.sub.b).
[0052] .sup.13C-NMR (151 MHz, CDCl.sub.3): δ/ppm=155.5 (C.sub.1′), 138.60, 138.23, 128.36, 128.28, 127.59, 127.56, 127.42 (C—Ar), 81.0 (C.sub.3), 79.2 (C.sub.2′), 73.3 (C.sub.8), 71.9 (C.sub.5), 71.3 (C.sub.7), 50.4 (C.sub.1), 44.9 (C.sub.4), 39.6 (C.sub.2), 35.0 (C.sub.6), 28.6 (C.sub.2′).
[0053] FTIR (ATR):={tilde over (ν)}/cm .sup.−13339 (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).
(1R, 3S, 4R)-1-Amino-3-benzyloxy-4-(benzyloxymethyl)cyclopentane (4)
[0054] ##STR00010##
[0055] 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 CH.sub.2Cl.sub.2 (28 mL) was stirred for 1 h at rt. After removing the solvent in vacuo, the residue was resuspended in sat. Na.sub.2CO.sub.3 (100 mL) and stirred for 10 min. The mixture was extracted with EtOAc (3×150 mL) and the combined organic phases were dried over Na.sub.2SO.sub.4. Removing the solvent in vacuo resulted in the free amine 4 as viscous brown oil, which was used without further purification due to its instability towards O.sub.2.
[0056] Analytical data of purified 4:
[0057] HRMS [ESI.sup.+]: [C.sub.23H.sub.29N.sub.4O.sub.4Na].sup.+, [M+Na.sup.+].sup.+ calculated: 312.1958, found: 312.1955
[0058] .sup.1.sub.H-NMR (400 MHz, CDCl.sub.3): δ/ppm=7.38-7.20 (m, 10H, C—Ar), 4.57-4.36 (m, 4H, C.sub.7, C.sub.8), 3.95-3.85 (m, 1H, C.sub.1), 3.62-3.50 (m, 1H, C.sub.3), 3.50-3.39 (m, 2H, C.sub.5), 2.41-2.28 (m, 1H, C.sub.4), 2.28-2.13 (m, 1H, C.sub.6—H.sub.a), 2.13-1.96 (m, 3H, NH.sub.2, C.sub.2—H.sub.a), 1.65-1.50 (m, 1H, C.sub.2—H.sub.b), 1.19-1.08 (m, 1H, C.sub.6—H.sub.a),
[0059] .sup.13C-NMR (101 MHz, CDCl.sub.3): δ/ppm=138.8, 138.4, 128.5, 128.5, 127.79, 127.75, 127.7, 127.6 (C—Ar), 81.82 (C.sub.3), 73.23 (C.sub.7), 72.58 (C.sub.5), 71.10 (C.sub.s), 51.40 (C.sub.1), 45.63 (C.sub.4), 42.07 (C.sub.2), 38.08 (C.sub.6).
2-Methyl-1-(1 -imidazoylcarbonyl)-isourea (5)
[0060] ##STR00011##
[0061] To a solution of O-methylisourea hydrochloride 6 (5.00 g, 45.65 mmol, 1.0 eq.) in Et.sub.2O:H.sub.2O (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 filtrated, and the residue was washed with ice-cold Et.sub.2O (3×50 mL). The combined organic phases were concentrated to 20 mL in vacuo. Cooling to −20° C. resulted in precipitation. Vacuum filtration under N.sub.2 flow resulted in 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 for 3 h at rt. After 1 h, a colorless precipitate was observed. The reaction mixture was concentrated to 3 mL in vacuo and filtrated. The residue was washed with ice-cold THF (2×3 mL) and the combined organic phases were lyophilized, which resulted in colorless solid 5 (1.946 g, 11.57 mmol, 69%).
[0062] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ/ppm=8.60 (br s, 1H, N—H), 8.35 (s, 1H, Ar—H), 7.58 (s, 1H, Ar—H), 7.04 (s, 1H, Ar—H), 6.04 (br s, 1H, N—H), 3.96 (s, 3H, C.sub.2—H).
[0063] .sup.13C-NMR (101 MHz, CDCl.sub.3): δ/ppm=165.7 (C.sub.1), 157.3 (C3), 137.5(Ar), 130.0 (Ar), 117.2 (Ar), 55.3 (C.sub.2).
1-[(1′R,3′S,4′R)-3′-Benzyloxy-4′-(benzyloxymethyl)-cyclopentyl]-methylisobiuret (8)
[0064] ##STR00012##
[0065] The crude product 4 was dissolved in dry acetonitrile (75 mL) and the carbonyl imidazole 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.fwdarw.1:1) to obtain the methylisobiurete 8 as a yellow oil (2.613 g, 6.35 mmol, 78%).
[0066] R.sub.f=0.51 (DCM/MeOH=19:1)
[0067] HRMS [ESI.sup.+]: [C.sub.23H.sub.29N.sub.4O.sub.4Na].sup.+, [M+Na.sup.+].sup.+ calculated: 434.2050, found: 434.2049
[0068] .sup.1H-NMR (599 MHz, CDCl.sub.3): δ/ppm=7.35-7.16 (m, 10H, Ar—H), 5.42 (d,.sup.3J.sub.1′,N1 =7.8 Hz, 1H, N.sub.1—H), 4.50-4.34 (m, 4H, C.sub.7′—H.sub.a,b, C.sub.8′—H.sub.a,b), 4.33-4.16 (m, 1H, C.sub.1′—H), 3.93-3.81 (m, 1H, C.sub.3—H), 3.62 (s, 3H, C.sub.6—H), 3.43-3.31 (m, 2H,C.sub.5′—H), 2.34-2.18 (m, 2H, C.sub.6′—H.sub.a, C.sub.4′—H), 2.06 (ddd, .sup.2J.sub.2′a,2′b=13.0 Hz, .sup.3J.sub.2′a,1′/3′=6.9 Hz, .sup.3J.sub.2′a,1′/3′=4.6 Hz, 1H, C.sub.2′—H.sub.a), 1.73 (ddd, .sup.2J.sub.2′a,2b′=13.0 Hz, .sup.3J.sub.2′b,1′/3′=7.0 Hz, .sup.3J.sub.2′b,1′/4′=7.0 Hz, 1H, C.sub.2—H.sub.b), 1.28-1.11 (m, 1H,C.sub.6′—H.sub.b)
[0069] .sup.13C-NMR (151 MHz, CDCl.sub.3): δ/ppm=163.8 (C.sub.2), 162.3 (C.sub.4), 138.9 (C.sub.Ar), 138.5 (C.sub.Ar), 128.51 (C.sub.Ar), 128.41 (C.sub.Ar), 127.76 (C.sub.Ar), 127.74 (C.sub.Ar), 127.70 (C.sub.Ar), 127.56 (C.sub.Ar), 81.1 (C.sub.3′), 73.3 (C.sub.7′), 71.9 (C.sub.5′), 71.2 (C.sub.8′), 53.8 (C.sub.6), 49.5 (C.sub.1′), 45.0 (C.sub.4′), 39.5 (C.sub.2′), 35.0 (C.sub.6′).
