CN1744903A - Process for the production of 2'-branched nucleosides - Google Patents

Process for the production of 2'-branched nucleosides Download PDF

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CN1744903A
CN1744903A CNA2003801095761A CN200380109576A CN1744903A CN 1744903 A CN1744903 A CN 1744903A CN A2003801095761 A CNA2003801095761 A CN A2003801095761A CN 200380109576 A CN200380109576 A CN 200380109576A CN 1744903 A CN1744903 A CN 1744903A
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methyl
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lactone
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理查德·斯托勒
阿德尔·穆萨
纳拉扬·乔杜里
弗兰克·瓦利戈拉
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Idenix Cayman Ltd
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Abstract

The present invention provides an improved process for preparing ss-D and ss-L 2'-C-methyl-nucleosides and 2'-C-methyl-3'-O-ester nucleosides.

Description

2 '-preparation method of branched nucleosides
Cross reference
It number is 60/432,766 that the application requires provisional application, and submitting in 12 days U.S. Patent application of December in 2002 and U.S. Provisional Patent Application number is 60/466,194 to submit in the priority of the U.S. Patent application on April 28th, 2003.
Technical field
The present invention relates to have the preparation method of the substituent saccharide of oxygen of protection like chemical compound; particularly about 2; 3; 5-(hydroxyl of independent optional protection)-2-C-methyl-β-D-ribofuranose and 2; 3, the preparation method of 5-(hydroxyl of independent optional protection)-2-C-methyl D-ribonic acid-gamma lactone.The present invention also comprises the method for preparing nucleoside; particularly by optional use according to the sugar in the method preparation of this proposition prepare 3 '; 5 '-(independent optional protection hydroxyl)-2 '-method of C-methyl-β-D-cytidine, special, be synthetic about the medical compounds precursor.More specifically, the invention describes synthetic, special as the prodrug of anti-viral nucleoside and the administration of nucleoside analog derivative compound, be 2 '-C-methyl-β-D-cytidine '-O-L-valine ester synthetic.
Background technology
In the preparation that is used for the synthetic sugar analogue of nucleoside and vitamin, a kind of intermediate of key is 2-C-methyl D-ribonic acid-lactone.As far back as 1880, Scheibler has just introduced the method (JohnSowden for preparing lactone, " The Saccharinic Acids " in Adv.Carbohydrate Chem.12:43-46 (1957) quotes C.Scheibler, Berichte 13:2212 (1880)).Yet the productive rate of product but has only about 10% (Id.).Approximately at one time, H.Kiliani utilizes calcium hydroxide to handle D-fructose, synthesized 2-methyl D-ribose acid lactone (H.Kiliani, Berichte, 15:2953 (1882), at F.J.Lopez-Herrera etal., J Carbohydrate Chemistry, 13 (5): 767-775 is cited in (1994)).Yet this method needs the time of several months to finish, and productive rate only has 10% (Id.at 768).But, the method of Kiliani make he can determine the position of important functional group on chemical compound (John Sowden, " The Saccharinic Acids " in Adv.Carbohydrate Chem.12:43-46 (1957) quotes H.Kiliani, Ann., 213:361 (1883)).
Early stage in the sixties in 20th century, Whistler and BeMiller attempted improving synthetic (the RoyL.Whistler and J.N.BeMiller of Kiliani, " a-D-Glucosaccharino-1; 4-lactone " in Methods inCarbohydrate Chemistry, 2:484-485 (1963)).Whistler and BeMiller add boiling water and calcium hydroxide in D-fructose, feed nitrogen in this system, repeat identical process.After two weeks; Again mixture is placed 6-8 week reuse CO 2Handle with two oxalic acid hydrates, pressure filters down.The repeated washing residue, until becoming uniform serosity shape, merging filtrate, decompression is solvent distillation down, makes products therefrom cooling crystallization (Id.) down.Ultimate yield still only is about 10% (Id.at 485).
In the trial that improves productive rate, Lopez-Aparicio etc. have reported by 2,3-O-isopropylidene-D-glyceraldehyde Synthetic 2-C-methyl D-ribonic acid-1, the 4-lactone substitutes synthetic (the Lopez-Aparicio et al. of Kiliani, Carbohydrate Res., 129:99 (1984) is at F.J.Lopez-Herrera et al., J.CarbohydrateChemistry, 13 (5): be cited in 767-775 (1994) the 768-769 pages or leaves).The method of Lopez-Aparicio comprises makes 2, and 3-O-isopropylidene-D-glyceraldehyde and the condensation of (1-methoxyl group-carbonyl-acetal) triphenyl phosphorane produce methyl E-(S)-4,5-dihydroxy-4,5-O-isopropylidene-2-methyl-2-pentenoate; Water carries out photoisomeric change with (in HCl) and with pentenoate; The pentenoate product is lactonized, produce butenolide; By with the reaction of trityl chloride and pyridine, at C-5 butenolide is carried out tritylation, in the presence of crown ether, carry out cis hydroxyl groups with potassium permanganate and dichloromethane then and turn usefulness into.By with the reaction of TFA (trifluoroacetic acid) (Id.at 768), finally removed trityl.According to Lopez-Aparicio etc., the productive rate of ribose acid lactone is about 80%, but other people are according to the quality (gram) of the material that provides in the experimental section of delivering at them, but can not obtain above-mentioned data.On the contrary, the yield percentage that shows ribose acid lactone as calculated is about 36%.And, the method of Lopez-Aparicio etc. is synthetic more complex compared with Kiliani, need to use toxic reagent and special equipment as potassium permanganate be used for radiation, and (Id.at 768,770-772) 60 hours response time of minimum needs with the acquisition photoisomeric change.
Walton etc. reported by 2-C-methyl D-ribonic acid-lactone Synthetic 2 '-C-methyladenosine (Walton etal., J.Am.Chem.Soc., 88 (19): 4524-5 (1966)).Here, lactone be converted into its 2,3,5-three-O-benzoyl derivative is used two (3-methyl-2-butyl) borane reduction then, obtains 2,3, the end group heterogeneous mixture of 5-three-O-benzoyl-2-C-methyl D-ribofuranose (Id.).When attempting to separate the end group heterogeneous mixture on pickling aluminium oxide and silica gel, causing resetting is 1,3,5-four-O-benzoyl-2-C-methyl-α-D-ribofuranose (Id).For fear of rearrangement, need to increase with Benzenecarbonyl chloride. and in pyridine, handle blended anomer obtaining 1,2,3, the step of 5-four-O-benzoyl-2-C-methyl-(α)/(β)-D-ribofuranose, and with the step (Id.) of chromatographic isolation end product.After this, Walton etc. reported by Hilbert-Johnson reaction synthesized 2 '-C-methyl-5-fluorine cytidine, 2 '-C-methyl-5-floxuridine and 2 '-and 3 '-C-methylcytidine (Walton et al., AntiviralNucleosides 12:306-309 (1969)).Yet, unexpectedly, becoming 2 by N-acetylcytosine-amalgamation '-during the C-methylcytidine, produced a large amount of O-glycosides, and hydrargyrum itself is toxic reagent, wish to avoid using (Id.).In two kinds of synthetic methods of reports such as Walton, the productive rate of end product all only has an appointment 11%.
In 1997, Harry-O ' Kuru etc. reported preparation '-synthetic route (Harry-O ' Kuru et al., J Org.Chem., 62:1754-9 (1997)) of C-side chain ribonucleotide.Can be from 1,3 of commercial acquisition, 5-three-O-benzoyl-α-D-ribofuranose is used as initiation material, and it is to be made by D-ribose or D-arabinose (D-arabopyranose).1,3,5-three-O-benzoyl-α-D-ribofuranose is oxidized under 2-OH and the effect of Dess-Martin periodinane reagent freely, produces 1,3,5-three-O-benzoyl-2-ketone-ribofuranose with and corresponding hydrate.Product and the hydrate and the excessive MgSO of expectation 4Stir, and place and spend the night.Filtering mixt also concentrates and produces a large amount of pure ketone products then.Use MeMgBr/TiCl 4(perhaps also available MeTiCl 3, CH 2=CHMgBr/CeCl 3, or TMSC=CLi/CeCl 3) handle the 2-ketose obtain, with near 5: 3 the expectation product and the ratio of isomeric form (Id.at 1755), produce 1 of expectation, 3, alkyl, alkenyl or alkynyl ribose furanoside (ribofuranoside) and its ester exchange reaction that 5-three-O-benzoyl-2-replaces the end group heterogeneous mixture of isomer, and α-and β-2,3, alkyl, alkenyl or the alkynyl ribose furanoside that 5-three-O-benzoyl-2-replaces ( Id.at 1755).Then, handle by Benzenecarbonyl chloride., DMAP and triethylamine, the ratio (Id.) of productive rate with about 70% and 4: 1 beta/alpha is converted into independent expectation product 1,2,3 with the alkylating ribose furanoside of 2-, 5-four benzoyls-2-alkyl ribose furanoside.
