Patent Application
20250163095
The present disclosure relates to bis-protected, activated guanine monomers, methods of their synthesis and their use in the production of antisense oligonucleotides.
Antisense oligonucleotides (ASO) are used in the modulation of gene expression in a sequence-specific manner. They have been developed for target validation and therapeutic purposes. Antisense technology has the potential to cure diseases caused by the expression of harmful genes, including diseases caused by viral infections, cancer growth, neuronal degradation (i.e., Alzheimer's) and inflammatory diseases. Optimized antisense oligonucleotides (ASOs) can be used to target primary gene transcripts, mRNA product(s), spliced and unspliced coding and noncoding RNAs.
ASOs modulate RNA function by two broad mechanisms. A steric blocking mechanism that could lead to splicing modulation, non-sense mediated decay (NMD) and translation blocking. And RNase H-mediated degradation that results in cleavage of the target RNA by making an RNA-ASO heteroduplex.
Phosphorodiamidate morpholino oligomers (PMO) are short single-stranded DNA analogs that contain a backbone of morpholine rings connected by phosphorodiamidate linkages. They have been reported to be useful in certain therapies.
One nucleic acid monomer that is modified for the production of PMOs is guanine. The guanine base of these monomers can be modified to protect the guanine base from participating in side reactions during the production of PMOs. When the guanine base of guanine monomers is mono-protected, stability issues begin to arise. In light of this feature of the guanine monomers, we provide bis-protected, activated guanine monomers with improved stability relative to mono-protected guanine monomers. These more stabilized guanine monomers can be used to produce PMOs with a lower incidence of by-product formation and in higher yields.
One embodiment is a bis-protected, activated guanine monomer or a pharmaceutically acceptable salt thereof. A monomer is said to be “activated” when that monomer has been prepared for use in further steps leading to synthesis of a dimer or oligomer.
In some embodiments, the bis-protected, activated guanine monomer includes an activated morpholine ring according to Formula I
and where R7 may be a C1-C6 alkyl, isopropyl, 2,2,2-trichloroethyl, benzyl or aryl.
In some embodiments, R1 and R2 can be linked together to form a C3 to C7 cycloalkyl ring or a heterocycle ring comprising oxygen and/or nitrogen, all of which may be saturated or unsaturated, and may be substituted at one or more carbon atoms with a C1-C6 alkyl.
In other embodiments, the bis-protected, activated guanine monomer is a stereoisomer of Formula I. Without being limited to the following structures, some embodiments of the bis-protected, activated guanine monomer comprise a stereoisomeric structure according to one of Formula (Ia) and (Ib).
In other embodiments, the bis-protected, activated guanine monomer comprises an activated tetrahydrofuran ring according to Formula II:
and where R7 may be a C1-C6 alkyl, isopropyl, 2,2,2-trichloroethyl, benzyl or aryl;
In other embodiments, R5 and R6 can be linked together to form a C3 to C7 cycloalkyl ring or heterocycle ring comprising oxygen and/or nitrogen, all of which may be saturated or unsaturated, and may be unsubstituted or substituted with C1-C6 alkyl. For example, in one embodiment, R5 and R6 can be linked together to form S-cEt, as depicted (in the context of the overall molecule) below:
In another embodiment R5 and R6 form LNA, which has the structure depicted (in the context of the overall molecule) below:
In other embodiments, R1 and R2 can be linked together to form a C3 to C7 cycloalkyl ring or heterocycle ring comprising oxygen and/or nitrogen, all of which may be saturated or unsaturated, and may be unsubstituted or substituted with C1-C6 alkyl.
In some embodiments, the bis-protected, activated guanine monomer is a stereoisomer of Formula II. Without being limited to the following structures, some embodiments of the bis-protected, activated guanine monomer comprise a stereoisomeric structure according to Formulas (IIa) and (IIb).
In other embodiments, the bis-protected, activated guanine monomer could be represented by the following structures:
In some embodiments, the bis-protected, activated guanine monomer possesses the following structure:
The bis-protected, activated guanine monomers described herein may be produced from a process comprising:
and
In some embodiments, the reagent used in step (i) is N-methylpyrrolidine, 1,4-diazabicyclo[2.2.2]octane (DABCO), quinuclidine, trimethylamine and any combination thereof.
In other embodiments, the base used in step (i) is 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU), diisopropyl ethylamine, potassium carbonate, potassium tert-butoxide, sodium hydride, Na2CO3, CsCO3, pyrrolidine, triethylamine, pyridineand any combination thereof.
In some embodiments, the alcohol used in step (i) may be 3-hydroxy-2-methylpropanenitrile, 3-hydroxy-3-methylpropanenitrile or 2,3-dimethyl-3-hydroxymethylpropanenitrile. The alcohol used in step (i) may also be any stereoisomer of 3-hydroxy-2-methylpropanenitrile, 3-hydroxy-3-methylpropanenitrile or 2,3-dimethyl-3-hydroxymethylpropanenitrile.
In some embodiments, the second activating agent used in step (iii) is DBU, 2,6-lutidine, N-methyl imidazole, 1H-tetrazole, 4,5-dichloroimidazole, 4,5-dicyanoimidazole, LiHMDS, 4-ethylmorpholine, DMAP, triethylamine, pyridine, Hunig's base and any combination thereof.
In some embodiments, the reaction of step (i), (ii) or (iii) may further comprise a solvent. The solvent may be DCM, ethyl acetate, acetonitrile, THF, toluene, dimethylsulfoxide, dimethylacetamide, DMFor any combination thereof.