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)
[0070] ##STR00013##
[0071] 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.fwdarw.1:1.fwdarw.1:2) to obtain 9a (1.774 g, 4.07 mmol, 64%, colorless solid) and 9b (268.0 mg, 0,64 mmol, 10%, colorless oil).
[0072] 9a:
[0073] R.sub.f=0.71 (iHex/EtOAc, 1:3)
[0074] HRMS [ESI.sup.+]: [C.sub.25H.sub.30N.sub.3O.sub.4H].sup.+, [M+H.sup.+].sup.+ calculated: 436.2232, found.: 436.2231.
[0075] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ/ppm=8.23 (s, 1H, C.sub.6—H), 7.38-7.24 (m, 10H, Ar—H), 5.07-4.89 (m, 1H, C.sub.1′—H), 4.56-4.39 (m, 6H, C.sub.7′—H.sub.a,b, C.sub.8′—H.sub.a,b, C.sub.7—H.sub.a,b), 4.07-3.08 (m, 1H, C.sub.3′—H), 3.61-3.46 (m, 2H, C.sub.5′—H.sub.a,b), 2.52-2.36 (m, 2H, C.sub.6′—H.sub.a, C.sub.4—H), 2.28 (ddd, 1H, .sup.2J.sub.2′a,2′b=13.3 Hz, .sup.3J=7.6 Hz, .sup.3J=2.7 Hz, C.sub.2′—H.sub.a), 2.11 (ddd, 1H, .sup.2J.sub.2′a,2′b=13.3 Hz, .sup.3J=10.0 Hz, .sup.3J=6.3 Hz, C.sub.2′—H.sub.b), 1.84-1.68 (m, 1H, C.sub.6′—H.sub.b), 1.40 (t, 3H, .sup.3J.sub.8, 7=7.1 Hz, C.sub.8—H),
[0076] .sup.13C-NMR (101 MHz, CDCl.sub.3): δ/ppm=169.3 (C.sub.4), 158.4 (C.sub.6), 155.0 (C.sub.2), 138.2 (C.sub.Ar), 138.1 (C.sub.Ar), 128.6 (C.sub.Ar), 128.6 (C.sub.Ar), 127.93 (C.sub.Ar), 127.85 (C.sub.Ar), 127.82 (C.sub.Ar), 80.4 (C.sub.3′), 73.4 (C.sub.7′/8′), 71.26 (C.sub.7′/8′), 71.25 (C.sub.5′), 65.1 (C.sub.7), 56.8 (C.sub.1′), 44.9 (C.sub.4′), 36.9 (C.sub.2′), 32.6 (C.sub.6′), 14.2 (C.sub.8).
[0077] 9b:
[0078] R.sub.f=0.63 (iHex/EtOAc, 1:3)
[0079] HRMS [ESI.sup.+]: [C.sub.24H.sub.28N.sub.3O.sub.4].sup.+, [M+H.sup.+].sup.+ calculated: 422.2074 , found: 422.2075.
[0080] .sup.1H-NMR (599 MHz, CDCl.sub.3): δ/ppm==8.23 (s, 1H, C.sub.6—H), 7.38-7.25 (m, 10H, Ar—H), 5.04-4.93 (m, 1H, C.sub.1′—H), 4.57-4.43 (m, 6H, C.sub.7′—H.sub.a,b, C.sub.8′—H.sub.a,b), 4.09-3.98 (m, 4H, C.sub.3′—H, C.sub.7—H), 3.60-3.50 (m, 2H, C.sub.5′—H.sub.a, b), 2.50-2.39 (m, 2H, C.sub.6′—H.sub.a, C.sub.4′—H), 2.28 (ddd, 1H, .sup.2J.sub.2′a,2′b=13.1 Hz, .sup.3J=7.4 Hz, .sup.3J=2.7 Hz, C.sub.2′—H.sub.a), 2.11 (ddd, 1H, .sup.2J.sub.2′a,2′b=13.1 Hz, .sup.3J=10.1 Hz, .sup.3J=6.3 Hz, C.sub.2′—H.sub.b), 1.80-1.68 (m, 1H, C.sub.′6—H.sub.b).
[0081] .sup.13C-NMR (101 MHz, CDCl.sub.3): δ/ppm=169.9 (C.sub.4), 158.5 (C.sub.6), 155.0 (C.sub.2), 138.3 (C.sub.Ar), 138.0 (C.sub.Ar), 128.63 (C.sub.Ar), 128.57 (C.sub.Ar), 127.93 (C.sub.Ar), 127.84 (C.sub.Ar), 127.82 (C.sub.Ar), 80.4 (C.sub.3′), 73.5 (C.sub.7′/8′), 71.3 (C.sub.7′/8′), 56.9 (C.sub.1′), 56.0 (C.sub.7), 44.9 (C.sub.4′), 36.9 (C.sub.2′), 32.6 (C.sub.6′).
4-Amino-1 -[(1′R,3′S,4′R)-3′-benzyloxy-4′-(benzyloxymethyl)-cyclopentyl]-1H -[1,3,5]triazin-2-one (10)
[0082] ##STR00014##
[0083] Ethoxytriazine 9a (1.630 g, 3.74 mmol, 1.0 eq.) was dissolved in methanolic NH.sub.3 (7 N in MeOH, 60 mL) and stirred for 3 h at rt. The mixture was diluted with H.sub.2O (340 mL), resulting in a colorless precipitate, which was extracted with EtOAc (3×200 mL). The organic phases were combined, dried and the solvent was removed in vacuo, which is resulted in the benzyl-protected cAzadC 10 (1.478 g, 3.63 mmol, 97%) as a colorless foam. Synthesis of 10 was also successful starting from the methoxytriazine 9b and following the same procedure.