Beigelman etc. reported by D-glucose and D-ribose Synthetic 2 '-C-methyl-nucleoside (Beigelman etal., Carbohydrate Research, 166:219-232 (1987)).When using the D-glucose, prepared 1,2 as initiation material; 5; 6-two-O-isopropylidene-3-C-methyl-α-D-allofuranose, by 5, the insertion p-methyl benzoyl on 6-O-dibutyl stannylidene derivatives selectively ground and transform (Id.).Then, the trifluoroacetic acid with 90% is handled and is carried out periodate oxidation, removes the formyl on the chemical compound, carries out acetylation (Id.).Ultimate yield is about 77% (Id.).When using D-ribose as initiation material, make 2 of 5-position protection, 3-dimethyl-isopropylidene derivant and formaldehyde carry out aldol condensation, handle (Id.) with excessive toluene p-sulfonic acid chloride in pyridine then.Then, according to known condition in the document, for example, Kuhn methylates, uses LiAlH 4Reduction, acid-catalyzed hydrolysis in THF, and in pyridine excessive Ac 2Acetylation (Id.) is carried out in boiling among the O, with the various products of this compound formation.
Novak and Sorm play-by-play by sodium borohydride reduction, prepare crystallization 2-C-methyl D-ribose and derivative compound (J.J.K.Novak﹠amp thereof by 2-C-methyl D-ribose acid lactone; F.Sorm, CollectionCzechoslov.Chem.Commun., 34:857-866 (1969)).They have described the character at the hydroxyl of the 2-position of 2-C-methyl-ribose furanoside, and particularly the hydroxyl with the similar position that is positioned at corresponding lactone compares (Id.).Though the hydroxyl on the lactone is acetylation under condition known in the art easily and produces 2; 3; 5-three-O-acetyl group and 2; 3; 5-three-O-benzoyl-2-C-methyl D-ribose acid lactone, but under similar condition, only produce 3 by 2-C-methyl-ribose furanoside; 5-two-O-acetyl group-and 3,5-two-O-benzoyl-2-C-methyl D-ribose acid lactone (Id.).
After this; Novak has described the 2-C-methyl isophthalic acid; chirality-the photoactive characteristics of 4-lactone; the 2-C-methyl isophthalic acid; the 4-lactone is to be made by the hypoiodite oxidation by D-lyxose and D-xylose; and on the C3 of lactone and C5, p-toluoyl protecting group (J.J.K.Novak, Collection Czechoslov.Chem.Commun., 39:869-882 (1974)) is arranged.Particularly, by 3,5-p-toluoyl-2-Br, 2-CH 3-ribonic acid-1, the 4-lactone has synthesized 2-CH by hydrolysis 3-ribonic acid-1,4-lactone (Id.).Yet Novak has mentioned when separating the lactone of protection difficult, and has generated the product (Id.at 871) of pulpous state when trial is cut off lactone by the alcoholysis of alkali.
Tokyo Tanabe Co., Ltd. (JP 61-212592) and BASF Aktiendgesellschaft (EP 0 288847) have reported the arabinose by D-, a kind of initiation material of production ribose commonly used prepares the method for unprotected D-ribose.
Tokyo Tanabe Co.; Ltd.; instructed moisture D-arabinose in organic solvent; the preferred epimerism in the presence of molybdenum (VI) acid and boronic acid compounds; collect and pass through reactant liquor by the cation exchange material (preferably changing the multi-styrene sulfonic acid type strong-acid ion exchange resin of Ca-type into) of 2-or 3-valency; the water elution separates unprotected ribose, and collects ribose chemical compound (JP 61-212592, summary).
Water/alcoholic solution that BASF has instructed the D-arabinose load is arranged molybdenum (VI) chemical compound base exchanger in the presence of, the continuous processing that in solvent, heats.Eluate is collected and is dry, adds methanol or ethanol in exsiccant eluate, and mixture is cooled to about 0 ℃, makes the crystallization of unprotected D-arabinose, and then to its separation and recovery.According to road known in this field method, concentrate and the remaining filter liquor of purification by the form of highly acid ion exchanger, and at crystallization stage, any arabinose/ribose that breaks away from product all is recycled to (EP 0288847) in the arabinose with Ca2+.
Tokyo Tanabe Co., the method for Ltd. and BASF all requires complicated and expensive equipment and reagent, and the product chemical compound that obtains still needs to add protecting group.
Japan Tobacco; Inc.; be positioned at 5-OH on γ-ribose acid lactone by protection, made the 3-DPA-lactone, wherein; having used hydrochloric acid or anhydride and tertiary amine makes 3-OH that β-elimination take place; between carbon 2 and 3, form two keys, make 2-OH that acetylation takes place simultaneously, finally the two keys between C-2 and the C-3 are carried out catalytic hydrogenation; and remove protecting group, generate 5-OH.Referring to EP O 526,655A1, EP O 553,358 A1 and EP O 553,358B1, and their U.S. patent families U.S.5,322,955 and U.S.5,391,769.
Also comprise following about synthetic substituent ribose acid lactone and synthetic other related works of sugar analogue that have protection:
Li et al., Organic Letters, 3 (7): 1025-28 (2001) is by 1,3,5-three-O-benzoyl-α-D-ribofuranose synthesized '-C-β-trifluoromethyl pyrimidine nucleoside, convert it into 3 then, 5-two-O-benzoyl-2-C-β-trifluoromethyl-α-D-1-ribofuranose bromide.This bromination derivative compound is found to be in the effecting reaction intermediate when forming nucleoside.
Beigelman et al., Bioorg Khim., 12 (10): 1359-65 (1986), by 1,2,5, the benzyl of 6-two-O-isopropylidene-3-C-methyl-α-D-allofuranose has been combined to 2-C-methyl D-ribose derivative compound, forms first intermediate; This first intermediate of hydrolysis and highly selective acylation forms 3-O-Bian Ji-1,2-O-isopropylidene-3-C-methyl-6-O-toluoyl-c-D-allofuranose; Continue to remove isopropylidene, oxidation (using periodic acid), the piptonychia acidylate, Bian Jihua is taken off in acetylation, acetylation once more, thus obtain final products 1,2,3-three-O-acetyl-2-C-methyl-5-O-toluoyl-β-D-ribose furanose.
Feast et al., Acta Chemica Scandinavica 19:1127-34 (1965) has reported the preparation of α-D-gluconic acid, it is 2-C-methyl D-ribose-valeric acid, be by 1,4-lactone intermediate, the D-fructose that replaces through alkali treatment D-fructose or 1-O-obtains.
Kohn et al.; J.Am.Chem.Soc.; 87 (23): 5475-80 (1965) has described the brief path that obtains the furanose derivative of aldose, and it is to adopt the disiamyl borine as Reducing agent, and Fourth Ring hexyloxy-gamma lactone is reduced into its corresponding four acyl group hexose furanoses.This reaction is particularly important for the formation of the intermediate in synthetic C-1 ' furyl nucleoside.
Kempe et al., Nucleic Acids Res., 10 (21): 6695-6714 (1982) reported the ribonucleotide of protection along 2 ', 3 '-glycol optionally 2 '-benzoylation, and with 2 '-the benzoate isomery turns to 3 '-benzoate.The nucleoside of these protections is used to synthesis of oligonucleotides ribonucleotide on the solid silicone supporter, carries out deprotection then, and such benefit is that inner nucleoside decomposition is minimum.
The United States Patent (USP) U.S.4 of Schmidt etc., 294,766 describe in detail by the synthetic pure ribose acid lactone of the mixture of ribose acid lactone and arabic acid lactone.Ribose acid lactone is the intermediate when forming riboflavin (riboflavin (vitamin B2)).The mixture of arabic acid potassium and ribonic acid potassium " is lactonized ", and what obtain about 70% separates by fractional crystallization with diox or glycol monoethyl ether for the interior ester admixture of ribose acid lactone.Adopt known road method to lactonize, for example, by the use ion exchanger, or at H 2SO 4Or K 2SO 4Following concentrating lactones, and filter out precipitate.
Nucleoside coupling
Walton has described the synthetic of branched nucleosides, and it is by making 2,3,5-three-O-acyl group-2-(or 3)-C-alkyl ribose furanose halogenide and chlorine hydrargyrum purine or pyrimidine compound reaction and prepare (U.S.3,480,613).3-low alkyl group-D-ribose furanose halogenide intermediate is with 1, and 2-O-isopropylidene-5-O-acyl-alpha--D-is red-and penta furan-3-ketose is as initiation material, and introduce low alkyl group by making this chemical compound Yu Geshi reagent reacting at C3 and obtain.Then, undertaken by two approach: in article one approach, 5-O-acyl group-1,2-O-isopropylidene-3-low alkyl group-D-ribose furanose is carried out acidic alcoholysis, forms alkyl 5-O-acyl group-3-low alkyl group D-ribose furanoside; It is an alkyl 2,3 by acidylate again, 5-three-O-acyl group-3-low alkyl group-D-ribose furanoside; Basic solvent decomposes resultant ribose furanoside and further hydrolysis in the strong acid solution medium by carrying out then, and is converted into free sugar, perhaps by carrying out the halogen substitution reaction change halogeno-sugar in solvent suitable.In the second approach; 5-O-acyl group-1; in the 2-O-isopropylidene-3-low alkyl group-atent solvent of D-ribose furanose under alkali condition (pyridine) by acidylate; form 3; 5-two-O-acyl group-1; 2-O-isopropylidene-3-low alkyl group-D-ribose furanose, it is hydrolyzed in strong acid again, and then acidylate obtains the target intermediate.Then, by making 2,3; 5-three-O-acyl group-D-ribose furanose halogenide and chlorine hydrargyrum 2; 6-two substituted purins react in 100 ℃ to 140 ℃ toluene or dimethylbenzene equal solvent, and preparation replaces at the 2-that the two or three-digit of sugar moieties has side chain, 6-replaces or 2, the dibasic purine nucleosides of 6-.By with 2; 4-dialkoxy-pyrimidine reaction forms 1-(2; 3; 5-three-O-acyl group-2 (or 3)-C-low alkyl group-D-ribose furanose)-4-alkoxyl-2 (1H)-pyrimidone; by 2; 3; 5-three-O-acyl group-2 (or 3)-C-low alkyl group-D-ribose furanose halogenide is derived and has been obtained having the nucleoside of expectation pyrimidone base; it has amino-substituted compounds with the C-4 that ammonia or primary amine or secondary amine reaction obtain at pyrimidone again; perhaps hydrolysis under acidity or alkali condition obtains having at C-4 the pyrimidone alkali of hydroxyl.Yet, synthetic a plurality of steps, specific conditions and the multiple toxic reagent of comprising of Walton.