In some embodiments, the bis-protected, activated guanine monomers described herein may be produced from a process comprising any, and in some embodiments all, of the following steps:
vi) reacting the protected guanine monomer according to Formula (XI) with an electrophile to produce a protected guanine monomer according to Formula II.
In some embodiments, step iv) comprises reacting the protected guanine monomer of Formula (IX) with 4-(hydroxylmethyl)phenyl pivalate to produce a protected guanine monomer according to Formula (XII):
In other embodiments, step v) comprises reacting the protected guanine monomer according to Formula (XII) with a deprotecting agent to produce a protected guanine monomer according to Formula (XIII):
In some embodiments, step vi) comprises reacting the protected guanine monomer according to Formula (XIII) with an electrophile to produce a compound with the following structure:
In some embodiments, the first protecting agent can be trityl chloride, 4-monomethoxytrityl chloride, 4,4′-Dimethoxytrityl chloride, or a silyl chloride comprising the formula (Si(R6)3)Cl, wherein R6 is C1-C6 alkyl or aryl. In some embodiments, the silyl chloride is tert-Butyldimethylsilyl chloride.
In other embodiments, the second protecting agent can be trityl chloride, 4-monomethoxytrityl chloride, 4,4′-Dimethoxytrityl chloride, or a silyl chloride comprising the formula (Si(R6)3)Cl, wherein R6 is C1-C6 alkyl or aryl. In some embodiments, the silyl chloride is tert-Butyldimethylsilyl chloride.
In other embodiments, the activating agent is 2,4,6-Triisopropylbenzenesulfonyl chloride.
In some embodiments, A1 is
arylsulfonyl which may be substituted with 1 to 3 alkyl groups, trifluromethanesulfonyl, methylsulfonyl and the combination thereof.
In other embodiments, the alcohol in step iv) is
or 4-(hydroxymethyl)phenyl pivalate.
In other embodiments, the electrophile is
Additional reagents and/or solvents can be added to any one or more of steps i) to vi). These reagents can be selected from the either DBU, DMAP, triethylamine, N-methylpyrrolidine, LiBr, 2,6-Lutidine, N-methylimidazole or combinations thereof. The solvents added to any one or more of steps i) to vi) can be either DCM, THF, MeCN, toluene, DMF, water or combinations thereof.
An aspect of the present disclosure is directed to bis-protected, activated guanine monomers. The bis-protected, activated guanine monomers may comprise a morpholine ring and have a structure depicted in
The bis-protected, activated guanine monomers may also comprise a tetrahydrofuran ring and have a structure represented by Formula II, IIa or IIb.
One utility of the bis-protected, activated guanine monomers described herein is improvement of synthesis of PMO by reducing the occurrence of side-reactions between de-protected guanine residues and thymine residues (see
When conventional cyanoethyl protecting groups are installed onto a guanine base, the deprotection of that cyanoethyl group generates acrylonitrile, which can react with a thymine residue in a PMO to give an alkylated impurity (see
Additionally, use of a β-methylated or an α-methylated cyanoethyl protecting group for guanine monomers has been found to reduce the occurrence in the loss of the cyanoethyl protecting group during activation of the guanine monomers described herein.
The structures of the bis-protected, activated guanine monomers described herein can also comprise stereoisomers of the Formulas I and II, in addition to the structures depicted in
While the terms used herein are believed to be well understood by one of ordinary skill in the art, definitions are set forth herein to facilitate explanation of the subject matter disclosed herein.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter disclosed herein belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the presently disclosed subject matter, representative methods, devices, and materials are described herein.
All combinations of method or process steps as used herein can be performed in any order, unless otherwise specified or clearly implied to the contrary by the context in which the referenced combination is made.
The methods and devices of the present disclosure, including components thereof, can comprise, consist of, or consist essentially of the essential elements and limitations of the embodiments described herein, as well as any additional or optional components or limitations described herein or otherwise useful.
Unless otherwise indicated, all numbers expressing physical dimensions, quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.
“R” and “S” as terms describing isomers are descriptors of the stereochemical configuration at asymmetrically substituted atoms, including but not limited to: carbon, sulfur, phosphorous and quaternary nitrogen. The designation of asymmetrically substituted atoms as “R” or “S” is done by application of the Cahn-Ingold-Prelog priority rules, as are well known to those skilled in the art, and described in the International Union of Pure and Applied Chemistry (IUPAC) Rules for the Nomenclature of Organic Chemistry. Section E, Stereochemistry.
“Pharmaceutically acceptable salt” as used herein refers to acid addition salts or base addition salts of the compounds in the present disclosure. A pharmaceutically acceptable salt is any salt which retains the activity of the parent compound and does not impart any unduly deleterious or undesirable effect on a subject to whom it is administered and in the context in which it is administered. Pharmaceutically acceptable salts include, but are not limited to, metal complexes and salts of both inorganic and carboxylic acids. Pharmaceutically acceptable salts also include metal salts such as aluminum, calcium, iron, magnesium, manganese, sodium and complex salts. In addition, pharmaceutically acceptable salts include, but are not limited to, acid salts such as acetic, aspartic, alkylsulfonic, arylsulfonic, axetil, benzenesulfonic, benzoic, bicarbonic, bisulfuric, bitartaric, butyric, calcium edetate, camsylic, carbonic, chlorobenzoic, citric, edetic, edisylic, estolic, esyl, esylic, formic, fumaric, gluceptic, gluconic, glutamic, glycolic, glycolylarsanilic, hexamic, hexylresorcinoic, hydrabamic, hydrobromic, hydrochloric, hydroiodic, hydroxynaphthoic, isethionic, lactic, lactobionic, maleic, malic, malonic, mandelic, methanesulfonic, methylnitric, methylsulfuric, mucic, muconic, napsylic, nitric, oxalic, p nitromethanesulfonic, pamoic, pantothenic, phosphoric, monohydrogen phosphoric, dihydrogen phosphoric, phthalic, polygalactouronic, propionic, salicylic, stearic, succinic, sulfamic, sulfanlic, sulfonic, sulfuric, tannic, tartaric, teoclic, toluenesulfonic, and the like.
