Patent Application
20220332737
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.
5-Aza-2′-deoxycytidine (decitabine, AzadC) and 5-aza-cytidine (vidaza, AzaC) are nucleoside analogues that act as an antimetabolite by manipulating epigenetic information [1-5]. Epigenetic information in DNA is associated with the formation of 5-methyl-2′-deoxycytidine (mdC) from 2′-deoxycytidine (dC) with the help of DNA methyltransferases (DNMTs) and S-adenosylmethionine (SAM) as the methylating 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 inside cells converted into the corresponding active triphosphate and subsequently incorporated into the genome during cell division. AzaC is a prodrug as well, that is 2′-deoxygenated and subsequently converted into the corresponding 5′-triphosphate for incorporation into DNA. AzaC is also converted into the 5′-triphosphate without prior 2′-deoxygenation and incorporated into RNA. The mode of action of Aza(d)C involves reaction of its electrophilic C6 positions with a DNMT active site thiol nucleophile as shown in
As a result of administering Aza(d)C, a large drop of mdC levels, i.e. a hypomethylating effect is observed, which leads to the reactivation of silenced tumor suppressor genes in cancer cells [1], which allows re-differentiating cancer cells back into normally proliferating cells or gives rise to cell death. Aza(d)C is currently in use as a medicament for the treatment of myelodysplastic syndromes (MDS) [2] and acute myeloid leukemia (AML) [4]. Clinically, it is administered in several cycles, with each cycle involving one week of treatment and three weeks of pausing.
A problem associated with Aza(d)C is that it hydrolyses in aqueous solution following the path depicted in
The present inventors have overcome this problem by providing analogues of Aza(d)C that can demethylate (and hence react with an S-nucleophile), while hydrolysis (reaction with an O-nucleophile) is blocked. Surprisingly, they have found that replacing the oxygen of the deoxyribose by a CH2-group has a large remote effect on the reactivity of the heterocycle. These analogues, particularly a carbocyclic version of Aza(d)C still inhibit methylation by DNA methyltransferases but are hydrolytically stable as shown in
A first aspect of the present invention relates to a compound of Formula I:
wherein R1 is H, a free or protected, modified or unmodified phosphate group, or a hydroxyl-protecting group,
In the compounds of Formula I, R1 may be selected from H, a free or protected, modified or unmodified phosphate group, e.g. a monophosphate, diphosphate or triphosphate group, or a hydroxyl-protecting group.
The term “phosphate group” includes an unmodified phosphate group, e.g. an unmodified monophosphate, unmodified diphosphate or unmodified triphosphate group or a modified phosphate group, e.g. a modified monophosphate, modified diphosphate or modified triphosphate group, wherein 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, e.g. phosphate esters including monoesters and diesters, and amidates including monoamidates, diamidates and amidate esters. Detailed reviews of protected phosphate and phosphonate groups are found in [28] and [29], the contents of which are herein incorporated by reference. Thus, in pharmaceutical applications, R1 may be a group that is a protected phosphate, diphosphate or triphosphate group to liberate a phosphate, diphosphate or triphosphate group upon cleavage in the cell (prodrug concept).
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 beta-methoxyethoxym ethyl.
R2 may be selected from H, an unmodified or modified, free or protected phosphate group as described above, e.g. a monophosphate, diphosphate or triphosphate group, or a hydroxyl-protecting group as described above. Thus, in pharmaceutical applications, R2 may be a group that is a protected phosphate diphosphate or triphosphate group to liberate a phosphate, diphosphate or triphosphate group upon cleavage in the cell (prodrug concept).
In certain embodiments, R3 may be H. In such a case, the 5-membered carbocyclic ring in Formula I can be considered as a deoxyribose wherein the oxygen atom at positon 1′ of the ring has been replaced by a CH2-group. In further embodiments, R3 may be OH or OR6, wherein R6 is a hydroxyl-protecting group as described above. In these embodiments, the 5-membered carbocyclic ring in Formula I may be considered as a ribose wherein the oxygen atom at positon 1′ of the ring has been replaced by a CH2-group. R3 may also be CH3, CH2F, CHF2, CF3 or F In these embodiments, the 5-membered carbocyclic ring in Formula I may be considered as a ribose analogue wherein the oxygen atom at positon 1′ of the ring has been replaced by a CH2-group.