As shown in Figure 5, prior art has been instructed and used BSA in acetonitrile, makes 1,2,3,5-four-O-benzoyl-2-C-methyl-β-D-ribofuranose ( 4) and N 4The coupling of-benzoyl cytosine.Reactant mixture is heated and refluxed about 30 minutes, adds lewis acid SnCl then 4, solution heats once more and refluxed about 3.5 hours, obtain 4-NH-benzoyl-2 ', 3 ', 5 '-three-O-benzoyl-β-D-2 '-C-methyl-cytidine ( 5a).By with the washing of ethyl acetate and saturated sodium bicarbonate aqueous solution and carry out abundant chromatogram purification obtain chemical compound ( 5a).With the in advance full methanol solution of ammonia to ( 5a) spend the night and handle to remove the benzoyl protecting group, obtain β-D-2 '-C-methyl-cytidine ( 6).
Prodrug
Reactive compound pharmaceutically is sometimes with the prodrug forms administration of esterification.Wherein commonly used with carboxylate, and phosphonate ester and phosphate ester be because hydrolysis and may produce toxic byproduct in vivo, thereby less use.(referring to the United States Patent (USP) U.S.6 of Erion etc., 312,662).The acyloxy Arrcostab is used as phosphonate ester and phosphate compound sometimes, as annular phosphonate and aromatic ester particularly prodrug (Farquhar et al., J.Pharm.Sci., (1983), 72 (3): 324 of phenyl and Bian Ji ester; The United States Patent (USP) U.S.6 of Erion etc., 312,662).As nucleoside, phosphonic acids is phosphine formic acid and PMEA (Adefovir for example; 9-(2-phosphono methoxyl group-ethyl) adenine) the same with the carboxylic acid or the ether lipid prodrug of nucleoside, all demonstrate antiviral activity (the United States Patent (USP) U.S.6 of Gosselin etc., 458,773).
For a long time, the synthetic and design of typical prodrug includes 5 of nucleoside or nucleoside analog ' position.In the nucleoside of above-mentioned Gosselin etc. report, 5 '-H of OH replaced by following any group: acyl group comprises that the non-carbonyl moiety of ester group wherein is selected from straight chain, side chain or cyclic C 1-C 20Alkyl, phenyl or Bian Ji; Natural and non-natural aminoacid; 5 '-ether lipid or 5 '-phosphoric acid ether lipid; Alkoxyalkyl comprises methoxy; Aralkyl comprises Bian Ji; Aryloxy alkyl, as, phenoxymethyl; Aryl comprises phenyl, and is optional with halogen, C 1-C 4Alkyl or C 1-C 4Alkoxyl replaces; Dicarboxylic acids is as succinic acid; Sulphonic acid ester, as, alkyl sulfonate esters or sulfonic acid aralkyl ester comprise methylmesylate; Or single-, two-or triguaiacyl phosphate.
Matulic-Adamic etc. (U.S.6,248,878) have reported the synthetic of nucleoside analog.This analog comprises the ribofuranose ring, and pyrimidine base by oxygen atom and replacement is connected with phosphorus-containing groups in 3 ' position on it.Phosphorus-containing groups comprises dithioates or phosphoramidite, perhaps can be the part of oligomeric nucleoside.The further reaction and the nucleoside and the nucleoside analog of final expectation are provided of these preceding drug compounds.These chemical compounds are synthetic by multistep, comprise that coupling has hydroxyl or acetate at C-1, the ribofuranose and the 4-OSiMe that have the benzoyl protecting group at C-2-, C-3 and C-5 as initiation material 3Pyrimidine produces 1-(2,3,5-three-O-benzoyl-ribose-furanose)-pyrimidin-4-one; Then, in the product of first reaction, add the methanol that contains ammonia, to remove the benzoyl protecting group; Then, the reaction of DMT-Cl/Pyr and unprotected chemical compound, 5 of ribofuranose '-O position addition DMT; Then, with TBDMS-Cl, AgNO 3The ribofuranose that replaces with Pyr/THF and 5 '-O-DMT reacts; Carry out the phosphitylation (phosphitylation) of standard at last, 3 '-O produces phosphorus-containing groups.Each synthesizes and comprised at least 4～7 steps.
The prodrug of descriptions such as Chu is azide derivatives and compositions, comprises the nucleoside analog (U.S.6,271,212) of nucleoside and phosphorylation.The advantage of this azide prodrug is to pass through blood-brain barrier, and longer half-life and the bioavailability of Geng Gao are provided, and the stability that increases the pharmaceutically active form.Yet Chu etc. have reported to be that their azide prodrug of preparation needs the synthetic of long-term and multistep.
The antiviral prodrug of descriptions such as Borretzen is nucleoside and nucleoside analog.They have reported some fatty acid ester of anti-viral nucleoside and nucleoside analog, wherein unsaturated C18 of some monobasic or C20 fatty acid by acylation process be connected 5 of nucleoside and nucleoside analog '-position (U.S.6,153,594).This process was carried out in the presence of catalyst 24-60 hour.Finish product through organic solvent extraction and separate, by selecting appropriate solvent to carry out chromatograph and/or recrystallization carries out purification.The yield percentage of product changes between 15-82%.But Borretzen etc. do not use term " prodrug ".
1999, McCormick etc. described and have used unprotected ribose as initiation material, 3 of guanosine '-OH forms carbonic ester.(McCormick et al.，J Am.Chem.Soc.1999，121(24)：5661-5)。McCormick connects, utilizes some protecting group, sulfonation and last deprotection by introducing O-and N-glycoside continuously, step by step, has finished synthetic.As one of step in their method, McCormick etc. make unprotected guanosine and BOC-acid anhydride, DMAP, Et 3N and DMSO at room temperature reacted 4 hours, directly obtain 3 of guanosine '-carbonic ester of OH.
Equally in 1999, Tang etc. disclose preparation '-method of the phosphoramidite prodrug of C-Beta-methyl-cytidine ribonucleotide.(Tang et al.，J.Org.Chem.，1999，64：747-754)。Go together equally with they many, Tang etc. are 1,2,3,5-four-O-benzoyl-2-C-methyl-β-D ribofuranose with through the 4-N-of complete silylanization deactivation benzoyl cytosine at lewis acid and SnCl 4Down reaction of existence, with it as first step in synthesizing.(Id.at 748，SchemeI a)。
2000, Novirio Pharmaceuticals (now for Idenix) found by using the amino-acid ester form of anti-viral nucleoside, the stability and the bioavailability of anti-viral nucleoside be improved (U.S. serial number No.09/864,078, in the examination; U.S. serial number No.10/261,327, in the examination; WO01/90121; And U.S. provisional application 60/377,983 and 60/392,351).The method for preparing the amino-acid ester of these nucleoside and nucleoside analog at first is with suitable can being begun by the side chain β-D of suitable protecting group such as silicyl selective protection or β-L nucleoside, then, adopt known method to carry out deprotection (Zhang et al., Tetrahedron Letters, 1992,33:1177-80; Greene et al., Protective Groups in Organic Synthesis, John Wiley﹠amp; Sons, 2 NdEdition (1991); Kerr et al., J.Pharmaceutical Sciences, 1994,83:582-6; Tang et al., J.Org.Chem., 1999,64 (3): 747-754; And Cavelier et al., TetrahedronLetters, 1996,37:5131-4).Then, branched nucleosides of being protected by selectivity and suitable acry radical donor, as acyl chlorides and/or acyl acid anhydride or active acid coupling, be coupled in suitable proton solvent or the aprotic solvent and under suitable reaction temperature, carry out, obtain 1 ', 2 ', 3 ' or 2 ' or 3 ' prodrug of 4 ' side chain β-D or β-L nucleoside, choose wantonly in the presence of suitable coupling agent and carry out (referring to Synthetic Communications, 1978,8 (5): 327-33; J.Am.Chem.Soc., 1999,121 (24): 5661-5; Bryant et al., Antimicrob.Agents Chemother., 2001,45,229-235; Standring et al., Antiviral Chem.﹠amp; Chemother., 2001,12 (Suppl.1), 119-129; Benzaria et al., Antiviral Res., 2001,50, A79; Pierra et al., Antiviral Res., 2001,50, A79; And Cretton-Scott et al., Antiviral Res., 2001,50, A44).Available coupling reagent is that those can include but not limited to various carbon imidodicarbonic diamide, CDI, BOP and carbonyl diurethane acid imide with chemical compound and wherein a part of material that is connected on other parts.For example, for 2 '-branched nucleosides 3 '-prodrug, nucleoside is preferably protected, but by carbon imidodicarbonic diamide coupling agent, directly is coupled on alkanol acid or the amino acid residue.