The term “pharmaceutical composition” includes preparations suitable for administration to mammals, e.g., humans. When the compounds of the present invention are administered as pharmaceuticals to mammals, e.g., humans, they can be given per se or as a pharmaceutical composition containing, for example, 0.1% to 99.9% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
The term “alkyl” includes branched, straight chain and cyclic, substituted or unsubstituted saturated aliphatic hydrocarbon groups. Examples of C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, cyclopropylmethyl and neohexyl radicals.
The term “aryl” includes a 6- to 14-membered monocyclic, bicyclic or tricyclic aromatic hydrocarbon ring system. Examples of an aryl group include phenyl and naphthyl.
The halogen can be F, Cl, Br or I.
The term “cycloalkyl” includes a cycloalkyl ring containing 5 to 12 carbon atoms.
Examples include cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl and cyclododecyl.
The following abbreviations may be used throughout the examples.
A solution of 9-((2R,6S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-4-tritylmorpholin-2-yl)-2-isobutyramido-9H-purin-6-yl 2,4,6-triisopropylbenzenesulfonate (5.50 g, 5.73 mmol) (prepared by the procedure described in CA2813183) in CH2Cl2 (57.3 ml, 5.733 mmol) was and cooled to 0° C. and treated with 1-methylpyrrolidine (2.38 ml, 22.93 mmol) in CH2Cl2 (5.0 ml). After being stirred at 0° C. for 1 h, a solution of 3-hydroxy-2-methylpropanenitrile (1.95 g, 22.93 mmol) and DBU (1.12 ml, 7.45 mmol) in CH2Cl2 (5.0 ml) was added and continue stirring at 0° C. for 2-3 h (reaction was monitored by LCMS). The reaction mixture was diluted with 1.0 M aq. NaH2PO4 (50 ml) and water (50 ml) and stirred at rt for 30 min. The CH2Cl2 layer was separated and the aqueous layer was washed twice with CH2Cl2 (30 ml). The combined organic layer was washed with brine (30 ml), dried over Na2SO4, filtered, and concentrated in vacuo. The crude residue was purified by column chromatography (ethyl acetate in n-heptane=0% to 80%) to give the N-(9-((2R,6S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-4-tritylmorpholin-2-yl)-6-(2-cyanopropoxy)-9H-purin-2-yl)isobutyramide (4.42 g, 5.816 mmol) contaminated with 3-hydroxy-2-methylpropanenitrile, which was used for next step without further purification.
The above obtained compound was dissolved in CH2Cl2 (58.2 ml, 5.81 mmol) in a plastic container and cooled to 0° C. Triethylamine trihydrofluoride (9.57 g, 58.15 mmol) was added to the reaction solution in drop-wise. The reaction solution was stirred at 0° C. for 7-8 h. The reaction mixture was poured into a ice-cold solution of sodium bicarbonate (7.33 g, 87.234 mmol) in water (60 ml) and stirred ar RT for 1 h. Then the CH2Cl2 layer was separated and the aqueous layer was extracted twice with CH2Cl2 (50 ml). The combined organic layer was dried over Na2SO4, filtered, concentrated under reduced pressure and purified by a silica gel column chromatography to give 2.59 g (4.01 mmol, 70%) of the title compound as white solid. 1H NMR (400 MHz, CDCl3) δ 7.88 (s, 1H), 7.82 (s, 1H), 7.58-7.44 (m, 6H), 7.33 (t, J=7.6 Hz, 6H), 7.26-7.16 (m, 3H), 6.28 (dd, J=9.9, 2.4 Hz, 1H), 4.80-4.68 (m, 1H), 4.66-4.52 (m, 1H), 4.41-4.30 (m, 1H), 3.64 (qd, J=11.8, 5.0 Hz, 2H), 3.47 (dt, J=11.4, 2.4 Hz, 1H), 3.41-3.28 (m, 1H), 3.22-3.14 (m, 1H), 3.14-3.10 (s, 1H), 1.84 (t, J=10.6 Hz, 1H), 1.62 (t, J=11.2 Hz, 1H), 1.49 (d, J=7.1 Hz, 3H), 1.39 (d, J=6.8 Hz, 3H), 1.36 (d, J=6.8 Hz, 3H).
Using the same procedure and same volumes as for the preparation of N-(6-(2-cyanopropoxy)-9-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-9H-purin-2-yl)isobutyramide, 20.0 g of 9-((2R,6S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-4-tritylmorpholin-2-yl)-2-isobutyramido-9H-purin-6-yl 2,4,6-triisopropylbenzenesulfonate gave 8.5 g of the title compound (13.16 mmol, 63%) as a white solid.