[0084] R.sub.f=0.50 (CH.sub.2Cl.sub.2/MeOH, 9:1)
[0085] HRMS [ESI.sup.+]: [C.sub.23H.sub.27N.sub.4O.sub.3].sup.+, [M+H.sup.+] calculated: 407.2078, found: 407.2081.
[0086] .sup.1H-NMR (599 MHz, CDCl.sub.3): δ/ppm=8.01 (s, H, C.sub.6—H), 7.35-7.23 (m, 10H, Ar—H), 5.78 (5, 2H, NH), 4.92-4.76 (m, 4H, C.sub.7′—H and C.sub.8′—H), 4.59-4.46 (m, 1H, C.sub.1′—H), 4.07-3.95 (m, 1H, C.sub.3′—H), 3.55-3.45 (m, 2H, C.sub.5′—H), 2.44-2.28 (m, 2H, C.sub.6′—H.sub.a, C.sub.4′—H), 2.24-2.16 (m, 1H, C.sub.2′—H.sub.a), 2.14-2.05 (m, 1H, C.sub.2′—H.sub.b), 1.78-1.67 (m, 1H, C.sub.6′—H.sub.b)
[0087] .sup.13C-NMR (151 MHz, CDCl.sub.3): δ/ppm=165.7 (C.sub.4), 157.1 (C.sub.6), 154.2 (C.sub.2), 138.3, 138.3, 128.6, 128.5, 127.84, 127.78 (C—Ar), 80.4 (C.sub.3′), 73.4 (C.sub.8′), 71.4 (C.sub.5′), 71.2 (C.sub.8′), 56.7 (C.sub.1′), 44.9 (C.sub.4′), 36.8 (C.sub.2′), 32.5 (C.sub.6′).
4-Amino-1-[(1′R,3′S,4′R)-3′-hydroxy-4′-(hydroxymethyl)-cyclopentyl]-1H -[1,3,5]triazin-2-one (cAzadC, 1)
[0088] ##STR00015##
[0089] A solution of benzyl-protected 10 (1.478 g, 3.63 mmol, 1.0 eq) in CH.sub.2Cl.sub.2 (100 mL) was cooled to −78° C. and BCl.sub.3 (1 M in DCM, 12.7 mL, 12.7 mmol, 3.5 eq.) was added dropwise. The mixture was stirred for 1 h at −78° C. and stirred for additional 2 h at rt. The reaction was quenched by 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 CH.sub.2Cl.sub.2/MeOH (9:1.fwdarw.7:3) to obtain cAzadC (1, 0.740 g, 3.27 mmol, 90%). Recrystallization from hot MeOH resulted in 57% cAzadC (1, 465.3 mg, 2.06 mmol) as colorless acicular monocrystals.
[0090] R.sub.f=0.1 (CH.sub.2Cl.sub.2/MeOH, 9:1)
[0091] Mp.: 198-200° C.
[0092] HRMS [ESI.sup.+]: [C.sub.9H.sub.15N.sub.4O.sub.3].sup.+, [M+H.sup.+].sup.+ calculated: 227,1138, found: 227,1139.
[0093] .sup.1H-NMR (400 MHz, D.sub.2O): δ/ppm=8.29 (s, 1H, C.sub.6—H), 4.90-4.62 (m, 1H, C.sub.1′—H, Overlap with D.sub.2O signal), 4.29-4.16 (m, 1H, C.sub.3′—H), 3.73 (dd, .sup.5J.sub.5′a,5′b=11.2 Hz, .sup.3J.sub.5′a,4′=5.7 Hz, 1H, C.sub.5′a—H ), 3.62 (dd, .sup.5J.sub.5′a,5′b=11.2 Hz, .sup.3J.sub.5′b,4′=6.8 Hz, 1H, C.sub.5′b—H), 2.41-2.29 (m, 1H, C.sub.6′a—H), 2.28-2.18 (m, 1H, C.sub.2′a—H), 2.17-2.02 (m, 2H, C.sub.2′, b—H, C.sub.4′b—H), 1.77-1.55 (m, 1H, C.sub.6′b—H).
[0094] .sup.13C-NMR (101 MHz, CDCl.sub.3): δ/ppm=165.5 (C.sub.4), 158.5(C.sub.6), 156.4(C.sub.2), 72.2 (C.sub.3′), 62.8 (C.sub.5′), 56.2 (C.sub.1′), 48.11 (C.sub.4′), 38.0 (C.sub.2′), 32.0 (C.sub.6′.).
[0095] FTIR (ATR): {tilde over (ν)}/cm .sup.−=3194 (m), 1653 (m), 1540 (s), 1437 (s), 1278 (s), 1036 (s).
Example 2—X-Ray Crystal Structure Analysis of cAzadC
[0096] The cAzadC monocrystals were analysed by X-ray crystallography. Table 1 shows the solved structure of cAzadC 1 in two different cyclopentane conformations in the crystal. One conformer adopts a C6′-endo (P=88.2°, max=47.8°) conformation (
[0097] Further figures were generated with the program Mercury 3.5.1. (Cambridge Crystallographic Data Center). The data are deposited under CCDC 1910952.
TABLE-US-00001 TABLE 1 Crystallographic data for cAzadC net formula C.sub.9H.sub.14N.sub.4O.sub.3 transmission factor 0.9087- range 0.9580 M.sub.r/g mol.sup.−1 226.24 refls. measured 34579 crystal size/mm 0.100 × 0.080 × R.sub.int 0.0373 0.030 T/K 100(2) mean σ(/)// 0.0197 radiation MoKα θ range 3.031-25.40 diffractometer ′Bruker observed refls. 3474 D8Venture′ crystal system orthorhombic x, y (weighting 0.0372, scheme) 0.4868 space group ′P 21 21 21′ hydrogen refinement mixed a/Å 8.4451(3) Flack parameter 0.3(3) b/Å 11.0958(4) refls in refinement 3787 c/Å 21.9635(8) parameters 321 α/° 90 restraints 0 β/° 90 R(F.sub.obs) 0.0284 γ/° 90 R.sub.w(F.sup.2) 0.0699 V/Å.sup.3 2058.09(13) S 1.054 Z 8 shift/error.sub.max 0.001 calc. density/ 1.460 max electron density/e 0.180 g cm.sup.−3 Å.sup.−3 μ/mm.sup.−1 0.112 min electron density/e −0.173 Å.sup.−3 absorption correction multi-scan
Example 3—Test for Hydrolytic Stability
[0098] We next investigated the hydrolytic stability of cAzadC 1 in direct comparison to the pharmaceutical AzadC.