The method of the prior art shown in Fig. 5 comprise in the following order reaction prepare 3 of cytidine '-the L-valine ester nucleoside prodrugs: with 1,2,3,5-four-O-benzoyl-2-C-methyl-β-D-ribofuranose ( 4) join BSA and N in the acetonitrile 4In the mixture of-benzoyl cytosine, be heated to and refluxed about 30 minutes, then, in solution, add lewis acid SnCl 4, solution is heated to once more and refluxed about 3.5 hours, generation 4-NH-benzoyl-2 ', 3 ', 5 '-three-O-benzoyl-β-D-2 '-C-methyl-cytidine ( 5a).Chemical compound ( 5a) obtain by washing and carry out abundant chromatogram purification with ethyl acetate and saturated sodium bicarbonate aqueous solution.With the pre-saturated methanol solution of ammonia to ( 5a) spend the night and handle to remove the benzoyl protecting group, obtain β-D-2 '-C-methyl-cytidine ( 6).Chemical compound ( 6) DMF solution and N, the dinethylformamide dimethylacetal at room temperature reacted about 1.5 hours, obtained having at C4 the cytidine N of the amino of protection 4-[(dimethylamino) methylene]-β-D-2 '-C-methyl-cytidine ( 7); Then, the cytidine of aminoacid-protection ( 7) dried pyridine solution and imidazoles and TBDPSCl at room temperature reacted about 6 hours, obtain 5 '-cytidine of O silicyl protection ( 8); Then, in the presence of DEC, DMAP and THF/DMF, with the N-Boc-L-valine at room temperature join 4-and 5 '-protection β-D-2 '-C-methylcytidine ( 8), approximately two days later, obtain the β-D-2 '-C-methyl-cytidine 3 of 4-and 5 '-protection '-the O-L-N-BOC-L-valine ester ( 9); 3 of the β-D-2 '-C-methyl-cytidine of 4-and 5 '-protection '-the O-L-N-BOC-L-valine ester ( 9) dried methanol solution in add amine fluoride, then mixture reflux with remove 5 '-silicyl and 4-amino protecting group, generate 3 of β-D-2 '-C-methylcytidine '-O-L-N-(uncle-butoxy carbonyl) L-valine ester ( 10), it is purified by chromatographic column; At last, to 3 of β-D-2 '-C-methylcytidine '-O-L-N-(uncle-butoxy carbonyl) L-valine ester ( 10) dry ethyl acetate solution in, add 20% HCl/ ethyl acetate solution, with mixture stir about 2 hours, remove the BOC-protecting group, generate end product β-D-2 '-C-methylcytidine '-hydrochlorate of O-L-valine ester ( 11).The prior art of representing among Fig. 6 synthetic used uracil replace benzoyl cytosine prepare chemical compound ( 11) β-D-2 '-C-methylcytidine.
In sum, if can have a kind of effective method to prepare nucleoside or nucleoside analog will be favourable.Such nucleoside or nucleoside analog for example be 2 '-methyl-nucleoside or 2 '-methyl-3 '-O-valine-nucleoside, their intermediate comprises 2-C-methyl-ribose acid lactone and 2-C-methyl D-ribofuranose, and their salt and/or prodrug.
Another object of the present invention provide 3 of nucleoside '-method of OH selectivity addition, to derive preceding drug compound.
A further object of the present invention provides the method for a kind of saccharide of effective preparation protection like chemical compound, and this method comprises the minimized number step, and uses cheap initiation material.
Another purpose of the present invention is to compare with synthetic similar product, significantly reduces the time of the sugared intermediate of preparation protection.
In addition, one of purpose of the present invention provides a kind of method, and it can be finished in a few hours, and provides end product with high yield and high-purity.
Another object of the present invention provides a kind of method, and this method has adopted wieldy nontoxic reagent, and its final products are easy to by routine techniques separatedly, and enlarges scale easily.
Another purpose of the present invention is high yield and the high-purity acquisition end product to surpass at least 90 or 95%.
That a further object of the present invention is to use is nontoxic, the reagent of easy operating.
Summary of the invention
The invention discloses a kind of new change improved be used to prepare nucleoside and nucleoside analog, as β-D and β-L2 '-C-methyl-nucleoside and 2 '-C-methyl-3 '-method of O-ester nucleoside and their salt and/or prodrug, this method is by using one or more consumptions reagent still less in the shorter time, by more simple purification step, obtain the productive rate higher than prior art.In addition, method of the present invention satisfy in the industrial requirement highly beneficial.
In the specific embodiment of the present invention, particularly including the method that contains following steps: (a), obtain 2-C-methyl D-ribonic acid-gamma lactone with D-fructose and CaO reaction; And/or (b) make 2-C-methyl D-ribonic acid-gamma lactone and the appropriate reductant such as the Red-Al of optional protection, and choose wantonly in solvent such as ethanol and react, obtain optional 2-C-methyl D-ribofuranose of protecting; And/or 2-C-methyl D-ribofuranose and the unprotected base that (c) will choose protection wantonly carry out coupling, coupling in this way with cytosine in the presence of activator such as silylation reagent (for example BSA), choose wantonly at lewis acid such as SnCl 4Exist and to carry out down, obtain 2 of optional protection '-C-methyl-nucleoside, for example, 2 '-C-methyl-cytidine; And/or (d) under reagent, reaction condition (solvent, response time etc.) and the extraction/purification technique of optimizing, obtain 2 '-C-methyl-nucleoside, for example 2 '-C-methyl-cytidine 3 '-ester.In a concrete embodiment of the present invention, course of reaction is shown in Fig. 1 and 4.
Also provide a kind of effectively can enlarge scale, with the method for produced in high yields nucleoside prodrugs, this nucleoside 3 of nucleoside '-there is the part that can split the position.
Further provide the nontoxic reagent of employing of saving cost to prepare the method for nucleoside, nucleoside analog, its salt and prodrug.Relatively be shown in the method for the present invention and the method that is shown in the prior art of Fig. 5 of Fig. 4, as can be seen, economic benefit increases to some extent in improved method.Relatively be shown in the method for the present invention among Fig. 4, and the method that is shown in the prior art among Fig. 5, can prove the economy of the process that in improved method, improved.
In addition; provide a kind of preparation 2-C-methyl effective, that can enlarge scale sugared intermediate again; as independently 2; 3; 5-(independent optional protection) and unprotected 2-C-methyl D-ribonic acid-gamma lactone (being also referred to as 2-C-methyl-ribose acid lactone) and independently 1; 2,3, the synthetic method of 5-(independent optional protection) and unprotected 2-C-methyl D-ribofuranose.Compared with the method for prior art, this method has adopted one or more cheap reagent, and required time is shorter, and purification process is more easy, and productive rate is higher.
The distinguished one side of the present invention has been to use the reagent of particular combinations, thereby has saved the separation in the synthesis step, segregation and/or purification.The selection of particular agent makes most initiation materials be converted into product, and has reduced the racemization effect, can easily remove from end product, thereby the ratio more efficiently synthetic method of method in the past is provided.The long and is to shorten the time that obtains final prodrug product, and the yield percentage of expected product improves.
In addition, need less time and reagent still less to improve whole cost efficiency and the industrial method that enlarges scale and safety.
In one embodiment; method of the present invention is a kind of preparation of nucleoside, its 2 '-two the replacement, take place on the C, as 2 '-methyl-nucleoside or 2 '-methyl-3 '-O-valine-nucleoside; its intermediate; for example 2,3,5-(independent optional protection) and unprotected 2-C-methyl D-ribonic acid-gamma lactone (being also referred to as 2-C-methyl-ribose acid lactone) and 1; 2; 3,5-(independent optional protection) and unprotected 2-C-methyl D-ribofuranose and its salt and/or prodrug.One preferred embodiment in, the present invention is used to prepare 2,3,5-(independent optional protection) or unprotected 2-C-methyl D-ribonic acid-gamma lactone.Another preferred embodiment in, the present invention is used to prepare 1,2,3,5-(independent optional protection) or unprotected 2-C-methyl D-ribofuranose.Another preferred embodiment in, the present invention is used to prepare β-D-2 '-C-methyl-cytidine (4-amino-1-(3,4-dihydroxy-5-methylol-3-C-methyl-oxolane-2-yl)-1H-pyrimid-2-one).Another preferred embodiment in, the present invention be used to prepare 3 of β-D-2 '-C-methyl-cytidine (2-amino-3-methyl-butanoic acid 5-(4-amino-2-oxygen-2H-pyrimidine-1-yl)-4-hydroxyl-4-C-methyl-2-methylol-oxolane-3-base ester) '-O-amino-acid ester (including but not limited to valyl ester) or its preferred hydrochloride form.Nucleoside, nucleoside analog, salt or ester prodrugs by the present invention's preparation can be used as the intermediate for preparing various other nucleoside analogs, perhaps directly as antiviral and/or anti-tumor agent comprising salmosin.