1H NMR (400 MHz, CDCl3) δ 7.83 (s, 1H), 7.82 (s, 1H), 7.57-7.43 (m, 6H), 7.33 (t, J=7.6 Hz, 6H), 7.26-7.18 (m, 3H), 6.27 (dd, J=9.9, 2.3 Hz, 1H), 5.73-5.62 (m, 1H), 4.41-4.31 (m, 1H), 3.70-3.56 (m, 2H), 3.51-3.43 (m, 1H), 3.22-3.14 (m, 1H), 3.08 (s, 1H), 2.97 (dd, J=5.9, 2.5 Hz, 2H), 1.86 (t, J=10.6 Hz, 2H), 1.66-1.57 (m, 4H), 1.39 (d, J=6.9 Hz, 3H), 1.36 (d, J=6.8 Hz, 3H).
Using the same procedure and same volumes as for the preparation of N-(6-(2-cyanopropoxy)-9-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-9H-purin-2-yl)isobutyramide, 3.00 g of 9-((2R,6S)-6-(((tert-butyldimethylsilyl)oxy)methyl)-4-tritylmorpholin-2-yl)-2-isobutyramido-9H-purin-6-yl 2,4,6-triisopropylbenzenesulfonate gave 1.38 g of the title compound (1.87 mmol, 68%) as a white solid.
1H NMR (400 MHz, CDCl3) δ 7.88 (s, 1H), 7.82 (s, 1H), 7.58-7.42 (m, 6H), 7.33 (t, J=7.6 Hz, 6H), 7.24-7.18 (m, 3H), 6.27 (dd, J=9.9, 2.4 Hz, 1H), 5.74-5.62 (m, 1H), 4.40-4.30 (m, 1H), 3.71-3.56 (m, 2H), 3.52-3.43 (m, 1H), 3.22-3.13 (m, 1H), 3.08 (s, 1H), 3.00 (dd, J=16.9, 6.1 Hz, 1H), 2.92 (dd, J=16.8, 5.5 Hz, 1H), 1.83 (t, J=10.6 Hz, 1H), 1.67-1.56 (m, 4H), 1.38 (t, J=7.2 Hz, 6H).
To a solution of N-(6-(2-cyanopropoxy)-9-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-9H-purin-2-yl)isobutyramide (890 mg, 1.37 mmol) in CH3CN (7990 μl, 152.98 mmol)/CH2Cl2 (7981 μl, 124.03 mmol) was added lithium bromide (395 mg, 4.54 mmol) at room temperature and stirred until a clear solution was obtained. Then, the reaction mixture was cooled to 0° C. and was added a solution of DBU (686 μl, 4.54 mmol) in CH3CN (1.0 ml) followed by addition of N,N-Dimethylphosphoramic dichloride (262 μl, 2.205 mmol) in CH3CN (1.0 ml) at 0° C. After 2 h, the reaction was quenched with 10% aq. Citric acid solution (20 ml) and diluted with ethylacetate (30 ml). After 30 min, ethylacetate layer was separated and the aqueous layer was extracted twice with ethylacetate (30 ml). The combined organic layer was eashed with water, brine, dried over Na2SO4, filtered and concentrated under reduced vaccum, The crude residue was purified by silica gel column to give 820 mg (1.06 mmol, 77%) of title compound.
Under HPLC conditions, the titled compound gave two peaks due to methyl stereochemistry adjacent to cyano group.
1H NMR (400 MHz, Acetone-d6) δ 9.17 (s, 1H), 7.94 (s, 1H), 7.53-7.34 (m, 6H), 7.22 (t, J=7.6 Hz, 6H), 7.08 (t, J=7.4 Hz, 3H), 6.27 (dd, J=9.9, 2.4 Hz, 1H), 4.61-4.45 (m, 3H), 4.06 (dd, J=8.5, 5.0 Hz, 2H), 3.48-3.37 (m, 2H), 3.21 (d, J=11.7 Hz, 1H), 3.09-2.97 (m, 1H), 2.48 (s, 3H), 2.45 (s, 3H), 1.87-1.83 (m, 1H), 1.55 (t, J=11.2 Hz, 1H), 1.30 (d, J=7.1 Hz, 3H), 1.13 (d, J=5.1 Hz, 3H), 1.10 (d, J=7.1 Hz, 3H); 31P NMR (162 MHz, Acetone-d6) δ 17.53.
1H NMR (400 MHz, Acetone-d6) δ 9.19 (s, 1H), 7.94 (s, 1H), 7.55-7.35 (m, 6H), 7.21 (t, J=7.7 Hz, 6H), 7.07 (t, J=7.4 Hz, 3H), 6.27 (dd, J=9.9, 2.4 Hz, 1H), 4.61-4.48 (m, 3H), 4.05 (dd, J=8.5, 5.0 Hz, 2H), 3.47-3.34 (m, 2H), 3.21 (dt, J=12.0, 2.5 Hz, 1H), 3.10-2.99 (m, 1H), 2.48 (s, 3H), 2.44 (s, 3H), 1.91-1.80 (m, 1H), 1.55 (t, J=11.2 Hz, 1H), 1.29 (d, J=7.1 Hz, 3H), 1.15 (d, J=6.8 Hz, 3H), 1.12 (d, J=6.8 Hz, 3H); 31P NMR (162 MHz, Acetone-d6) δ 17.56.
Under HPLC conditions, the titled compound gave two peaks due to methyl stereochemistry adjacent to cyano group.