[0099] Both nucleosides were dissolved in aqueous KH.sub.2PO.sub.4 buffer (100 mM, pH=7.4, 5.5 and 8.5) in 100 mM concentration. The solutions were immediately analysed via HPLC (t=0 h) with subsequent HPLC analyses every 1.5 h or at indicated time points. As stationary phase an EC 250/4 NUCLEOSIL 120-3 C18 (Macherey-Nagel) chromatography column was used. The mobile phase consisted of water (buffer A) and acetonitrile (buffer B) at a flow-rate of 0.5 mL/min as follows: 0.fwdarw.25 min, 0.fwdarw.5% buffer B; 25 min.fwdarw.28 min, 5%.fwdarw.80%; 28 min.fwdarw.38 min, 80%; 38 min.fwdarw.43 min 80%.fwdarw.0%; 43 min.fwdarw.45 min, 0%.
[0100] Since one treatment cycle for AzadC goes over four weeks we decided to measure the stability at a time point related to a half cycle (14 d). NMR spectra were measured after keeping the solutions at r.t.
[0101] As tumour cells often provide an acidic micro-environment [16], the stability under slightly acidic pH is particularly informative. As evident from the data shown in
[0102] In contrast to these results, we observed for cAzadC 1 surprisingly no degradation at all tested pH values, including pH=5.5. This result led to the surprising discovery that the simply O.fwdarw.CH.sub.2 exchange causes a strong remote disarming effect that seems to change the properties of the triazine ring so that reaction with water is stopped.
Example 4—Test for Biological Functions
Cell Culture of mESC for cAzadC Treatment
[0103] 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 passaged when applicable in DMEM supplemented with FBS and LIF as above but lacking the inhibitors. For drug treatment, cells were moved into the primed state by removing 2i from the medium. Cells were incubated 2 d in DMEM supplemented with FBS and LIF in 6-well plates (VWR). After splitting, 2×10.sup.5 cells were transferred into a 6-well plate culture dish and supplemented with either 1 μM (in 0.01% DMSO) or 5 μM cAzadC (in 0.05% DMSO) and treated for 72 h. After removal of the medium and washing the cells with DPBS, they were directly lysed with RLT.sup.+ buffer as described in a previous publication [18].
gDNA Isolation, Total Enzymatic Digest and UHPLC-MS.SUP.2
[0104] The gDNA was isolated as described previously [18]. The gDNA was directly subjected to a total enzymatic digest and analyzed using UHPLC-MS.sup.2 as described in a previous publication [19]. The external calibration curve was generated 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 it in technical triplicates prior to each measurement. Linear regression was done by OriginPro 2016G.
Results
[0105] We investigated if the disarming effect described in Example 3 would influence the biological functions. We used for this purpose mouse embryonic stem cells (mESC) that were primed in serum/Lif as a model system, since mdC levels increase from naïve to primed state [17]. We added cAzadC 1 in two different concentrations (1 μM and 5 μM) to mESC that have been primed for 48 h and allowed the cells to further proliferate under priming conditions in the presence of cAzadC 1 for additional 72 h. After the 72 h, we harvested the cells, isolated the DNA and digested the DNA down to the nucleoside level using our described protocol [18].
[0106] The levels of mdC were finally precisely quantified using isotope dilution UHPLC-MS.sup.2. To this end, isotopically labelled standards of the nucleosides were spiked-in for exact quantification [19, 18]. In addition to mdC, we quantified the levels of 5-hydroxymethyl-2′-deoxycytidine (hmdC), which is formed from mdC by the action of TET enzymes [20-21]. The absolute levels of hmdC are in mESC more than ten times lower than the mdC levels [20, 22]. The consequence is that even after a substantial reduction of mdC, there should be sufficient mdC to keep the hmdC levels constant. The question if and by how much the hmdC level is affected can therefore inform us about how epigenetic reprogramming is organized.
[0107] Parallel to the quantification of mdC and hmdC we also quantified to which extend cAzadC 1 itself was incorporated into the genome of the mESC. Detection of AzadC in the genome of treated cells is only possible after treatment of the DNA with NaBH.sub.4. Application of NaBH.sub.4 reduces the C(5)=C(6) double bond, which stabilizes the compound so that its quantification becomes possible [23, 19]. To our delight, we noted that the stability of cAzadC 1 allowed its quantification without this pre-treatment. We also noted that the applied enzymatic digestion protocol allowed to digest genomic DNA (gDNA) even in the presence of large amounts of cAzadC 1. Taken together, quantification of cAzadC 1 by UHPLC-MS.sup.2 using an external calibration curve was possible in parallel to quantification of canonical and epigenetic bases.
[0108] At 1 μM cAzadC 1 concentration, we detected a cAzadC 1 level of 5×10.sup.−4 cAzadC per dN (
[0109] Importantly, after exposing the mESC for 72 h at 1 M cAzadC in the medium, we detected a reduction of the mdC values by almost 30% (
[0110] The data show that the carbocyclic version cAzadC 1 needs simply more time to affect the mdC levels by the same amount. We believe that this effect is caused by a potentially slower conversion of cAzadC 1 into the triphosphate. The slower kinetics of cAzadC 1, however, is not necessarily a disadvantage given the long treatment times that are applied in the clinic.
[0111] Very interesting is also the discovery that the hmdC levels were reduced to about 50% already in the 1 μM experiment. At 5 μM, we were even unable to detect hmdC above background levels using 0.5 μg of genomic DNA. The result shows that the hmdC level dropped even faster than the mdC levels, although hmdC is more than ten times less abundant in the genome. This result is interesting. It indicates that hmdC might be potentially predominantly generated in the mdC maintenance process during cell division. We see here that compound cAzadC 1 is a perfect tool molecule that now allows to gain further insight into the interplay between methylation of dC to mdC and oxidation of mdC to hmdC. With the new compound cAzadC 1 in hand we can cleanly correlate demethylation of the genome with the corresponding cellular effects without compromising DNA damaging effects. Finally, cAzadC 1 may not only be a valuable tool compound but even a next generation epigenetic pharmaceutical.
SUMMARY
[0112] We show that replacement of the in-ring O-atom by a CH.sub.2-unit stabilizes the pharmaceutical so that its nucleophilic reaction with water is stopped. The new nucleoside cAzadC 1 is accepted by the phosphorylating enzymes in cells and the corresponding cAzadC-triphosphates are efficiently incorporated into the genome. cAzadC 1 is incorporated in the genome with several million nucleotides and it causes the mdC level to decrease to 70% relative to the control levels.