In one embodiment, improved method of the present invention comprises makes cytosine and activator, as BSA, chooses wantonly at lewis acid such as SnCl 4Exist down, and 1,2; 3; 5-(independent optional protection) or unprotected 2-C-methyl-β-D-ribofuranose reaction, generation 4-amino-1-(3,4-(independent optional protection-hydroxyl)-5-O-protection-hydroxyl methylene-3-methyl-oxolane-2-yl)-1H-pyrimid-2-one (see figure 4)
Figure A20038010957600241
Wherein, each P 1, P 2, P 3And P 4Be hydrogen or suitable oxygen protecting group independently, for example, acyl group is preferably benzoyl;
Then,
If desired; then optionally with the 4-amino-1-(3 in the preceding step reaction; 4-(independent optional protection-hydroxyl)-5-(optional O-protection-hydroxyl methylene)-3-methyl-oxolane-2-yl)-the 1H-pyrimid-2-one sloughs protection; formation 4-amino-1-(3,4-dihydroxy-5-methylol-3-methyl-oxolane-2-yl)-1H-pyrimid-2-one ( 6).
For example, if intermediate ( V) P 1, P 2And P 3Be benzoyl, then this chemical compound can react with NaOMe/MeOH, obtain 4-amino-1-(3,4-dihydroxy-5-methylol-3-methyl-oxolane-2-yl)-1H-pyrimid-2-one ( VI), it is also referred to as β-D-2 '-C-methyl-cytidine, can choose recrystallization from ethanol etc. wantonly, obtains the purified form of β-D-2 '-C-methyl-cytidine.This chemical compound can be used as anti-virus formulation as required, perhaps further derives and becomes prodrug.
Then, intermediate ( VI) selectively by optional protection; as 3 '-position is esterified; use known method to choose deprotection wantonly; 3 of acquisition β-D-2 '-C-methyl-cytidine '-ester prodrugs; for example; 3 of β-D-2 '-C-methyl-cytidine (2-amino-3-methyl-butanoic acid 5-(4-amino-2-oxygen-2H-pyrimidine-1-yl)-4-hydroxyl-4-C-methyl-2-methylol-oxolane-3-base ester) '-O-L-valine ester, perhaps its preferred hydrochloride form.
As a non-limiting instance of the present invention, if 3 '-L-valine ester is preferred, then esterification can comprise the represented step that goes out among Fig. 4,, makes β-D-2 '-C-methyl-cytidine and Me that is 2NCH (OMe) 2In DMF, react, form ( 7), N[1-(3,4-dihydroxy-5-methylol-3-methyl-oxolane-2-yl)-2-oxygen-1,2-dihydro-pyrimidine-4-yl]-N, dinethylformamide, its be ( VI) the form of amino-protection; Make ( 7) react in DCM with TBDPSCl and imidazoles, generate ( 7) 5 '-form of silicyl-protection, N '-1-[5-(tert-butyl group-diphenyl-siloxy methyl)-3,4-dihydroxy-3-methyl-oxolane-2-yl]-2-oxygen-1,2-dihydro-pyrimidine-4-yl }-N, dinethylformamide ( 8); Make ( 8) react in DCM with N-Boc-L-valine, EDC and DMAP, form 2-t-butoxycarbonyl amino-3-methyl-butanoic acid 2-(tert-butyl group-diphenyl-siloxy methyl)-5-[4-(dimethylamino-methene amido)-2-oxygen-2H-pyrimidine-1-yl]-4-hydroxy-4-methyl-oxolane-3-base ester ( 9); Make ( 9) and NH 4F reacts in MeOH; and add ethyl acetate (for the ammonia that prevents to separate out cut off 3 '-the O-L-valine ester); to remove silicyl and amino protecting group; and the backflow mixture, obtain 2-t-butoxycarbonyl amino-3-methyl-butanoic acid 5-(4-amino-2-oxygen-2H-pyrimidine-1-yl)-4-hydroxyl-2-methylol-4-methyl-oxolane-3-base ester ( 10), it is purification by simple crystallization; Make at last, ( 10) react in EtOH with HCl, obtain 2-amino-3-methyl-butanoic acid 5-(4-amino-2-oxygen-2H-pyrimidine-1-yl)-4-hydroxyl-2-methylol-4-methyl-oxolane-3-base ester, dihydrochloride ( 11) as final products.
The benzoyl cytosine of using in the use cytosine replacement prior art or the cytosine of other protections have improved economy and have simplified purge process.
Method of the present invention is that with the advantage that similar prior art is compared the reagent of its use can reduce to about 50%.Even if use reagent still less, to compare with immediate prior art, the gross production rate of method of the present invention but is improved, and such as in an example, productive rate brings up to 38% by 12%.Further advantage is embodied in to have shortened and finishes the synthetic cycle of prodrug.Compare with the synthetic of prior art, the method for improvement of the present invention with the cycle brief about 80%.This mainly is for following four reasons: i) along with the load increase, reduced required reaction batch; The ii) raising of yield percentage; Iii) use easy-to-handle solvent and reagent; And iv) avoided the chromatographic purification step that expends man-hour.
The distinguished aspect of the present invention has been to use the reagent of particular combinations, thereby has saved the separation in the synthesis step, segregation and/or purification.The selection of particular agent makes most initiation materials be converted into product, and has reduced the racemization effect, can easily remove from end product, thereby the ratio more efficiently synthetic method of method in the past is provided.The long and is to shorten the time that obtains final prodrug product, and the yield percentage of expected product improves.In addition, need less time and reagent still less to improve whole cost efficiency and industrial upgradeable and safe method.
In another embodiment of the present invention; by making 2; 3; 5-(independent optional protection)-2-C-methyl D-ribose acid lactone and Reducing agent are as two (2-methoxy ethoxy) sodium aluminum hydrides (Red-Al); choose wantonly in solvent such as ethanol and react, obtained 1,2; 3,5-(independent optional protection)-2-C-methyl-β-D-ribofuranose.
Figure A20038010957600271
Wherein, each P 1, P 2And P 3Be hydrogen or suitable oxygen protecting group independently, for example, acyl group is preferably benzoyl;
Then,
If desired, then the ribofuranose derivative compound in the reaction of preceding step of protection (for example benzoylation) optionally forms 1,2,3,5-(independent optional protection)-2-C-methyl-β-D-ribofuranose,
Figure A20038010957600272
Wherein, P 4Be hydrogen or suitable oxygen protecting group independently, for example, acyl group is preferably benzoyl.
Use Red-Al as Reducing agent of the present invention, provide unexpectedly to have specific stereochemical product, this has just realized effective separation.Thereby simplified the separation of final expected product.
In another embodiment of the invention, 2,3,5-(independent optional protection)-2-C-methyl D-ribose acid lactone obtains by making D-fructose and CaO reaction;
Figure A20038010957600273
Then, as needs, then optional protection lactone for example, with Benzenecarbonyl chloride. (or other suitable acyl chlorides of working as), forms 2,3,5-(independent optional protection)-2-C-methyl D-ribose acid lactone;
Figure A20038010957600281
Wherein, each P 1, P 2And P 3Be hydrogen or suitable oxygen protecting group independently, for example, acyl group is preferably benzoyl.
In addition, in order to separate pure single end group isomerized products (for example, the pure form of essence refers at least 95%), can increase purification step as required.
Method of the present invention has adopted cheap D-fructose as initiation material, has therefore saved a large amount of costs for Producer.This point is even more important for needs with when estimate enlarging commercial Application.
Except using the great economic benefit that D-fructose brings as initiation material, the distinguished one side of the present invention also is to use calcium oxide (CaO) as the reagent in the first step of method.CaO is added in the aqueous solution of D-fructose, preparation 2-C-methyl D-ribonic acid-gamma lactone.This step finishes soon itself, has improved productive rate 30-40% compared with similar step of the prior art.In addition, CaO is nontoxic, be easy to use, and with fructose and water good mixing.
Can utilize precipitant to come from solution, to remove calcium.In one embodiment, in reactant mixture, add CO 2With the acid stronger than ribonic acid, one preferred embodiment in, adding be organic acid, form calcium carbonate.The organic acid that is fit to includes but not limited to: oxalic acid, malonic acid, succinic acid, 1,3-propanedicarboxylic acid, adipic acid, suberic acid, decanedioic acid, Azelaic Acid, maleic acid, acetic acid, propanoic acid, isopropylformic acid., acrylic acid, methacrylic acid, butanoic acid, valeric acid caproic acid or caproic acid.
In addition, the advantage of total process that Fig. 1 and Fig. 4 combine is, compares with art methods, and the reagent of its use can reduce to about 50%.Even if use reagent still less, to compare with immediate prior art, the gross production rate of method of the present invention but is improved, and such as in an example, productive rate brings up to 38% by 12%.Further advantage is embodied in to have shortened and finishes the synthetic cycle of prodrug.Compare with the synthetic of prior art, the method for improvement of the present invention with the cycle brief about 80%.This mainly is for following four reasons: i) along with the load increase, reduced required reaction batch; The ii) raising of yield percentage; Iii) use easy-to-handle solvent and reagent; And iv) avoided the chromatographic purification step that expends man-hour.