1H NMR (400 MHz, Acetone-d6) δ 9.18 (s, 1H), 7.95 (s, 1H), 7.54-7.33 (m, 6H), 7.21 (t, J=7.7 Hz, 6H), 7.08 (t, J=7.4 Hz, 3H), 6.26 (dd, J=9.9, 2.4 Hz, 1H), 4.60-4.44 (m, 3H), 4.15-4.03 (m, 2H), 3.49-3.36 (m, 2H), 3.20 (dt, J=11.8, 2.4 Hz, 1H), 3.10-2.99 (m, 1H), 2.48 (s, 3H), 2.45 (s, 3H), 1.91-1.79 (m, 1H), 1.59 (t, J=11.2 Hz, 1H), 1.29 (d, J=7.1 Hz, 3H), 1.15 (d, J=6.8 Hz, 3H), 1.12 (d, J=6.8 Hz, 3H); 31P NMR (162 MHz, Acetone-d6) δ 17.17.
1H NMR (400 MHz, Acetone-d6) δ 9.17 (s, 1H), 7.95 (s, 1H), 7.52-7.31 (m, 6H), 7.21 (t, J=7.7 Hz, 6H), 7.08 (t, J=7.4 Hz, 3H), 6.26 (dd, J=9.9, 2.4 Hz, 1H), 4.91 (s, 1H), 4.60-4.47 (m, 3H), 4.15-4.03 (m, 2H), 3.47-3.34 (m, 2H), 3.20 (dt, J=11.8, 2.4 Hz, 1H), 3.10-2.99 (m, 1H), 2.48 (s, 3H), 2.45 (s, 3H), 1.90-1.78 (m, 1H), 1.59 (t, J=11.2 Hz, 1H), 1.29 (d, J=7.1 Hz, 3H), 1.15 (d, J=6.9 Hz, 3H), 1.12 (d, J=6.8 Hz, 3H); 31P NMR (162 MHz, Acetone-d6) δ 17.17.
Using the same procedure as for the preparation of ((2S,6R)-6-(6-(2-cyanopropoxy)-2-isobutyramido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl dimethyl-phosphoramidochloridate, 3.3 g of N-(6-(((R)-1-cyanopropan-2-yl)oxy)-9-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-9H-purin-2-yl)isobutyramide gave 2.56 g of the title compound (3.32 mmol, 65% yield).
1H NMR (400 MHz, Acetone-d6) δ 9.22 (s, 1H), 7.93 (s, 1H), 7.54-7.33 (m, 6H), 7.21 (t, J=7.7 Hz, 6H), 7.12-7.03 (m, 3H), 6.25 (dd, J=9.9, 2.4 Hz, 1H), 5.56-5.44 (m, 1H), 4.59-4.49 (m, 1H), 4.09-4.01 (m, 2H), 3.40 (dt, J=11.6, 2.5 Hz, 1H), 3.21 (dt, J=12.0, 2.5 Hz, 1H), 3.07 (dd, J=17.1, 5.4 Hz, 1H), 3.04-2.98 (m, 1H), 2.93 (dd, J=17.1, 5.2 Hz, 1H), 2.47 (s, 3H), 2.44 (s, 3H), 1.90-1.82 (m, 1H), 1.54 (t, J=11.2 Hz, 1H), 1.42 (d, J=6.3 Hz, 3H), 1.14 (d, J=6.9 Hz, 4H), 1.11 (d, J=6.8 Hz, 3H); 31P NMR (162 MHz, Acetone-d6) δ 17.56.
1H NMR (400 MHz, Acetone-d6) δ 9.22 (s, 1H), 7.94 (s, 1H), 7.51-7.36 (m, 6H), 7.21 (t, J=7.7 Hz, 6H), 7.11-7.03 (m, 3H), 6.25 (dd, J=9.8, 2.4 Hz, 1H), 5.56-5.44 (m, 1H), 4.58-4.48 (m, 1H), 4.16-4.00 (m, 2H), 3.40 (dt, J=11.5, 2.5 Hz, 1H), 3.20 (dt, J=11.9, 2.5 Hz, 1H), 3.07 (dd, J=17.1, 5.4 Hz, 1H), 3.04-2.98 (m, OH), 2.93 (dd, J=17.1, 5.2 Hz, 1H), 2.48 (s, 3H), 2.44 (s, 3H), 1.90-1.83 (m, 1H), 1.58 (t, J=11.2 Hz, 1H), 1.42 (d, J=6.3 Hz, 3H), 1.15 (d, J=6.9 Hz, 4H), 1.12 (d, J=6.8 Hz, 3H); 31P NMR (162 MHz, Acetone-d6) δ 17.18.
Using the same procedure as for the preparation of ((2S,6R)-6-(6-(2-cyanopropoxy)-2-isobutyramido-9H-purin-9-yl)-4-tritylmorpholin-2-yl)methyl dimethyl-phosphoramidochloridate, 1.0 g of N-(6-(((S)-1-cyanopropan-2-yl)oxy)-9-((2R,6S)-6-(hydroxymethyl)-4-tritylmorpholin-2-yl)-9H-purin-2-yl)isobutyramide gave 0.810 g of the title compound (3.32 mmol, 68% yield).