R4 and R5 each may be H, thus providing a primary amino group NH2 or they may form an amino-protecting group, e.g. wherein one or both H-atoms are replaced by the 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 groups, carbobenzyloxy groups, tosyl groups, 9-fluorenyl methyloxycarbonyl groups or t-butyloxycarbonyl groups.
In certain embodiments, R1 is H.
In certain embodiments, R2 is H or an unmodified or modified phosphate group, e.g. a monophosphate, diphosphate or triphosphate group including a free or protected monophosphate, diphosphate or triphosphate group.
In certain embodiments, R3 is H or OH.
In certain particular embodiments R1, R2, R3, R4 and R5 each are H, or R1, R2, R4, and R5 each are H and R3 is OH or OR6. In an especially particular embodiment, R1, R2, R3, R4 and R5 are H. This compound is a carbocyclic analogue of AzadC (decitabine) which—as indicated above—has a surprisingly high hydrolytic stability while maintaining inhibitory activity towards methyltransferases, particularly DNA methyltransferases. In a further especially particular embodiment, R1, R2, R4 and R5 are H and R3 is OH. This compound is a carbocyclic analogue of AzaC (vidaza).
In further particular embodiments, the invention also relates to prodrugs of AzadC and AzaC, particularly to compounds, where R1 and/or R2 are different from H.
The present invention also encompasses salts of the compounds of Formula I, in particular pharmaceutically acceptable salts. The compounds of the present invention are capable of forming acid and/or base salts by means of the presence of amino and/or phosphate groups or groups similar thereto. Acid addition salts may be formed with inorganic acids and organic acids and include, but are not limited to, acetate, benzoate, hydrobromide, hydrochloride, citrate, lactate, phosphate, tosylate and trifluoroacetate salts. Base addition salts can be formed within inorganic bases and organic bases and include, but are not limited to, ammonium, sodium, potassium, calcium, magnesium salts or salts derived from primary, secondary and tertiary amines.
The compounds of the present invention act as antimetabolites by reducing the level of the epigenetic modification 5-methyl-2′-deoxycitidine (mdC). They may be incorporated into the genome and/or the RNA of proliferating cells and inhibit DNA- and/or RNA-methyltransferases leading to a 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, which elicits an anti-hyperproliferative effect.
Thus, a further aspect of the further invention relates to a compound of Formula I as described above or a pharmaceutically acceptable salt thereof for the use in medicine, e.g. in veterinary or human medicine.
In certain embodiments, the compound of the present invention is used for the treatment of a hyperproliferative disorder including malignant or non-malignant hyperproliferative disorders. The term “hyperproliferative disorder” relates to disorders caused by, associated with and/or accompanied by a dysfunctionally increased proliferation of cells, tissues and/or organs within an organism.
Thus, the invention also relates to a method of treating a hyperproliferative disorder, comprising administering to a subject in need thereof, particularly to a human subject, a therapeutically effective dose of a compound of Formula I or a pharmaceutically acceptable salt thereof.
In particular 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, e.g. acute myeloid leukemia (AML) or a myelodysplastic syndrome (MDS) including previously treated or untreated, primary or in secondary MDS such as refractory anemia, refractory anemia with ringed sideroblasts, refractory anemia with excess blasts, refractory anemia with excess blasts in transformation and chronic myelomonocytic leukemia as well as intermediate-1, intermediate-2 and high risk MDS.
In further embodiments, the compounds of the invention may be used for the treatment of renal insufficiency based on results reported for decitabine [26]. In certain embodiments, the compounds of the present invention may be used for the treatment of atherosclerosis based on the observation that decitabine prevents atherosclerosis lesion formation and reduces the production of inflammatory cytokines by macrophages [27].
A compound of Formula I or a pharmaceutically acceptable salt thereof may be administered to a subject in need thereof as a pharmaceutically composition comprising the active agent and a pharmacologically acceptable carrier. The composition may be administered in any suitable way, e.g. orally, parenterally, by inhalation and/or by dermal application. The carrier may be any suitable pharmaceutical carrier. The compound of Formula I is administered in a therapeutically effective dose which may be determined by the skilled practitioner based on the subject and the disorder to be treated.
In pharmaceutical applications, the composition may be in the form of a solid dosage form, e.g. a tablet, a capsule etc. or a liquid dosage form, e.g. a solution, emulsion, suspension or the like.
A compound of Formula I may be administered in one or several treatment cycles, wherein each treatment cycle may involve at least 1, 2, 3, 4, 5, 6, 7 or 10 days.