Therefore, in an embodiment of the invention, provide by D-fructose preparation 2 '-method of C-methyl D-cytidine, it may further comprise the steps:
(a) make D-fructose CaO reaction, obtain 2-C-methyl D-ribonic acid-gamma lactone;
(b) as needs, then optional protection lactone for example, with Benzenecarbonyl chloride. (or other suitable acyl chlorides of working as), forms 2,3,5-(independent optional protection)-2-C-methyl D-ribose acid lactone;
Figure A20038010957600292
Wherein, each P 1, P 2And P 3Be hydrogen or suitable oxygen protecting group independently, for example, acyl group is preferably benzoyl;
(c) make 2,3,5-(independent optional protection)-2-C-methyl D-ribose acid lactone and Reducing agent are chosen wantonly in solvent such as ethanol and are reacted as two (2-methoxy ethoxy) sodium aluminum hydrides (Red-Al);
Figure A20038010957600293
Wherein, each P 1, P 2And P 3Be hydrogen or suitable oxygen protecting group independently, for example, acyl group is preferably benzoyl;
(d) if desired, then the ribofuranose derivative compound in the reaction of preceding step of protection (for example benzoylation) optionally forms 1,2,3,5-(independent optional protection)-2-C-methyl-β-D-ribofuranose,
Figure A20038010957600301
Wherein, P 4Be hydrogen or suitable oxygen protecting group independently, for example, acyl group is preferably benzoyl,
(e) make 1,2,3,5-(independent optional protection)-2-C-methyl-β-D-ribofuranose and cytosine and activator as BSA, chosen wantonly at lewis acid such as SnCl 4There is reaction down, generation 4-amino-1-(3,4-(independent optional protection-hydroxyl)-5-O-protection-hydroxyl-methylene-3-methyl-oxolane-2-yl)-the 1H-pyrimid-2-one
Wherein, each P 1, P 2, P 3And P 4Be hydrogen or suitable oxygen protecting group independently, for example, acyl group is preferably benzoyl;
Then,
(f) if desired; then optionally with the 4-amino-1-(3 in the preceding step reaction; 4-(independent optional protection-hydroxyl)-5-O-protection-hydroxyl methylene-3-methyl-oxolane-2-yl)-the 1H-pyrimid-2-one sloughs protection; formation 4-amino-1-(3,4-dihydroxy-5-methylol-3-methyl-oxolane-2-yl)-1H-pyrimid-2-one ( VI),
Figure A20038010957600311
(g) if desired, optional protection/deprotection, then to 4-amino-1-(3,4-dihydroxy-5-methylol-3-methyl-oxolane-2-yl)-1H-pyrimid-2-one ( VI) 3 '-position carries out esterification with L-valine etc., obtain 3 of β-D-2 '-C-methyl-cytidine (2-amino-3-methyl-butanoic acid 5-(4-amino-2-oxygen-2H-pyrimidine-1-yl)-4-hydroxyl-4-C-methyl-2-methylol-oxolane-3-base ester) '-the O-L-valine ester, optional form with salt exists.
Description of drawings
Fig. 1 is preparation 1,2,3,5-four-O-benzoyl-2-C-methyl-β-D-ribofuranose and 2,3, the sketch map of the method for optimizing of 5-three-O-protection-2-C-methyl D-ribonic acid-gamma lactone.
Fig. 2 is the sketch map of the process for selective of preparation 2-C-methyl-β-D-ribose acid lactone.
Fig. 3 is preparation 1,2,3, the sketch map for choosing method of 5-four-O-benzoyl-2-C-methyl-β-D-ribofuranose.
Fig. 4 is the preferred sketch map for preparing the method for nucleoside, nucleoside analog or its salt or prodrug of the present invention.
Fig. 5 be 3 of preparation β-D-2 '-C-methyl-cytidine '-the pharmaceutically acceptable salt that arrives of O-L-valine ester for choosing method.
Fig. 6 shows the confession routing footpath that road known in the state of the art prepares β-D-2 '-C-methyl-cytidine.
The specific embodiment
A kind of preparation nucleoside and nucleoside analog are provided, as 2 '-C-methyl-nucleoside and 2 '-C-methyl-3 '-method of O-valine nucleoside and their salt and/or prodrug, product has comprised all spatial chemistry and tautomeric form.Compared with prior art, this method is used reagent still less, has obtained higher productive rate in shorter time.And, saved the chromatographic purification step that takes time and effort, and the racemization that will not expect is suppressed to the level that can receive.The method of this improvement comprise form expectation nucleoside as prodrug the intermediate in synthetic, and be expected to the expansion scale for meeting industrial requirement.
In addition; a kind of scale preparation 2-C-methyl sugar intermediate that effectively can enlarge also is provided; as 2; 3; 5-(independent optional protection) and unprotected 2-C-methyl D-ribonic acid-gamma lactone (being also referred to as 2-C-methyl-ribose acid lactone) and 1; 2,3, the synthetic method of 5-(independent optional protection) and unprotected 2-C-methyl D-ribofuranose.Compared with the method for prior art, this method has adopted cheap reagent, and required time is shorter, and purification process is more easy, and productive rate is higher.
In one embodiment; method of the present invention is a kind of preparation of nucleoside, its 2 '-two the replacement, take place on the C, as 2 '-methyl-nucleoside or 2 '-methyl-3 '-O-valine-nucleoside; its intermediate; for example 2,3,5-(independent optional protection) and unprotected 2-C-methyl D-ribonic acid-gamma lactone (being also referred to as 2-C-methyl-ribose acid lactone) and 1; 2; 3,5-(independent optional protection) and unprotected 2-C-methyl D-ribofuranose and its salt and/or prodrug.One preferred embodiment in, the present invention is used to prepare 2,3,5-(independent optional protection) or unprotected 2-C-methyl D-ribonic acid-gamma lactone.Another preferred embodiment in, the present invention is used to prepare 1,2,3,5-(independent optional protection) or unprotected 2-C-methyl D-ribofuranose.Another preferred embodiment in, the present invention is used to prepare β-D-2 '-C-methyl-cytidine (4-amino-1-(3,4-dihydroxy-5-methylol-3-C-methyl-oxolane-2-yl)-1H-pyrimid-2-one).Another preferred embodiment in, the present invention be used to prepare 3 of β-D-2 '-C-methyl-cytidine (2-amino-3-methyl-butanoic acid 5-(4-amino-2-oxygen-2H-pyrimidine-1-yl)-4-hydroxyl-4-C-methyl-2-methylol-oxolane-3-base ester) '-O-L-valine ester or its preferred hydrochloride form.Nucleoside, nucleoside analog, salt or ester prodrugs by the present invention's preparation can be used as the intermediate for preparing various other nucleoside analogs, perhaps directly as antiviral and/or anti-tumor agent comprising salmosin.
In the first embodiment, method of the present invention uses D-fructose as initiation material, has made 1,2,3 with brief synthesis path, 5-(independent optional protection)-2-C-methyl-β-D-ribofuranose.
In second embodiment, method of the present invention be a kind of nucleoside, nucleoside analog with and the preparation of salt or prodrug, they 2 '-two the replacement, take place on the C.
In the 3rd embodiment, method of the present invention is used to prepare β-D-2 '-C-methyl-cytidine (4-amino-1-(3,4-dihydroxy-5-methylol-3-C-methyl-oxolane-2-yl)-1H-pyrimid-2-one).
In the 4th embodiment, method of the present invention be used to prepare 3 of β-D-2 '-C-methyl-cytidine (2-amino-3-methyl-butanoic acid 5-(4-amino-2-oxygen-2H-pyrimidine-1-yl)-4-hydroxyl-4-C-methyl-2-methylol-oxolane-3-base ester) '-the O-L-valine ester, or its preferred hydrochloride form.
Nucleoside, nucleoside analog, salt or ester prodrugs by the present invention's preparation can be used as the intermediate for preparing various other nucleoside analogs, perhaps directly as antiviral and/or anti-tumor agent comprising salmosin.
Method of the present invention is that with the advantage that similar prior art is compared the reagent of its use lacks 50% than prior art.Even if like this, to compare with immediate prior art, the gross production rate of method of the present invention but brings up to 38% by 12%.Further advantage is embodied in to have shortened and finishes the synthetic cycle of prodrug.An advantage is the safety and the easy upgradability of this new method again, is expected to become the method that meets industrial requirement.
The distinguished aspect of the present invention has been to use the reagent of particular combinations, thereby has saved the separation of intermediate in the synthesis step, segregation and/or purification.The selection of particular agent makes most initiation materials be converted into product, and has reduced the racemization effect of L-threonine derivatives of high therapeutic index part, thereby can easily remove from end product, thereby the ratio more efficiently synthetic method of method in the past is provided.The long and is to shorten the time that obtains final prodrug product, and the yield percentage of expected product improves.In addition, owing to need less time and reagent still less, generally speaking, be favourable from saving cost consideration.Cost efficiency and industrial upgradeable and safe method.