N-(9-((2R,4S,5R)-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (2.5 g, 7.411 mmol) was co-evaporated with anhydrous pyridine once before it was dissolved in DMF (25 mL) in a flask, into which was added imidazole (2.52 g, 37.055 mmol) followed by addition of TBS-Cl (2.79 g, 18.528 mmol) portionwise at room temperature. The reaction mixture was kept stirring at room temperature for 48 hr. Into the mixture was then added water (200 mL), and the solid precipitate was collected and rinsed with water. The solid was redissolved in DCM, was washed subsequently with sat. sodium bicarbonate (aq.) and half sat. brine, dried over Na2SO4 and concentrated. Silica gel column purification of the concentrate with MeOH in DCM afforded 4.11 g of product. 1H NMR (400 MHz, CHLOROFORM-d) δ ppm 11.95 (s, 1H), 8.18 (s, 1H), 7.96 (s, 1H), 6.22 (dd, J=6.4, 6.8 Hz, 1H), 4.52-4.61 (m, 1H), 3.98 (m, 1H), 3.76 (d, J=3.2 Hz, 2H), 2.61 (m, 1H), 2.28-2.51 (m, 2H), 1.27 (br d, J=7.0 Hz, 3H), 1.28 (br d, J=6.8 Hz, 3H), 0.91 (s, 9H), 0.90 (s, 9H), 0.10 (s, 6H), 0.07 (s, 3H), 0.07 (s, 3H).
MS (ESI) m/z: calculated for C26H47N5O5Si2 [M+H]+: 566.3; found 566.2.
N-(9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (4.11 g, 7.263 mmol) was co-evaporated with anhydrous MeCN twice before it was dissolved in DCM (41.1 mL) in a flask at room temperature, into which was added DMAP (0.089 g, 0.726 mmol) and triethylamine (3.04 mL, 21.79 mmol), followed by addition of 2,4,6-triisopropylbenzenesulfonyl chloride (3.30 g, 10.895 mmol). The reaction mixture was stirred at room temperature for 20 hr before it was cooled in an ice bath, and quenched with sodium dihydrogen phosphate aqueous solution (105 mL, 87.159 mmol, 10 wt %). After phase separation, it was back extracted multiple times with DCM (100 mL). The combined DCM layers were washed with brine (10 wt %), dried over Na2SO4, and concentrated.
The residue was co-evaporated with anhydrous toluene three times before it was redissolved in DCM (60.4 mL, 938.783 mmol) in a flask in an ice bath, into which was added N-methylpyrrolidine (1.509 mL, 14.515 mmol). The mixture was first stirred at 0° C. for 1 hr and then stirred at ambient temperature for another before it was cooled back to 0° C. Into the mixture was then added a solution of (R)-3-hydroxybutanenitrile (0.772 g, 9.072 mmol) and DBU (1.367 mL, 9.072 mmol) in DCM (6 mL) via a cannula. It was kept stirring at 0° C. for 4 hr and at ambient temperature for 1 hr before additional (R)-3-hydroxybutanenitrile (0.579 g, 6.804 mmol) and DBU (0.273 mL, 1.814 mmol) was added. It was stirred at ambient temperature for 1 hr before it was quenched with sodium dihydrogen phosphate (174 mL, 145.147 mmol) (aq. 10 wt %) at 0° C. The mixture was extracted with DCM multiple times (150 mL×2). The combined DCM layers were washed subsequently with water (80 mL×2) and brine (80 mL, 10%), dried over Na2SO4, and concentrated. Silica gel column purification of the concentrate with heptane-ethyl acetate afforded 1.58 g of product.
1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.16 (s, 1H), 7.79 (s, 1H), 6.39 (dd, J=6.8, 6.4 Hz, 1H), 5.67 (m, 1H), 4.60 (m, 1H), 4.00 (m, 1H), 3.85 (dd, J=11.2, 4.0 Hz, 1H), 3.77 (dd, J=11.2, 2.8 Hz, 1H), 3.05 (br s, 1H), 2.96 (d, J=5.8 Hz, 2H), 2.57 (m, 1H), 2.40 (ddd, J=13.0, 6.1, 3.9 Hz, 1H), 1.64 (d, J=6.5 Hz, 3H), 1.29 (d, J=7.0 Hz, 6H), 0.92 (s, 9H), 0.91 (s, 9H), 0.10 (s, 6H), 0.09 (s, 6H).
MS (ESI) m/z: calculated for C30H52N6O5Si2 [M+H]+: 633.4; found 633.4.
To a mixture of N-(9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-6-(((R)-1-cyanopropan-2-yl)oxy)-9H-purin-2-yl)isobutyramide (1.58 g, 2.496 mmol) in THF (10.64 mL, 129.907 mmol) and water (3.55 mL, 196.958 mmol) was added TFA (0.577 mL, 7.489 mmol) at 0° C. It was stirred in an ice bath for 5 hr. Into the mixture was added water (50 mL), and the solid precipitate was collected by filtration, and rinsed with water (30 mL×3). The solid was suspended in MeCN (50 mL), and sonicated for 5 min. The solid was collected by filtration, and the filtrate was concentrated and subjected to silica gel column chromatography, eluted with DCM-MeOH to give additional product fractions. The combined product fractions was co-evaporated with pyridine once and then anhydrous MeCN twice to give total 1.13 g of product.
1H NMR (400 MHz, CD3OD) δ ppm 8.41 (s, 1H), 6.48 (dd, J=6.8, 6.4 Hz, 1H), 5.72 (m, 1H), 4.79 (m, 1H), 3.97 (m, 1H), 3.79 (dd, J=12.0, 4.0 Hz, 1H), 3.74 (dd, J=12.0, 4.4 Hz, 1H), 3.17 (dd, J=17.2, 5.2 Hz, 1H), 3.04 (dd, J=17.2, 5.6 Hz, 1H), 2.81 (m, 2H), 2.44 (m, 1H), 1.60 (d, J=6.4 Hz, 3H), 1.23 (d, J=6.8 Hz, 6H), 0.95 (s, 9H), 0.17 (s, 3H), 0.15 (s, 3H).