Example 5—Synthetic Procedures for cAzaC (20)
Tert-Butyl-(1R,4S,5R,6S)-5,6-dihydroxy-3-oxo-2-azabicyclo[2.2.1]heptane-2-carboxylate 12
[0113] ##STR00016##
[0114] 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 H.sub.2O 22.59 g N-methylmorpholino-N -oxide (167.2 mmol, 3.0 eq.) and 1.03 g K.sub.2OsO.sub.4 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 Na.sub.2SO.sub.3 (278.5 mmol, 5.0 eq.) were added and the mixture was stirred for another 1 h at room temperature. Then, the reaction mixture was extracted with EtOAc (3×300 mL). Combined organic layers were washed with saturated aq. NaCl (300 mL) and dried over Na.sub.2SO.sub.4. After evaporation of the solvents in vacuo the residue was purified by silica gel column chromatography (CH.sub.2Cl.sub.2:MeOH 40:1.fwdarw.CH.sub.2Cl.sub.2:MeOH 30:1) to yield 2.91 g of the exo-bishydroxylated product 12 (12.0 mmol, 22%) as a yellow foam.
[0115] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ/ppm=4.35-4.32 (m, 1H, 1-H), 4.23 (d, J=6.0 Hz, 1H, 5-H), 4.08 (d, J=6.1 Hz, 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, 1.5 Hz, 1H, 7-H), 1.97 (m, 1H, 7-H), 1.51 (s, 9H, 3′-H).
[0116] .sup.13C-NMR (101 MHz, CDCl.sub.3): 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).
[0117] IR (ATR): v (cm.sup.−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).
[0118] R.sub.f (CH.sub.2Cl.sub.2:MeOH 20:1): 0.24.
Tert-Butyl-((1R,2S,3R,4R)-2,3-dihydroxy-4-(hydroxymethyl)-cyclopentyl) -carbamate 13
[0119] ##STR00017##
[0120] 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 NaBH.sub.4 (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. Subsequently, 100 mL acetic acid (10% v/v in MeOH) and silica gel were added and the mixture was concentrated to dryness in vacuo. Purification via silica gel column chromatography (dry load, CH.sub.2Cl.sub.2:MeOH 5:1.fwdarw.CH.sub.2Cl.sub.2:MeOH 4:1) and coevaporation with dry toluene (2×150 mL) yielded product 313 as its acetate salt (2.96 g, 9.6 mmol, 81%) as a slightly brown oil.
[0121] .sup.1H-NMR (400 MHz, DMSO-d6): δ/ppm=6.70 (d, J=7.9 Hz, 1H, NH), 3.65-3.49 (m, 2H, 1-H, 3-H), 3.52 (dd, J=6.3, 5.2 Hz, 1H, 2-H), 3.37 (dd, J=10.5, 5.8 Hz, 1H, 5-H), 3.30 (dd, J=10.5, 6.2 Hz, 1H, 5-H), 1.98 (dt, J=13.1, 8.4 Hz, 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).
[0122] .sup.13C-NMR (101 MHz, DMSO-d6): δ/ppm=173.28 (OAc-C1), 155.23 (C-1′), 77.37 (2′-C), 75.93 (2-C), 72.07 (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).
[0123] R.sub.f (CH.sub.2Cl.sub.2:MeOH 8:1): 0.43.
Tert-Butyl-((1R,2S,3R,4R)-2,3-bis(benzyloxy)-4-((benzyloxy)methyl) -cyclopentyl)-carbamate 14
[0124] ##STR00018##
[0125] 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 3 portions. After stirring the mixture for 15 min at 0° C. 5.31 mL BnBr (44.6 mmol, 4.7 eq,) were added slowly and the reaction mixture was stirred for 2.5 h at 0° C. and for 3 h at room temperature. Then, 220 mg TBAI (0.6 mmol, 6 mol %) and another 3.18 mL BnBr (26.8 mmol, 2.8 eq.) were added and the mixture was warmed to 80° C. for 3.5 h. Subsequently, the mixture was cooled to room temperature, poured in 300 mL saturated aq. NaHCO.sub.3 and extracted with CH.sub.2Cl.sub.2 (3×300 mL). Combined organic layers were washed with saturated aq. NaHCO.sub.3 (300 mL), saturated aq. NaCl (300 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated in vacuo at 50° C. The residue was purified by column chromatography (iHex:EtOAc 10:1.fwdarw.iHex:EtOAc 8:1.fwdarw.iHex:EtOAc 7:1.fwdarw.iHex:EtOAc 6:1.fwdarw.iHex:EtOAc 4:1) to yield the tribenzyl-protected product 14 (2.47 g, 4.8 mmol, 51%) a colorless solid wax.
[0126] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ/ppm=7.45-7.09 (m, 15H, 3×OCH.sub.2Ph), 4.95 (s, 1H, NH), 4.76-4.25 (m, 6H, 3×OCH.sub.2Ph), 4.15-3.90 (m, 1H, 1-H), 3.82 (dd, J=6.7, 4.4 Hz, 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).
[0127] .sup.13C-NMR (101 MHz, CDCl.sub.3): 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 (OCH.sub.2-Ph), 71.52 (OCH.sub.2-Ph), 71.02 (OCH.sub.2-Ph), 70.41 (5-C), 52.85 (1-C), 41.49 (1-C), 30.34 (6-C), 28.46 (3′-C).
[0128] HRMS (ESI.sup.+): calculated for C.sub.32H.sub.40NO.sub.5.sup.+ [M+H].sup.+ 518.2901; found: 518.2911.
[0129] IR (ATR): v (cm.sup.−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).
[0130] R.sub.f (iHex:EtOAc 4:1): 0.69.
(1R,2S,3R,4R)-2,3-Bis(benzyloxy)-4-((benzyloxy)methyl)-cyclopentane-1-amine 15
[0131] ##STR00019##
[0132] Under Ar atmosphere 1.02 g of Boc-protected cyclopentane 14 (1.97 mmol, 1.0 eq.) were dissolved in 7.0 mL dry CH.sub.2Cl.sub.2, 3.0 mL TFA were added and the mixture was stirred at room temperature for 1 h. The red solution was concentrated to dryness, 20.0 mL saturated aq NaHCO.sub.3 were added and the mixture was stirred at room temperature for 10 min. Then, 60 mL EtOAc were added, phases were separated and the aqueous layer was further extracted with EtOAc (2×60 mL). Combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated to dryness in vacuo to yield deprotected amine 15 as a yellow oil (882 mg, 1.97 mmol, quant.) which was used for the next step without further purification due to its instability towards oxidation.