A compound of Formula I may be administered as a monotherapy or in is combination with a further medicament, particularly a medicament suitable for the treatment of hyperproliferative disorders as described above. In certain embodiments a compound of Formula I may be administered as a combination therapy together with an anti-cancer drug different from Formula I.
Further, the compounds of Formula I are also useful for non-medical applications, e.g. generally in the fields of basic research, diagnostics and/or drug screening. Thus, in certain embodiments, the invention relates to an in vitro use of a compound of Formula I for inhibiting the methylation of nucleic acids including DNA and RNA, particularly for inhibiting the methylation of DNA, more particularly for inhibiting the methylation of genomic DNA. Thus, the compounds are useful for a genome analysis in cells, e.g. eukaryotic cells such as animal cells including mammalian and human cells, particularly for an epigenetic analysis.
A general scheme for the preparation of the compounds of Formula I is depicted in the following scheme. The synthesis starts with a t-butyloxycarbonyl (Boc)-protected aminocyclopentane derivative 2, which has previously been used to synthesize DNA lesion analogues [12-15]. Compound 2 was first benzyl-protected to 3, Boc-deprotected to 4, and then reacted with carbimidazole 5, which was prepared in two steps from isomethylurea 6 after generation of the free base 7 with potassium hydroxide and reaction of 7 with carbonyldiimidazole. This provides the carbamoylurea-cyclopentane nucleoside analogue 8. Cyclization to the triazine base 9 was subsequently performed with triethylorthoformate. Reaction of 9 with NH3 in methanol and deprotection of the benzyl groups with BCl3 in dichloromethane furnished the final compound cAzadC 1 as the free nucleoside.
Recrystallization of compound cAzadC 1 from hot methanol gave colourless needles, which allowed us to solve the crystal structure that is depicted in
Another general scheme for the preparation of the compounds of Formula I is depicted in the following showing the synthesis of the carbacyclic version of azacitidine (vidaza) cAzaC, 20:
A general scheme for the preparation of prodrugs of the compounds of Formula I is depicted in the following showing the synthesis of a 5′ phoshoroamidate prodrug 24 of cAzaC, 20:
Further, the present invention shall be explained in more detailed by the following Figures and Examples.
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].
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 CH2Cl2 (30 mL) and the reaction was quenched with sat. is NaHCO3 (30 mL). The mixture was extracted with CH2Cl2 (500 mL). The organic phase was washed three times with sat. NaHCO3 (1×400 mL, 2×100 mL) and dried over Na2SO4. The solvent was removed in vacuo. The crude product was purified by column chromatography on silica gel with a stepwise gradient iHex/EtOAc (9:1→7:1→4:1) to afford product 3 as a colorless solid (3.39 g, 8.24 mmol, 61%).
Rf=0.67 (iHex/EtOAc=2:1)
Mp=74-76° C.
HRMS [ESI+]: [C25H33NO4Na]+, [M+Na+]+ calculated.: 434.2292, found: 434.2299
1H-NMR (599 MHz, CDCl3): 5/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—Ha, C4—H), 2.11-2.05 (m, 1H, C2—Ha), 1.77-1.68 (m, 1H, C2—Hb), 1.43 (s, 9H, C2′—H), 1.29-1.20 (m, 1 H7 C6—Hb).
13C-NMR (151 MHz, CDCl3): δ/ppm=155.5 (C1′), 138.60, 138.23, 128.36, 128.28, 127.59, 127.56, 127.42 (C—Ar), 81.0 (C3), 79.2 (C2′), 73.3 (C8), 71.9 (C5), 71.3 (C7), 50.4 (C1), 44.9 (C4), 39.6 (C2), 35.0 (C6), 28.6 (C2′).
FTIR (ATR):={tilde over (ν)}/cm −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).
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 CH2Cl2 (28 mL) was stirred for 1 h at rt. After removing the solvent in vacuo, the residue was resuspended in sat. Na2CO3 (100 mL) and stirred for 10 min. The mixture was extracted with EtOAc (3×150 mL) and the combined organic phases were dried over Na2SO4. 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 O2.
Analytical data of purified 4:
HRMS [ESI+]: [C23H29N4O4Na]+, [M+Na+]+ calculated: 312.1958, found: 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—Ha), 2.13-1.96 (m, 3H, NH2, C2—Ha), 1.65-1.50 (m, 1H, C2—Hb), 1.19-1.08 (m, 1H, C6—Ha),
13C-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 (Cs), 51.40 (C1), 45.63 (C4), 42.07 (C2), 38.08 (C6).