MS (ESI) m/z: calculated for C24H38N6O5Si [M+H]+: 519.3; found 519.4.
Into a suspension of N-(9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-(((R)-1-cyanopropan-2-yl)oxy)-9H-purin-2-yl)isobutyramide (1.134 g, 2.186 mmol) in DCM (18.99 mL, 295.147 mmol) and acetonitrile (18.96 mL, 362.922 mmol) was added 1-methylimidazole (0.105 mL, 1.312 mmol) and 2,6-lutidine (1.528 mL, 13.118 mmol), followed by addition of dimethylphosphoramidic dichloride (0.781 mL, 6.559 mmol) at rt. The suspension was then stirred at room temperature for 1 day before it was quenched with aqueous citric acid solution (88 mL, 45.912 mmol, 10%) at 0° C. It was extracted with DCM (113 mL×2). The combined DCM layers were washed subsequently with water twice and half sat. brine, dried over Na2SO4, and concentrated. Silica gel column purification of the concentrate with heptane-ethyl acetate afforded 944 mg of a stereoisomer mixture. MS (ESI) m/z: calculated for C26H43ClN7O6PSi [M+H]+: 644.3; found 644.2.
The stereoisomer mixture was subjected to the following HPLC separation method to isolate the (R) and (S)-stereoisomers of the product:
1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.73 (br s, 1H), 7.90 (s, 1H), 6.31 (dd, J=6.8, 6.8 Hz, 1H), 5.67 (m, 1H), 4.76-4.91 (m, 2H), 4.11-4.27 (m, 2H), 3.05-3.29 (m, 2H), 2.94 (d, J=17.2, 5.2 Hz, 1H), 2.84 (m, 1H), 2.76 (s, 3H), 2.72 (s, 3H), 2.37 (ddd, J=13.6, 6.8, 2.9 Hz, 1H), 1.65 (d, J=6.3 Hz, 3H), 1.27 (d, J=6.8 Hz, 6H), 0.92 (s, 9H), 0.14 (s, 3H), 0.13 (s, 3H). 31P NMR (162 MHz, CHLOROFORM-d) δ ppm 19.17 (s, 1P).
1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.57 (s, 1H), 7.90 (s, 1H), 6.32 (dd, J=7.2, 6.8 Hz, 1H), 5.65 (m, 1H), 4.74-4.83 (m, 1H), 4.54-4.74 (m, 1H), 4.35-4.49 (m, 1H), 4.18-4.28 (m, 1H), 3.17 (dd, J=16.8, 6.0 Hz, 1H), 3.06 (m, 1H), 2.95 (dd, J=17.0, 4.8 Hz, 1H), 2.79-2.88 (m, 1H), 2.69 (s, 3H), 2.65 (s, 3H), 2.35 (ddd, J=13.4, 6.4, 2.6 Hz, 1H), 1.65 (d, J=6.5 Hz, 3H), 1.22-1.34 (m, 6H), 0.93 (s, 9H), 0.15 (s, 3H), 0.13 (s, 3H). 31P NMR (162 MHz, CHLOROFORM-d) δ ppm 19.27 (s, 1P).
N-(9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide (1.50 g, 2.651 mmol) was co-evaporated with anhydrous pyridine and then with anhydrous MeCN before it was dissolved in DCM (15.00 mL, 233.126 mmol) in a flask with a room temperature water bath. Into the solution was added DMAP (0.032 g, 0.265 mmol) and triethylamine (1.108 mL, 7.952 mmol), followed by addition of 2,4,6-triisopropylbenzenesulfonyl chloride (1.445 g, 4.771 mmol). It was stirred for 1.5 hr before it was cooled in ice bath and quenched with sodium dihydrogen phosphate aqueous solution (54.9 mL, 39.762 mmol, 10 wt %). It was extracted with DCM twice. The combined DCM layers were washed with 5 wt % brine, dried over Na2SO4, and concentrated. The residue was co-evaporated with toluene three times before it was re-dissolved in DCM (22.06 mL, 342.873 mmol), into which was then added 4-(hydroxymethyl)phenyl pivalate (1.548 g, 7.430 mmol), DBU (0.799 mL, 5.301 mmol) and N-methylpyrrolidine (0.551 ml, 5.301 mmol) at 0° C. After addition, the ice bath was removed and the reaction mixture was stirred at room temperature overnight. It was then cooled in ice bath and quenched with sodium dihydrogen phosphate aqueous solution (73.2 ml, 53.012 mmol, 10 wt %). It was extracted with DCM twice, and the combined DCM layers were washed with 5 wt % brine, dried over Na2SO4, and concentrated. Silica gel column purification of the concentrate with heptane-ethyl acetate afforded 2.08 g of the product.
1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.13 (s, 1H), 7.78 (s, 1H), 7.53 (d, J=8.5 Hz, 2H), 7.04 (dd, J=8.5, 2.3 Hz, 2H), 6.39 (dd, J=6.4, 6.4 Hz, 1H), 5.60 (m, 2H), 4.60 (m, 1H), 3.99 (m, 1H), 3.85 (dd, J=11.2, 4.0 Hz, 1H), 3.77 (dd, J=11.2, 3.2 Hz, 1H), 3.22 (br s, 1H), 2.56 (m, 1H), 2.40 (ddd, J=13.0, 6.0, 3.8 Hz, 1H), 1.35 (s, 9H), 1.28 (d, J=6.8 Hz, 6H), 0.91 (s, 9H), 0.91 (s, 9H), 0.10 (s, 6H), 0.08 (s, 6H).