[0133] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ/ppm=7.39-7.26 (m, 15H, 3×OCH.sub.2Ph), 4.63-4.33 (m, 6H, 3×OCH.sub.2Ph), 3.81 (dd, J=5.0, 3.1 Hz, 1H, 3-H), 3.49 (dt, J=9.1, 7.8 Hz, 1H, 1-H), 3.41 (dd, J=9.2, 5.1 Hz, 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).
[0134] .sup.13C-NMR (101 MHz, CDCl.sub.3): δ/ppm=138.66 (Ar—C), 138.52 (Ar—C), 138.48 (Ar—C), 128.72 (Ar—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 (OCH.sub.2-Ph), 72.27 (5-C), 71.86 (OCH.sub.2-Ph), 70.89 (OCH.sub.2-Ph), 54.32 (1-C), 41.49 (4-C), 32.19 (6-C).
[0135] HRMS (ESI.sup.+): calculated for C.sub.27H.sub.32NO.sub.3.sup.+ [M+H].sup.+ 418.2377; found: 418.2378.
1-[(1′R,2′S,3′R,4′R)-2′,3′-Bis(benzyloxy)-4′-((benzyloxy)methyl)-cyclopentyl]-methylisobiuret 16
[0136] ##STR00020##
[0137] Under Ar atmosphere 882 mg cyclopentyl amine 15 (1.87 mmol, 1.97 mmol, 1.0 eq.) were dissolved in 18.20 mL dry MeCN and 431 mg 2-methyl-1-(1-imidazoylcarbonyl)-isourea .sup.[1] (2.56 mmol, 1.3 eq.) were added. The mixture was refluxed for 2 h at 92° C., subsequently cooled to room temperature and evaporated to dryness. The residue was purified by column chromatography on silica gel (iHex:EtOAc 3:1.fwdarw.iHex:EtOAc 5:2.fwdarw.iHex:EtOAc 2:1.fwdarw.iHex:EtOAc 1:1) to yield methylisobiuret 16 as a colorless oil (886 mg, 1.17 mmol, 87%).
[0138] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ/ppm=7.43-7.22 (m, 15H, 3×OCH.sub.2Ph), 5.60 (s, 1H, 1-H), 4.80-4.61 (m, 2H, OCH.sub.2Ph), 4.55-4.41 (m, 3H, OCH.sub.2Ph), 4.32 (d, J=11.8 Hz, 1H, OCH.sub.2Ph), 4.24 (td, J=7.6, 3.4 Hz, 1H, 1′-H), 3.87 (dd, J=6.9, 4.5 Hz, 1H, 3′-H), 3.82 (dd, J=4.5, 3.1 Hz, 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).
[0139] .sup.13C-NMR (101 MHz, CDCl.sub.3): 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 (OCH.sub.2-Ph), 71.64 (OCH.sub.2-Ph), 71.20 (OCH.sub.2-Ph), 70.60 (5′-C), 52.56 (1′-C), 41.68 (4′-C), 31.07 (6-C), 30.57 (6′-C).
[0140] HRMS (ESI.sup.+): calculated for C.sub.30H.sub.36N.sub.3O.sub.5.sup.+ [M+H].sup.+ 518.2650; found: 518.2652.
[0141] IR (ATR): v (cm.sup.−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).
[0142] R.sub.f (iHex:EtOAc 2:1): 0.37.
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)rnethyl)-cyclopentyl]-1 H-[1,3,5]-triazin-2-one (18)
[0143] ##STR00021##
[0144] Methylisobiuret 16 (837 mg, 1.62 mmol, 1.0 eq.) was dissolved in 15.30 mL triethylorthoformate and 20 μL TFA were added. The mixture was heated to 90° C. for 3 h, subsequently 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.fwdarw.iHex:EtOAc 5:2.fwdarw.iHex:EtOAc 2:1.fwdarw.iHex:EtOAc 1:1.fwdarw.iHex:EtOAc 1:2) to yield 373 mg ethoxytriazine 18 (0.69 mmol, 43%) as a colorless wax and 389 mg methoxytriazine 17 (0.73 mmol, 46%) as a colorless solid.
[0145] Methoxytriazine 17:
[0146] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ/ppm=7.96 (s, 1H, 6-H), 7.38-7.17 (m, 15H, 3×OCH.sub.2Ph), 4.70-4.40 (m, 6H, 1′-H, 2.5×OCH.sub.2Ph), 4.36-4.24 (m, 2H, 2′-H, 0.5×OCH.sub.2Ph), 3.98 (s, 3H, 7-H), 3.93 (dd, J=4.9, 3.1 Hz, 1H, 3′-H), 3.54-3.40 (m, 2H, 5′-H), 2.51 (ddt, J=9.8, 4.4, 2.3 Hz, 1H, 4′-H), 2.35 (dt, J=13.3, 9.4 Hz, 1H, 6′-H), 1.82 (ddd, J=13.4, 9.0, 6.7 Hz, 1H, 6′-H).
[0147] .sup.13C-NMR (101 MHz, CDCl.sub.3): 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 (OCH.sub.2-Ph), 72.17 (OCH.sub.2-Ph), 71.41 (OCH.sub.2-Ph), 71.20 (5′-C), 63.34 (1′-C), 55.95 (7-C), 41.14 (4′-C), 26.75 (6′-C).
[0148] HRMS (ESI.sup.+): calculated for C.sub.31H.sub.34N.sub.3O.sub.5.sup.+ [M+H].sup.+ 528.2493; found: 528.2498.
[0149] IR (ATR): v (cm.sup.−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).
[0150] R.sub.f (iHex:EtOAc 2:1): 0.27.
[0151] Ethoxytriazine 18:
[0152] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ/ppm=7.97 (s, 1H, 6-H), 7.39-7.15 (m, 15H, 3×OCH.sub.2Ph), 4.66-4.21 (m, 10H, 1′-H, 2′-H, 7-H, 3×OCH.sub.2Ph), 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).
[0153] .sup.13C-NMR (101 MHz, CDCl.sub.3): 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 (OCH.sub.2-Ph), 72.28 (OCH.sub.2-Ph), 71.55 (OCH.sub.2-Ph), 71.20 (5′-C), 65.06 (7-C), 63.33 (1′-C), 41.15 (4′-C), 26.81 (6′-14.24 (8-C).