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 filtrated, and the residue was washed with ice-cold Et2O (3×50 mL). The combined organic phases were concentrated to 20 mL in vacuo. Cooling to −20° C. resulted in precipitation. Vacuum filtration under N2 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%).
1H-NMR (400 MHz, CDCl3): 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, C2—H).
13C-NMR (101 MHz, CDCl3): δ/ppm=165.7 (C1), 157.3 (C3), 137.5(Ar), 130.0 (Ar), 117.2 (Ar), 55.3 (C2).
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→1:1) to obtain the methylisobiurete 8 as a yellow oil (2.613 g, 6.35 mmol, 78%).
Rf=0.51 (DCM/MeOH=19:1)
HRMS [ESI+]: [C23H29N4O4Na]+, [M+Na+]+ calculated: 434.2050, found: 434.2049
1H-NMR (599 MHz, CDCl3): δ/ppm=7.35-7.16 (m, 10H, Ar—H), 5.42 (d,3J1′,N1 =7.8 Hz, 1H, N1—H), 4.50-4.34 (m, 4H, C7′—Ha,b, C8′—Ha,b), 4.33-4.16 (m, 1H, C1′—H), 3.93-3.81 (m, 1H, C3—H), 3.62 (s, 3H, C6—H), 3.43-3.31 (m, 2H,C5′—H), 2.34-2.18 (m, 2H, C6′—Ha, C4′—H), 2.06 (ddd, 2J2′a,2′b=13.0 Hz, 3J2′a,1′/3′=6.9 Hz, 3J2′a,1′/3′=4.6 Hz, 1H, C2′—Ha), 1.73 (ddd, 2J2′a,2b′=13.0 Hz, 3J2′b,1′/3′=7.0 Hz, 3J2′b,1′/4′=7.0 Hz, 1H, C2—Hb), 1.28-1.11 (m, 1H,C6′—Hb)
13C-NMR (151 MHz, CDCl3): δ/ppm=163.8 (C2), 162.3 (C4), 138.9 (CAr), 138.5 (CAr), 128.51 (CAr), 128.41 (CAr), 127.76 (CAr), 127.74 (CAr), 127.70 (CAr), 127.56 (CAr), 81.1 (C3′), 73.3 (C7′), 71.9 (C5′), 71.2 (C8′), 53.8 (C6), 49.5 (C1′), 45.0 (C4′), 39.5 (C2′), 35.0 (C6′).
Trifluoroacetic acid (75 μL) was added to a solution of 8 (2.613 g, 6.35 mmol, 1.0 eq) in triethyl orthoformate (60 mL). The mixture was refluxed for 3 h. The solvent was removed in vacuo. The residue was co-evaporated once with MeOH and the crude product was purified by column chromatography on silica gel with a stepwise gradient of iHex/EtOAc (2:1→1:1→1:2) to obtain 9a (1.774 g, 4.07 mmol, 64%, colorless solid) and 9b (268.0 mg, 0,64 mmol, 10%, colorless oil).
9a:
Rf=0.71 (iHex/EtOAc, 1:3)
HRMS [ESI+]: [C25H30N3O4H]+, [M+H+]+ calculated: 436.2232, found.: 436.2231.
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, C1′—H), 4.56-4.39 (m, 6H, C7′—Ha,b, C8′—Ha,b, C7—Ha,b), 4.07-3.08 (m, 1H, C3′—H), 3.61-3.46 (m, 2H, C5′—Ha,b), 2.52-2.36 (m, 2H, C6′—Ha, C4—H), 2.28 (ddd, 1H, 2J2′a,2′b=13.3 Hz, 3J=7.6 Hz, 3J=2.7 Hz, C2′—Ha), 2.11 (ddd, 1H, 2J2′a,2′b=13.3 Hz, 3J=10.0 Hz, 3J=6.3 Hz, C2′—Hb), 1.84-1.68 (m, 1H, C6′—Hb), 1.40 (t, 3H, 3J8, 7=7.1 Hz, C8—H),
13C-NMR (101 MHz, CDCl3): δ/ppm=169.3 (C4), 158.4 (C6), 155.0 (C2), 138.2 (CAr), 138.1 (CAr), 128.6 (CAr), 128.6 (CAr), 127.93 (CAr), 127.85 (CAr), 127.82 (CAr), 80.4 (C3′), 73.4 (C7′/8′), 71.26 (C7′/8′), 71.25 (C5′), 65.1 (C7), 56.8 (C1′), 44.9 (C4′), 36.9 (C2′), 32.6 (C6′), 14.2 (C8).