The advantage that the present invention improves one's methods is embodied in: among Fig. 1, use the initiation material of D-fructose as cheapness, and use CaO, it has reduced the response time and has increased the yield percentage that lactone is produced; Adopt Red-Al/ ethanol to reduce, provide the zone of end group isomerized products chemical compound to select mixture, can adopt known method easily from this mixture with end product 1,2,3,5-four-O-benzoyl-2-C-methyl-β-D-ribofuranose is isolated; Among Fig. 4, adopt cytosine as initiation material, rather than adopt benzoyl-cytosine of the prior art (Fig. 5) or uracil (Fig. 6) as initiation material, more simply reach the lower cheap chemical compound of molecular weight owing to having used like this, improved " atom benefit (the atom economy) " of process; Compare with similar step in the existing method, need still less normal cytosine, SnCl among the present invention 4And BSA; Because reactant and reagent all are less than equivalent in the past, bring the benefit of reaction dual, that is, reaction was promptly finished with 3 to 4 hours, and the intermediate among Fig. 4 ( 2) purity so high, and need not further chromatographic isolation and purification step.
Fig. 1 is the sketch map of an embodiment of the invention.In this improved method, the time that is formed ribose acid lactone (chemical compound 1) by D-fructose is approximately 40 hours or is less than two days, and product yield is about 13.6%, and it is than the high 30-40% of immediate prior art.Compare it, synthetic each needs two months of the ribose acid lactone of Kiliani and Scheibler or longer time finish, and product yield is approximately 10% (Lopez-Herrera et al., J Carbohydrate Chemistry 1994,13 (5): 767-775 at 768).
Unexpectedly, calcium oxide (CaO) and water can react with the initiation material D-fructose of cheapness, preparation 1,2,3, and 5-four-O-protection-2-C-methyl-β-D-ribofuranose, productive rate was for acquiring high 30-40% than in the past.This method can prepare the 2-C-methyl-β-D-ribofuranose with protected hydroxyl in a large number, and it is the important intermediate in synthesis of biologically active nucleoside and some vitamin chemical compound.Find that thus the another benefit of bringing is to save the preparation cost of product, this is particular importance in large-scale industry is combined to.For example, 1,2,3,5-four-O-benzoyl-2-C-methyl-β-D-ribofuranose prepares as initiation material by using the D-arabinose normally by D-ribose.Synthetic needs at least 5 step and chromatogram purifications from the D-arabinose.And the cost of per kilogram D-arabinose is about 250 times of D-fructose! The improved method of the application of the invention only needs 4 steps and cheap reagent just can prepare the 2-C-methyl-ribose that protecting group is arranged at 1,2,3 and 5.Therefore, need not to carry out the product that cost that chromatogram purification promptly can be lower makes expectation effectively.
To one's surprise equally, when using CaO, significantly reduced forming 2-C-methyl-β-required time of D-ribose acid lactone as initial reagent.With Kiliani and the early stage use Ca (OH) of Scheibler 2Work as reaction reagent is compared, and this has just significantly reduced synthetic required total time.
Also find in addition, when with Red-Al (optional its alcoholic solution) as Reducing agent, 2,3 of generation, 5-three-O-benzoyl-1-hydroxyl-2-C-methyl-β-D-ribofuranose produces a large amount of single end group isomerized products during by acidylate, easier realization effectively separates.This has just simplified the separation to the end product of expectation.
Advantage of the present invention is embodied in following aspect: select D-fructose to have advantage as the initiation material of cheapness economically for the sugar analogue of the final protection of preparation; Use CaO to improve the product productive rate, reduced the response time that lactone produces; Provide the zone of end group isomerized products to select mixture with the Red-Al reduction, end product can be easily from wherein being separated by universal method.Otherwise advantage comprises, except that using cheap initial reagent, also used cheap reaction reagent, in whole process, handle the required step of intermediate minimum, only need to use usual way well known in the art and equipment, and avoided complicated step and expensive device.
Fig. 4 is the sketch map of another embodiment of the invention.This advantage of improving one's methods is embodied in following aspect.Adopt cytosine as initiation material, rather than adopt benzoyl-cytosine of the prior art (Fig. 5) or uracil (Fig. 6) as initiation material, more simply reach the lower cheap chemical compound of molecular weight owing to having used like this, improved " the atom benefit " of process.In addition, compare, need still less normal cytosine, SnCl among the present invention with similar step in the existing method 4And BSA; Because reactant and reagent all are less than equivalent in the past, bring the benefit of reaction dual, that is, reaction was promptly finished with 3 to 4 hours, and the intermediate among Fig. 4 ( 2) purity so high, and need not further chromatographic isolation and purification step.
Astonishing discovery is, in deprotection steps of the present invention (Fig. 4, ( 5) → ( 6), slough the benzoyl protecting group), use the NaOMe be dissolved in MeOH brought than use in the existing method ammonia (Fig. 5, ( 5a) → ( 6)) more economical, safely, be easy for the synthetic of industrial upgrades.Other benefit is to come from the shorter response time: use Feldalat NM to finish reaction needed about 1 hour, and same reaction uses ammonia then to need 1-2 days.In addition, in the present invention, handle by simple methanol or ethanol (preferred alcohol), obtained highly purified ( 6), thereby removed from very key in the conventional method but the chromatographic purification step that takes time and effort (see Fig. 5, ( 5a) → ( 6)).
Except more simple, cost is lower, the safety of the reactant that uses and reagent higher, total product yields in two step of the inventive method are 85%, and in the existing method shown in Figure 5 are 24%.Produce ( 7) sensitivity carbonamidine-protection step (Fig. 4, ( 6) → ( 7)) in, of the present invention 13% even reaction load (reaction loading) is increased to from 5% of prior art, the product productive rate still maintains about about 80+%.
On the prior art basis one simple the improvement to ensuing two steps, monosilane-protection and the coupling of BOC-ester have produced positive influence, generated as ( 8) and ( 9).At first, in the prior art as the more expensive and difficult pyridine of removing of reaction dissolvent (Fig. 5, ( 7) → ( 8)) replaced by dichloromethane (Fig. 4, ( 7) → ( 8)).Silylation in dichloromethane produced still less do not expect 3 ', 5 '-the dimethylamino silane radical derivative, thereby can control silicyl by-product ground better and form, and from ( 7) be converted into ( 8) conversion ratio surpass 99%.And because dichloromethane also is used as the link coupled solvent with BOC-Val-OH, therefore need not ( 8) obtain with the BOC-Val-OH coupling ( 9) separate before.( 8) obtain with the BOC-Val-OH coupling ( 9) before, only need simple extraction to collect to get final product (comparison diagram 4, ( 8) → ( 9) with prior art Fig. 5 in same steps as).
And then art methods has adopted N, dinethylformamide and acetonitrile as the BOC-Val-OH coupling solvent (Fig. 5, ( 8) → ( 9a)).These reagent are all very expensive, make that the rate of load condensate of reaction is lower, and are only about 3%, and, N particularly, dinethylformamide boiling point height is difficultly removed from reactant mixture.In addition, roughly need two days and excessive BOC-Val-OH, EDC and dimethyl aminopyridine (DMAP) to finish reaction.Need these excessive reagent to finish reaction on the one hand, their existence makes the product purification of back become complicated again but then.In addition, use excessive DMAP to bring out the racemization of the amino acid moiety of L-valine derivative.
Different with the method for prior art is, use in the present invention dichloromethane as the solvent of coupling reaction (Fig. 4, ( 8) → ( 9) make the rate of load condensate to be about 11%, and only used in the prior art reagent of half amount approximately, and be reflected at about 4-6 hour and promptly finish.Minimizing on response time and the DMAP that is used for control consumption of the present invention are reduced to the racemization of the amino acid moiety of L-valine derivative and are less than 0.2%, and this is about 6% in the prior art.Low raceme level like this further is in pharmaceutically acceptable scope as 0.2%, because this means that a kind of enantiomer for another enantiomer, has stronger activity.
The deprotection of the β-D-2 ' to esterification subsequently-C-methyl-cytidine can use the amine fluoride (NH that is dissolved in methanol 4F).Be dissolved in the amine fluoride (NH of methanol 4F) be by ( 9) reaction obtain ( 10) in, be used for chemical compound deprotection (promptly removing silicyl and dimethyl formamide base) and the reagent (Fig. 4) of selection.The method of prior art (Fig. 5, ( 9a) → ( 10)) adopted identical reagent, but used 10 normal amine fluorides, about 3% rate of load condensate, need chromatographic isolation obtain ( 10), Comparatively speaking, the present invention uses 4 normal amine fluorides, and about 10% rate of load condensate does not need chromatographic isolation.In this step of improved method, use still less amine fluoride and the benefit brought of the ethyl acetate of about 10 molar equivalents to be, the BOC-ester ( 10) be hydrolyzed to β-D-2 '-C-methyl-cytosine, also be defined as 4-amino-1-(3,4-dihydroxy-5-methylol-3-methyl-oxolane 2-yl)-lH-pyrimid-2-one ( 6) be lowered to minimum.Therefore, method of the present invention is used and is improved in its effective agents and has advantage on the rate of load condensate.