[0154] HRMS (ESI.sup.+): calculated for C.sub.32H.sub.36N.sub.3O.sub.5.sup.+ [M+H].sup.+ 542.2650; found: 542.2656.
[0155] IR (ATR): v (cm.sup.−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).
[0156] R.sub.f (iHex:EtOAc 2:1): 0.30.
4-Amino-1-[(1′R,2′S,340 R,4′R)-2′,3′-bis(benzyloxy)-4′-((benzyloxy)methyl) -cyclopentyl]-1H-[1,3,5]-triazin-2-one 19
[0157] ##STR00022##
[0158] A mixture of ethoxytriazine 18 (358 mg, 0.66 mmol) and methoxytriazine 17 (363 mg, 0.69 mmol) were dissolved in 26.8 mL 7 N NH.sub.3 in MeOH and stirred at room temperature for 3 h. Subsequently, 125 mL H.sub.2O were added to the reaction mixture, which was then extracted with EtOAc (3×200 mL). Combined organic layers were dried over Na.sub.2SO.sub.4 and evaporated to dryness in vacuo yielding 593 mg of the aminated product 19 (1.16 mmol, 86%) as a colorless foam which was used for the next step without further purification.
[0159] .sup.1H-NMR (400 MHz, CDCl.sub.3): δ/ppm=7.80 (s, 1H, 6-H), 7.26 (m, 15H, 3×OCH.sub.2Ph), 5.70 (s, 1H, NH), 4.63-4.42 (m, 6H, 1′-H, 2.5×OCH.sub.2Ph), 4.39-4.28 (m, 2H, 2′-H, 0.5×OCH.sub.2Ph), 3.91 (dd, J=5.0, 3.5 Hz, 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, 9.3 Hz, 1H, 6′-H), 1.86 (ddd, J=13.3, 9.2, 7.1 Hz, 1H, 6′-H)
[0160] .sup.13C-NMR (101 MHz, CDCl.sub.3): 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 (OCH.sub.2-Ph), 72.18 (OCH.sub.2-Ph), 71.39 (OCH.sub.2-Ph), 71.3 (5′-C)7, 63.53 (1′-C), 41.21 (4′-C), 26.79 (6′-C).
[0161] HRMS (ESI.sup.+): calculated for C.sub.30H.sub.33N.sub.4O.sub.4.sup.+ [M+H].sup.+ 513.2496; found: 513.2496.
[0162] IR (ATR): v (cm.sup.−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).
[0163] R.sub.f (CH.sub.2Cl.sub.2:MeOH 15:1): 0.42.
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
[0164] ##STR00023##
[0165] A solution of benzyl protected 5-aza-carbaC 19 (576 mg, 1.12 mmol, 1.0 eq.) was dissolved in 30.8 mL dry CH.sub.2Cl.sub.2 under Ar atmosphere and cooled to −78° C. Then, 5.88 mL BCl.sub.3 (1 M in CH.sub.2Cl.sub.2, 5.88 mmol, 5.25 eq.) were added dropwise, the mixture was stirred 1 h at −78° C. and 2 h at room temperature. Subsequently, 40 mL dry MeOH were added, 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 load, CH.sub.2Cl.sub.2:MeOH 8:1.fwdarw.CH.sub.2Cl.sub.2:MeOH 5:1.fwdarw.CH.sub.2Cl.sub.2:MeOH 4:1.fwdarw.CH.sub.2Cl.sub.2:MeOH 7:3) to yield 228 mg of the deprotected compound 20 (0.94 mmol, 84%) as a colorless solid.
[0166] For cell biological application 34.5 mg of the compound were further purified by reversed phase HPLC (0% to 3% MeCN in H.sub.2O in 45 min, flow 5 mL/min) to yield 25.3 mg of HPLC-purified 20 (73%).
[0167] .sup.1H-NMR (400 MHz, D.sub.2O): δ/ppm=8.32 (s, 1H, 6-H), 4.57-4.33 (m, 2H, 1′-H, 2′-H), 4.04 (dd, J=5.3, 4.4 Hz, 1H, 3′-H), 3.76-3.57 (m, 2H, 5′-H), 2.29 (dt, J=13.0, 8.2 Hz, 1H, 6′-H), 2.23-2.15 (m, 1H, 4′-H), 1.76-1.59 (m, 1H, 6′-H).
[0168] .sup.13C-NMR (101 MHz, D.sub.2O): 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).
[0169] HRMS (ESI.sup.+): calculated for C.sub.9H.sub.15N.sub.4O.sub.4.sup.+ [M+H].sup.+ 243.1088; found: 243.1086.
[0170] HRMS (ESI.sup.−): calculated for C.sub.9H.sub.13N.sub.4O.sub.4.sup.− [M−H].sup.− 241.0942; found: 241.0941.
[0171] R.sub.f (CH.sub.2Cl.sub.2:MeOH 7:3): 0.29.
[0172] In an HPLC stability test it was shown that the carbacyclic compound cAzaC 20 is much more stable than azacitidine (decitabine) (
Example 6
Synthetic Procedures for a cAzaC Prodrug 24
[0173] For an improved delivery into cells we also prepared a 5′-phosphoroamidate prodrug 24 of cAzaC 20.
4-Amino-1-((3aS,4R,6R,6aR)-6-(hydroxymethyl)-2,2-dimethyltetrahydro-4H -cyclopenta-[d][1,3]dioxo1-4-yl)-1,3,5-triazin-2(1H)-one 21
[0174] ##STR00024##
[0175] In a heat-dried flask 5-aza-carbaC 20 (64 mg, 264 μmol, 1.0 eq.) was suspended in 6.60 mL 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 18 h at room temperature. To the resulting solution 150 μL NEt.sub.3 (1.08 mmol) were added and the mixture was stirred another 10 min at room temperature. Subsequently, the mixture was concentrated to dryness and the obtained residue was purified by column chromatography on silica gel (CH.sub.2Cl.sub.2:MeOH 10:1, containing 1% NEt.sub.3) to yield 81 mg of the isopropylidene -acetal protected compound 21 (182 μmol, 69%, adduct with 1.6 eq. NEt.sub.3) as a colorless solid.
[0176] .sup.1H-NMR (400 MHz, D.sub.3COD): δ/ppm=8.29 (s, 1H, 6-H), 4.94 (dd, J=7.0, 4.9 Hz, 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=7.3 Hz, 10H, N(CH.sub.2CH.sub.3).sub.3), 2.32-2.07 (m, 3H, 4′-H, 6′-H), 1.50 (s, 3H, 1″-H), 1.32 (t, J=7.3 Hz, 15H, N(CH.sub.2CH.sub.3).sub.3), 1.29 (s, 3H, 1″-H).