9b:
Rf=0.63 (iHex/EtOAc, 1:3)
HRMS [ESI+]: [C24H28N3O4]+, [M+H+]+ calculated: 422.2074 , found: 422.2075.
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, C1′—H), 4.57-4.43 (m, 6H, C7′—Ha,b, C8′—Ha,b), 4.09-3.98 (m, 4H, C3′—H, C7—H), 3.60-3.50 (m, 2H, C5′—Ha, b), 2.50-2.39 (m, 2H, C6′—Ha, C4′—H), 2.28 (ddd, 1H, 2J2′a,2′b=13.1 Hz, 3J=7.4 Hz, 3J=2.7 Hz, C2′—Ha), 2.11 (ddd, 1H, 2J2′a,2′b=13.1 Hz, 3J=10.1 Hz, 3J=6.3 Hz, C2′—Hb), 1.80-1.68 (m, 1H, C′6—Hb).
13C-NMR (101 MHz, CDCl3): δ/ppm=169.9 (C4), 158.5 (C6), 155.0 (C2), 138.3 (CAr), 138.0 (CAr), 128.63 (CAr), 128.57 (CAr), 127.93 (CAr), 127.84 (CAr), 127.82 (CAr), 80.4 (C3′), 73.5 (C7′/8′), 71.3 (C7′/8′), 56.9 (C1′), 56.0 (C7), 44.9 (C4′), 36.9 (C2′), 32.6 (C6′).
Ethoxytriazine 9a (1.630 g, 3.74 mmol, 1.0 eq.) was dissolved in methanolic NH3 (7 N in MeOH, 60 mL) and stirred for 3 h at rt. The mixture was diluted with H2O (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.
Rf=0.50 (CH2Cl2/MeOH, 9:1)
HRMS [ESI+]: [C23H27N4O3]+, [M+H+] calculated: 407.2078, found: 407.2081.
1H-NMR (599 MHz, CDCl3): δ/ppm=8.01 (s, H, C6—H), 7.35-7.23 (m, 10H, Ar—H), 5.78 (5, 2H, NH), 4.92-4.76 (m, 4H, C7′—H and C8′—H), 4.59-4.46 (m, 1H, C1′—H), 4.07-3.95 (m, 1H, C3′—H), 3.55-3.45 (m, 2H, C5′—H), 2.44-2.28 (m, 2H, C6′—Ha, C4′—H), 2.24-2.16 (m, 1H, C2′—Ha), 2.14-2.05 (m, 1H, C2′—Hb), 1.78-1.67 (m, 1H, C6′—Hb)
13C-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 (C3′), 73.4 (C8′), 71.4 (C5′), 71.2 (C8′), 56.7 (C1′), 44.9 (C4′), 36.8 (C2′), 32.5 (C6′).
A solution of benzyl-protected 10 (1.478 g, 3.63 mmol, 1.0 eq) in CH2Cl2 (100 mL) was cooled to −78° C. and BCl3 (1 M in DCM, 12.7 mL, 12.7 mmol, 3.5 eq.) was added dropwise. The mixture was stirred 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 CH2Cl2/MeOH (9:1→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.
Rf=0.1 (CH2Cl2/MeOH, 9:1)
Mp.: 198-200° C.
HRMS [ESI+]: [C9H15N4O3]+, [M+H+]+ calculated: 227,1138, found: 227,1139.
1H-NMR (400 MHz, D2O): δ/ppm=8.29 (s, 1H, C6—H), 4.90-4.62 (m, 1H, C1′—H, Overlap with D2O signal), 4.29-4.16 (m, 1H, C3′—H), 3.73 (dd, 5J5′a,5′b=11.2 Hz, 3J5′a,4′=5.7 Hz, 1H, C5′a—H ), 3.62 (dd, 5J5′a,5′b=11.2 Hz, 3J5′b,4′=6.8 Hz, 1H, C5′b—H), 2.41-2.29 (m, 1H, C6′a—H), 2.28-2.18 (m, 1H, C2′a—H), 2.17-2.02 (m, 2H, C2′, b—H, C4′b—H), 1.77-1.55 (m, 1H, C6′b—H).
13C-NMR (101 MHz, CDCl3): δ/ppm=165.5 (C4), 158.5(C6), 156.4(C2), 72.2 (C3′), 62.8 (C5′), 56.2 (C1′), 48.11 (C4′), 38.0 (C2′), 32.0 (C6′.).