MS (ESI) m/z: calculated for C38H61N5O7Si2 [M+H]+: 756.4; found 756.3.
To a solution of 4-(((9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydrofuran-2-yl)-2-isobutyramido-9H-purin-6-yl)oxy)methyl)phenyl pivalate (2.08 g, 2.672 mmol) in THF (22.78 mL) and water (3.80 mL) was added TFA (0.618 ml, 8.016 mmol) dropwise at 0° C. The reaction mixture was stirred with ice bath overnight before it was quenched with sat. sodium bicarbonate aqueous solution (42.1 mL, 40.078 mmol). It was extracted with EtOAc (100 mL 2×), and combined EtOAc layers were washed with half sat. brine, dried over Na2SO4, and concentrated. Silica gel column purification of the concentrate with heptane-ethyl acetate afforded 0.83 g of product.
1H NMR (400 MHz, CHLOROFORM-d) δ ppm 7.87 (s, 1H), 7.76 (s, 1H), 7.55 (d, J=8.4 Hz, 2H), 7.04 (m, J=8.4 Hz, 2H), 6.24 (dd, J=8.8, 6.0 Hz, 1H), 5.55-5.70 (m, 2H), 5.01 (br dd, J=10.0, 2.8 Hz, 1H), 4.78 (br d, J=5.2 Hz, 1H), 4.10 (m, 1H), 3.94 (br d, J=12.4 Hz, 1H), 3.78 (m, 1H), 2.97 (ddd, J=13.2, 8.4, 5.2 Hz, 1H), 2.84 (m, 1H), 2.23 (ddd, J=11.6, 5.6, 1.6 Hz, 1H), 1.35 (s, 9H), 1.28 (d, J=6.8 Hz, 6H), 0.92 (s, 9H), 0.12 (s, 3H), 0.11 (s, 3H).
MS (ESI) m/z: calculated for C32H47N5O7Si [M+H]+: 642.3; found 642.3.
4-(((9-((2R,4S,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-2-isobutyramido-9H-purin-6-yl)oxy)methyl)phenyl pivalate (0.83 g, 1.293 mmol) was co-evaporated with anhydrous MeCN once before it was dissolved in MeCN (8.46 mL, 162.048 mmol) and DCM (8.46 ml, 131.539 mmol), into which lithium bromide (0.337 g, 3.879 mmol) and DBU (0.585 ml, 3.879 mmol) was added. The mixture was then cooled in an ice bath, into which dimethylphosphoramidic dichloride (0.200 mL, 1.681 mmol) was added. The reaction mixture was stirred in an ice bath for 1 hr before it was quenched with aqueous solution of citric acid (16.40 mL, 8.535 mmol, 10 wt %) at 0° C. It was extracted with DCM (42.3 mL 2×). The combined DCM layers were washed subsequently with water twice and half sat. brine, dried over Na2SO4, and concentrated. Silica gel column purification of the concentrate with heptane-ethyl acetate afforded 0.60 g of the product. MS (ESI) m/z: calculated for C34H52ClN6O8PSi [M+H]+: 767.3; found 767.0.
The stereoisomer mixture was subjected to the following HPLC separation method to isolate the (R) and (S)-stereoisomers of the product:
1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.44 (br s, 1H), 7.90 (s, 1H), 7.58 (d, J=8.4 Hz, 2H), 7.03 (d, J=8.4 Hz, 2H), 6.32 (dd, J=6.8, 6.8 Hz, 1H), 5.56-5.72 (m, 2H), 4.80 (m, 1H), 4.73 (m, 1H), 4.15-4.30 (m, 2H), 3.08 (m, 1H), 2.97 (m, 1H), 2.74 (s, 3H), 2.70 (s, 3H), 2.37 (ddd, J=13.2, 6.4, 3.2 Hz, 1H), 1.34 (s, 9H), 1.28 (d, J=6.8 Hz, 6H), 0.92 (s, 9H), 0.13 (s, 3H), 0.12 (s, 3H). 31P NMR (162 MHz, CHLOROFORM-d) δ ppm 19.04 (s, 1P).
1H NMR (400 MHz, CHLOROFORM-d) δ ppm 8.31 (s, 1H), 7.89 (s, 1H), 7.57 (d, J=8.4 Hz, 2H), 7.03 (d, J=8.4 Hz, 2H), 6.32 (dd, J=6.8, 6.8 Hz, 1H), 5.56-5.70 (m, 2H), 4.78 (m, 1H), 4.55 (m, 1H), 4.39 (m, 1H), 4.21 (m, 1H), 3.00 (m, 2H), 2.67 (s, 3H), 2.63 (s, 3H), 2.37 (ddd, J=13.2, 6.4, 3.2 Hz, 1H), 1.34 (s, 9H), 1.28 (d, J=6.8 Hz, 3H), 1.27 (d, J=6.8 Hz, 3H), 0.92 (s, 9H), 0.15 (s, 3H), 0.13 (s, 3H). 31P NMR (162 MHz, CHLOROFORM-d) δ ppm 19.13 (s, 1P).
This application claims the benefit of priority of U.S. Provisional Patent Application No. 63/315,280, filed on Mar. 1, 2022. That application is incorporated by reference as if fully rewritten herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2023/014247 | 3/1/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63315280 | Mar 2022 | US |