[0177] .sup.13C-NMR (101 MHz, D.sub.3COD): 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), 66.26 (1′-C), 64.17 (5′-C), 47.88 (N(CH.sub.2CH.sub.3).sub.3), 47.81 (4′-C), 33.72 (6′-C), 27.97 (1″-C), 25.53 (1″-C), 9.23 (N(CH.sub.2CH.sub.3).sub.3).
[0178] HRMS (ESI.sup.+): calculated for C.sub.12H.sub.19N.sub.4O.sub.4.sup.+ 283.1401 [M+H].sup.+; found: 283.1397.
[0179] IR (ATR): v (cm.sup.−1)=3346 (br w(, 2983 (w), 2658 (w), 1634 (s), 1472 (s), 1210 (m), 1160 (m), 1065 (m), 868 (w), 800 (m), 682 (w).
[0180] R.sub.f (CH.sub.2Cl.sub.2:MeOH 6:1): 0.36.
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-alaninate 22
[0181] ##STR00025##
[0182] Protected 5-aza-carbaC 21 (69 mg, 155 μmol, 1.0 eq., as a 1.6 eq. NEt.sub.3-adduct) was suspended in 2.40 mL dry THF and cooled to −78° C. Then 855 μL tBuMgCl-solution (1.0 M in THF, 855 μmol, 5.5 eq.) were added, the mixture was directly warmed to 0° C. and stirred for 30 min at this temperature. Subsequently, the mixture was again cooled to −78° C. and 538 μL of a solution of phosphoroxyaryl to chloridate [31] 23 (1.0 M in THF, 538 μmol, 3.5 eq.) were added dropwise. The reaction mixture was allowed to warm to room temperature slowly and stirred for 17 h. The resulting clear yellow solution was quenched by addition of H.sub.2O (20 mL) and the aqueous layer was extracted with EtOAc (3×25 mL). Combined organic phases were washed with saturated aq. NaCl (25 mL), dried over anhydrous Na.sub.2SO.sub.4 and concentrated to dryness in vacuo. The residue was purified by column chromatography on silica gel (CH.sub.2Cl.sub.2:MeOH 60:1.fwdarw.CH.sub.2Cl.sub.2:MeOH 50:1.fwdarw.CH.sub.2Cl.sub.2:MeOH 40:1) to yield 83 mg of product 12 (78 μmol, 51%, adduct with 5.0 eq. NEt.sub.3) as a mixture of both diastereoisomers on P as a colourless solid.
[0183] .sup.1H-NMR (800 MHz, CDCl.sub.3): 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(CH.sub.2CH.sub.3).sub.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(CH.sub.2CH.sub.3).sub.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).
[0184] .sup.13.sub.C-NMR (201 MHz, CDCl.sub.3): δ/ppm=173.10 (1″′-C), 165.39 (4-C), 158.57 (6-C), 153.13 (O—C.sub.Ar), 150.83 (2-C), 129.73 (CH.sub.Ar), 124.99 (CH.sub.Ar), 120.45 (CH.sub.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(CH.sub.2CH.sub.3).sub.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(CH.sub.2CH.sub.3).sub.3).
[0185] .sup.31P-NMR (162 MHz, CDCl.sub.3): δ/ppm=2.46, 2.36.
[0186] HRMS (ESI.sup.+): calculated for C.sub.24H.sub.35N.sub.5O.sub.8P.sup.+ 552.2218 [M+H].sup.+; found: 552.2211.
[0187] HRMS (ESI.sup.−): calculated for C.sub.24H.sub.33N.sub.5O.sub.8P.sup.+ 550.2072 [M−H].sup.−; found: 550.2074.
[0188] IR (ATR): v (cm.sup.−1)=3379 (m), 2980 (s), 2946 (m), 2606 (s), 2498 (s), 1640 (s), 1476 (s), 1398 (m), 1383 (m), 1212 (s), 1158 (m), 1107 (w), 1070 (m), 1037 (s), 929 (m), 802 (w), 730 (m).
[0189] R.sub.f (CH.sub.2Cl.sub.2:MeOH 20:1): 0.25.
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-alaninate 24
[0190] ##STR00026##
[0191] To isopropylidene acetal protected compound 22 (38 mg, 69 μmol, 1.0 eq.) 1.0 mL 80% TFA in ddH.sub.2O was added and the mixture was stirred 20 min at 30° C. Then, the mixture was poured into 5.0 mL ddH.sub.2O and lyophilized. The resulting residue was dissolved in a mixture of 500 μL MeCN, 500 μL DMSO and 4.0 mL ddH.sub.2O containing 0.1% TFA, the turbid solution was filtered and purified by reversed-phase HPLC (5% MeCN in ddH.sub.2O cont. 0.1% TFA to 60% MeCN in ddH.sub.2O cont. 0.1% TFA in 45 min, flow 5 mL/min) to yield 10.4 mg of the target compound 24 (20 μmol, 29%) as a colorless solid.
[0192] .sup.1H-NMR (400 MHz, D.sub.3CCN): δ/ppm=8.11 (s, 1H), 7.50 (s, 1H, NH.sub.2), 7.41-7.29 (m, 2H, OPh), 7.25-7.15 (m, 3H, OPh), 6.61 (s, 1H, NH.sub.2), 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, 3.8 Hz, 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, 3.4 Hz, 6H, 5″-H).
[0193] .sup.13C-NMR (101 MHz, D.sub.3CCN): δ/ppm=174.00 (1″-C), 165.92 (4-C), 159.92 (6-C), 153.84 (2-C), 151.97 (O—C.sub.Ar), 130.62 (CH.sub.Ar), 125.75 (CH.sub.Ar), 121.36 (CH.sub.Ar), 121.31 (CH.sub.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).
[0194] .sup.31P-NMR (162 MHz, D.sub.3CCN): δ/ppm=3.08, 2.92.
[0195] HRMS (ESI.sup.+): calculated for C.sub.21H.sub.31N.sub.5O.sub.8P.sup.+ 512.1905 [M+H].sup.+; found: 512.1901.
[0196] HRMS (ESI.sup.−): calculated for C.sub.21H.sub.29N.sub.5O.sub.8P.sup.− 510.1759 [M−H].sup.−; found: 510.1757.
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