FTIR (ATR): {tilde over (ν)}/cm −=3194 (m), 1653 (m), 1540 (s), 1437 (s), 1278 (s), 1036 (s).
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 (
Further figures were generated with the program Mercury 3.5.1. (Cambridge Crystallographic Data Center). The data are deposited under CCDC 1910952.
We next investigated the hydrolytic stability of cAzadC 1 in direct comparison to the pharmaceutical AzadC.
Both nucleosides were dissolved in aqueous KH2PO4 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→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%.
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.
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
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→CH2 exchange causes a strong remote disarming effect that seems to change the properties of the triazine ring so that reaction with water is stopped.
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, 2×105 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+ buffer as described in a previous publication [18].
The gDNA was isolated as described previously [18]. The gDNA was directly subjected to a total enzymatic digest and analyzed using UHPLC-MS2 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.
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].
The levels of mdC were finally precisely quantified using isotope dilution UHPLC-MS2. 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.
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 NaBH4. Application of NaBH4 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-MS2 using an external calibration curve was possible in parallel to quantification of canonical and epigenetic bases.
At 1 μM cAzadC 1 concentration, we detected a cAzadC 1 level of 5×10−4 cAzadC per dN (
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% (
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.
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.
We show that replacement of the in-ring O-atom by a CH2-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.
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. Subsequently, 200 mL water and 35.1 g Na2SO3 (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 Na2SO4. After evaporation of the solvents in vacuo the residue was purified by silica gel column chromatography (CH2Cl2:MeOH 40:1→CH2Cl2:MeOH 30:1) to yield 2.91 g of the exo-bishydroxylated product 12 (12.0 mmol, 22%) as a yellow foam.
1H-NMR (400 MHz, CDCl3): 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).
13C-NMR (101 MHz, 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): v (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).
Rf (CH2Cl2:MeOH 20:1): 0.24.
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. 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, CH2Cl2:MeOH 5:1→CH2Cl2: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.
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).
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).
Rf (CH2Cl2:MeOH 8:1): 0.43.
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. NaHCO3 and extracted with CH2Cl2 (3×300 mL). Combined organic layers were washed with saturated aq. NaHCO3 (300 mL), saturated aq. 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 yield the tribenzyl-protected product 14 (2.47 g, 4.8 mmol, 51%) a colorless solid wax.
1H-NMR (400 MHz, CDCl3): δ/ppm=7.45-7.09 (m, 15H, 3×OCH2Ph), 4.95 (s, 1H, NH), 4.76-4.25 (m, 6H, 3×OCH2Ph), 4.15-3.90 (m, 1H, 1-H), 3.82 (dd, J=6.7, 4.4 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).
13C-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 for C32H40NO5+ [M+H]+ 518.2901; found: 518.2911.
IR (ATR): v (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).
Rf (iHex:EtOAc 4:1): 0.69.
Under Ar atmosphere 1.02 g of Boc-protected cyclopentane 14 (1.97 mmol, 1.0 eq.) were dissolved in 7.0 mL dry CH2Cl2, 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 NaHCO3 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 Na2SO4 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.
1H-NMR (400 MHz, CDCl3): δ/ppm=7.39-7.26 (m, 15H, 3×OCH2Ph), 4.63-4.33 (m, 6H, 3×OCH2Ph), 3.81 (dd, J=5.0, 3.1 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).
13C-NMR (101 MHz, CDCl3): 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 (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+): calculated for C27H32NO3+ [M+H]+ 418.2377; found: 418.2378.
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 [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→iHex:EtOAc 5:2→iHex:EtOAc 2:1→iHex:EtOAc 1:1) to yield methylisobiuret 16 as a colorless oil (886 mg, 1.17 mmol, 87%).
1H-NMR (400 MHz, CDCl3): δ/ppm=7.43-7.22 (m, 15H, 3×OCH2Ph), 5.60 (s, 1H, 1-H), 4.80-4.61 (m, 2H, OCH2Ph), 4.55-4.41 (m, 3H, OCH2Ph), 4.32 (d, J=11.8 Hz, 1H, OCH2Ph), 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).
13C-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 C30H36N3O5+ [M+H]+ 518.2650; found: 518.2652.
IR (ATR): v (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).
Rf (iHex:EtOAc 2:1): 0.37.
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→iHex:EtOAc 5:2→iHex:EtOAc 2:1→iHex:EtOAc 1:1→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.
Methoxytriazine 17:
1H-NMR (400 MHz, CDCl3): δ/ppm=7.96 (s, 1H, 6-H), 7.38-7.17 (m, 15H, 3×OCH2Ph), 4.70-4.40 (m, 6H, 1′-H, 2.5×OCH2Ph), 4.36-4.24 (m, 2H, 2′-H, 0.5×OCH2Ph), 3.98 (s, 3H, 7-H), 3.93 (dd, J=4.9, 3.1 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).
13C-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 C31H34N3O5+ [M+H]+ 528.2493; found: 528.2498.
IR (ATR): v (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).
Rf (iHex:EtOAc 2:1): 0.27.
Ethoxytriazine 18:
1H-NMR (400 MHz, CDCl3): δ/ppm=7.97 (s, 1H, 6-H), 7.39-7.15 (m, 15H, 3×OCH2Ph), 4.66-4.21 (m, 10H, 1′-H, 2′-H, 7-H, 3×OCH2Ph), 3.96-3.87 (m, 1H, 3′-H), 3.54-3.36 (m, 2H, 5′-H), 2.56-2.41 (m, 1H, 4′-H), 2.33 (ddd, J=14.5, 9.5, 4.7 Hz, 1H, 6′-H), 1.88-1.74 (m, 1H, 6′-H), 1.25 (q, J=6.9 Hz, 3H, 8-H).
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′-14.24 (8-C).
HRMS (ESI+): calculated for C32H36N3O5+ [M+H]+ 542.2650; found: 542.2656.
IR (ATR): v (cm−1)=3064 (w), 3030 (w), 2924 (w), 2962 (w), 1694 (s), 1615 (s), 1514 (m), 1495 (m), 1452 (s), 1361 (m), 1326 (m), 1270 (m), 1206 (w), 1096 (s), 1070 (s), 1027 (m), 803 (w), 734 8s), 697 (s).
Rf (iHex:EtOAc 2:1): 0.30.
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 NH3 in MeOH and stirred at room temperature for 3 h. Subsequently, 125 mL H2O were added to the reaction mixture, which was then extracted with EtOAc (3×200 mL). Combined organic layers were dried over Na2SO4 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.
1H-NMR (400 MHz, CDCl3): δ/ppm=7.80 (s, 1H, 6-H), 7.26 (m, 15H, 3×OCH2Ph), 5.70 (s, 1H, NH), 4.63-4.42 (m, 6H, 1′-H, 2.5×OCH2Ph), 4.39-4.28 (m, 2H, 2′-H, 0.5×OCH2Ph), 3.91 (dd, J=5.0, 3.5 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)
13C-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)7, 63.53 (1′-C), 41.21 (4′-C), 26.79 (6′-C).
HRMS (ESI+): calculated for C30H33N4O4+ [M+H]+ 513.2496; found: 513.2496.
IR (ATR): v (cm−1)=3331 (br w), 3182 (br w), 3060 (w), 3028 (w), 2925 (w), 2857 (w), 1633 (s), 1506 (m), 1495 8m), 1470 (m), 1452 8s), 1361 (w), 1262 (w), 1092 (s), 1070 (s), 1027 (m), 912 (w), 799 (m), 734 (s), 696 (s).
Rf (CH2Cl2:MeOH 15:1): 0.42.
A solution of benzyl protected 5-aza-carbaC 19 (576 mg, 1.12 mmol, 1.0 eq.) was dissolved in 30.8 mL dry CH2Cl2 under Ar atmosphere and cooled to −78° C. Then, 5.88 mL BCl3 (1 M in CH2Cl2, 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, CH2Cl2:MeOH 8:1→CH2Cl2:MeOH 5:1→CH2Cl2:MeOH 4:1→CH2Cl2:MeOH 7:3) to yield 228 mg of the deprotected compound 20 (0.94 mmol, 84%) as a colorless solid.
For cell biological application 34.5 mg of the compound were further purified by reversed phase HPLC (0% to 3% MeCN in H2O in 45 min, flow 5 mL/min) to yield 25.3 mg of HPLC-purified 20 (73%).
1H-NMR (400 MHz, 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, 4.4 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).
13C-NMR (101 MHz, 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 for C9H15N4O4+ [M+H]+ 243.1088; found: 243.1086.
HRMS (ESI−): calculated for C9H13N4O4− [M−H]− 241.0942; found: 241.0941.
Rf (CH2Cl2:MeOH 7:3): 0.29.
In an HPLC stability test it was shown that the carbacyclic compound cAzaC 20 is much more stable than azacitidine (decitabine) (
For an improved delivery into cells we also prepared a 5′-phosphoroamidate prodrug 24 of cAzaC 20.
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 NEt3 (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 (CH2Cl2:MeOH 10:1, containing 1% NEt3) to yield 81 mg of the isopropylidene -acetal protected compound 21 (182 μmol, 69%, adduct with 1.6 eq. NEt3) as a colorless solid.
1H-NMR (400 MHz, D3COD): 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(CH2CH3)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(CH2CH3)3), 1.29 (s, 3H, 1″-H).
13C-NMR (101 MHz, 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), 66.26 (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+): calculated for C12H19N4O4+ 283.1401 [M+H]+; found: 283.1397.
IR (ATR): v (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).
Rf (CH2Cl2:MeOH 6:1): 0.36.
Protected 5-aza-carbaC 21 (69 mg, 155 μmol, 1.0 eq., as a 1.6 eq. NEt3-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 H2O (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 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 yield 83 mg of product 12 (78 μmol, 51%, adduct with 5.0 eq. NEt3) as a mixture of both diastereoisomers on P as a colourless solid.
1H-NMR (800 MHz, CDCl3): δ/ppm=8.05 (2×s, 1H), 7.32-7.27 (m, 2H, OPh), 7.23-7.17 (m, 2H, OPh), 7.15-7.07 (m, 1H, OPh), 5.04-4.87 (m, 2H, 2′-H, 4′″-H), 4.59-4.54 (m, 1H, 3′-H), 4.31-4.13 (m, 3H, 1′-H, 5′-H), 4.02-3.93 (m, 1H, 2′″-H), 3.78 (m, 1H, NH), 3.09 (q, J=7.4 Hz, 30H, N(CH2CH3)3), 2.53-2.17 (m, 3H, 4′-H, 6′-H), 1.49-1.47 (m, 3H, 1″-H), 1.39 (t, J=7.3 Hz, 45H, N(CH2CH3)3), 1.37-1.34 (m, 3H, 3″-H), 1.26-1.24 (m, 3H, 1″-H), 1.21 -0.18 (m, 6H, 5″′-H).
13
C-NMR (201 MHz, CDCl3): δ/ppm=173.10 (1″′-C), 165.39 (4-C), 158.57 (6-C), 153.13 (O—CAr), 150.83 (2-C), 129.73 (CHAr), 124.99 (CHAr), 120.45 (CHAr), 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).
31P-NMR (162 MHz, CDCl3): δ/ppm=2.46, 2.36.
HRMS (ESI+): calculated for C24H35N5O8P+ 552.2218 [M+H]+; found: 552.2211.
HRMS (ESI−): calculated for C24H33N5O8P+ 550.2072 [M−H]−; found: 550.2074.
IR (ATR): v (cm−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).
Rf (CH2Cl2:MeOH 20:1): 0.25.
To isopropylidene acetal protected compound 22 (38 mg, 69 μmol, 1.0 eq.) 1.0 mL 80% TFA in ddH2O was added and the mixture was stirred 20 min at 30° C. Then, the mixture was poured into 5.0 mL ddH2O and lyophilized. The resulting residue was dissolved in a mixture of 500 μL MeCN, 500 μL DMSO and 4.0 mL ddH2O containing 0.1% TFA, the turbid solution was filtered and purified by reversed-phase HPLC (5% MeCN in ddH2O cont. 0.1% TFA to 60% MeCN in ddH2O 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.
1H-NMR (400 MHz, 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, 3.8 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).
13C-NMR (101 MHz, D3CCN): δ/ppm=174.00 (1″-C), 165.92 (4-C), 159.92 (6-C), 153.84 (2-C), 151.97 (O—CAr), 130.62 (CHAr), 125.75 (CHAr), 121.36 (CHAr), 121.31 (CHAr), 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).
31P-NMR (162 MHz, D3CCN): δ/ppm=3.08, 2.92.
HRMS (ESI+): calculated for C21H31N5O8P+ 512.1905 [M+H]+; found: 512.1901.
HRMS (ESI−): calculated for C21H29N5O8P− 510.1759 [M−H]−; found: 510.1757.
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| Number | Date | Country | Kind |
|---|---|---|---|
| 19178928.8 | Jun 2019 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2020/065621 | 6/5/2019 | WO |