In addition, ( 10) by simple EtOAc/TBME/H 2O handles and purification has been removed chromatographic isolation and purification once more from, thereby has avoided the chromatographic purification step that takes time and effort once more.Silylation, coupling and deprotection after this 3 step pure ( 10) yield percentage be approximately 60%-99%.
In the final step of improving one's methods, (Fig. 4, ( 10) → ( 11)), with ethanol as solvent carry out from 3 of β-D-2 '-C-methyl-cytidine '-the L-valine ester derivant removes the BOC-protecting group, rate of load condensate is increased to 12% of this method from 2% of prior art.In improved method, use ethanol replace ethyl acetate of the prior art as solvent (Fig. 5, ( 10) → ( 11)), this change makes load increase.Select ethanol to increase rate of load condensate, and then it is of the present invention about 95% that reaction yield is brought up to from about 80% of prior art, and avoided the pollution that brings by the acetic acid that ethyl acetate produces as solvent.The end product that obtains ( 11)＞98% pure form, and L-valine racemization is suppressed to and is less than 0.2%.
Therefore, the prototype compound of the improved on the whole β-D-2 ' of the present invention-C-methyl-cytidine (2-amino-3-methyl-butanoic acid 5-(4-amino-2-oxygen-2H-pyrimidine-1-yl)-4-hydroxyl-2-methylol-4-methyl-oxolane-3-base ester) '-O-L-valine ester or its dihydrochloride synthetic, it is about 26% to make that total yield percentage has improved, and the cycle has reduced about 80%.The shortening of cycle mainly give the credit to take time and effort and wasteful chromatographic isolation and purification step avoid.Other factor comprises the increase of load, thus only need carry out still less batch, and use easy-to-handle solvent and reagent.Use safer more cheap solvent and reagent to bring extra benefit to the present invention.Yet, being lower than the productive rate that art methods provides as the yield percentage of fruit product, these benefits can be out in the cold.Method of the present invention provides the increase of about 26% productive rate, and is therefore very useful.
Definition and optional reagent
Term " essentially no anomer " or " not having anomer to exist basically " are meant and comprise at least 95%～98% (weight), the more preferably nucleotide composition of the specified anomer of the nucleoside of 99%～100% (weight) as used herein.One preferred embodiment in, essentially no anomer in method of the present invention and the chemical compound.
Similarly, term " separated " refers to and comprises at least 85% or 90% (weight), preferred 95%～98% (weight), and the more preferably nucleotide composition of 99%～100% (weight) nucleoside, remainder contains other chemical substances or anomer.
Term " ribonic acid-gamma lactone " and " ribose acid lactone " are used interchangeably in this article, are meant the chemical compound 1 among Fig. 1 or the derivant of its oxygen protection.
The term of Shi Yonging " protection " is meant the group that is added on oxygen, nitrogen or the phosphorus atoms unless specialize herein, to prevent the further reaction or for other purposes of these groups.The protecting group of a large amount of known various oxygen, nitrogen or phosphorus is known by people in organic synthesis.
The example of suitable protecting group includes but not limited to: benzoyl; Replacement or unsubstituted alkyl, replacement or unsubstituted acyl, replacement or unsubstituted silicyl; Replace or unsubstituted aromatics or aliphatic ester, for example aryl such as benzoyl, toluyl (for example toluoyl base), nitro benzoyl, chlorobenzene formacyl; Ether as-C-O-aralkyl ,-the C-O-alkyl or-the C-O-aryl; Fatty group such as acyl group or acetyl group, comprise any replacement or unsubstituted aromatic series or aliphatic acyl radical ,-(C=O)-aralkyl ,-(C=O)-alkyl or-(C=O)-aryl; Wherein, the aromatic portion of acyl group or fats portion can be straight or branched; All groups that all can further do not contained improved synthetic reaction and influence replace (see Greene et al., Protective Groups in Organic Synthesis, John Wiley and Sons, 2 NdEdition (1991)).For example, in an embodiment of the invention, the group of the Reducing agent that protecting group is not selected for use (preferred Red-Al) influence replaces.For using ether as for the protecting group, quote the U.S.6 of Saischek etc., 229,008 as a reference, wherein reported to use ether as protecting group the condition stable and process of reagent to be brought tangible benefit, particularly in 5 of penta furanoside ' position.This is just final to be that separation, segregation and purification end product are provided convenience, and therefore, has improved the yield percentage of product.
The non-limitative example of sugar hydroxyl protecting group comprises; silicyl, benzoyl, toluoyl base, p-nitrophenyl formoxyl, to chlorobenzene formacyl, acyl group, acetyl group ,-(C=O)-alkyl and-(C=O)-aryl, these substituent groups can be unsubstituted or be replaced by one or more groups that not influenced by selected Reducing agent.In one embodiment, sugared hydroxyl protecting group is a benzoyl.Amino protecting group is preferably BOC (butoxy carbonyl) ,-(C=O)-aralkyl ,-(C=O)-alkyl or-(C=O)-aryl.In an embodiment of the invention, the aminoacid protecting group is BOC (butoxy carbonyl).
In this application, term " replace " refers to the single or multiple replacement undertaken by one or more specified substituent groups.When open or claimed single replacements, chemical compound can be substituted the base replacement once or more than once.When open or claimed multiple replacements, substituted chemical compound can be independently by one or more disclosed or claimed replacement part single or multiples replacements.
Term used herein " alkyl " is meant typical C unless specialize 1To C 10Saturated straight chain, side chain or cyclic, one-level, secondary or three grades of hydrocarbon, specifically comprise: methyl, trifluoromethyl, ethyl, propyl group, isopropyl, cyclopropyl, butyl, isobutyl group, the tert-butyl group, amyl group, cyclopenta, isopentyl, neopentyl, hexyl, isohesyl, cyclohexyl, cyclohexyl methyl, methyl amyl and dimethylbutyl.This term comprises and replacing and unsubstituted alkyl.Can be selected from the part of one or more positions substituted alkyl by halogen (comprising fluorine, chlorine, bromine or iodine), hydroxyl (CH for example 2OH), amino (for example, CH 2NH 2, CH 2NHCH 3Or CH 2N (CH 3) 2), alkyl amino, arylamino, alkoxyl, aryloxy group, nitro, azido be (as CH 2N 3), cyano group (CH 2CN), sulfonic acid, sulfate radical, phosphoric acid, phosphate radical or phosphonate radical) group formed, if desired, any one in above-mentioned group or all can be unprotected or can be according to the prior art instruction and further protected, for example, at Greene et al., Protective Groups in Organic Synthesis, John Wiley and Sons, 2 NdThat is instructed among the Edition (1991) is such.
Term " alkyl amino " and " arylamino " comprise having the substituent amino of one or more alkyl or aryls respectively.
Term " alkaryl " and " alkylaryl " comprise the alkyl with aryl substituent.Term " aralkyl " and " aryl alkyl " comprise the aryl with alkyl substituent.
Term " halogen " comprises chlorine, bromine, iodine and fluorine.
Term used herein " aryl " unless specialize, is meant phenyl, xenyl or naphthyl.This term comprises and replacing or unsubstituted part.
Aryl can be replaced by one or more parts; it includes but not limited to hydroxyl, amino, alkyl amino, arylamino, alkoxyl, aryloxy group, nitro, cyano group, sulfonic acid, sulfate radical, phosphoric acid, phosphate radical or phosphonate radical; if desired; above-mentioned any one or all can be unprotected or can be according to the prior art instruction and further protected; for example; at Greene et al. Protective Groups in Organic Synthesis, JohnWiley and Sons, 2 NdThat is instructed among the Edition (1991) is such.
Term " acyl group " comprises-C (=O)-R; non-carbonyl moiety R wherein be straight chain, side chain or cyclic alkyl or low alkyl group, alkoxyalkyl (comprising methoxy), aralkyl (comprising Bian Ji), aryloxy alkyl such as phenoxymethyl, aryl, comprise optional phenyl, the C that is replaced by halogen 1～C 4Alkyl or C 1～C 4Alkoxyl, sulfonate radical; as sulfonic alkyl root or sulfonic acid aralkyl root; comprise methyl sulphonyl, list-, two-or three-phosphate radical, triphenylmethyl or mono methoxy triphenylmethyl, the Bian Ji of replacement, trialkylsilkl, as dimethyl-tert-butyl group silicyl) or diphenyl methyl silicyl.Aryl in the ester preferably comprises phenyl.Term " lower acyl " refers to the acyl group that its non-carbonyl moiety is a low alkyl group.
Term " carboxylic acid " and " carboxylate " comprise structure RC (=O) OH and RC (=O) O-R ' respectively.The non-carbonyl moiety here, no matter be R or R ', for example can be straight chain, side chain or cyclic alkyl or low alkyl group, alkoxyalkyl (comprising methoxy), aralkyl (comprising Bian Ji), aryloxy alkyl such as phenoxymethyl, aryl, comprise optional phenyl, the C that is replaced by halogen 1～C 4Alkyl or C 1～C 4Alkoxyl.Sulfonate radical; as sulfonic alkyl root or sulfonic acid aralkyl root; comprise methyl sulphonyl, list-, two-or three-phosphate radical, triphenylmethyl or mono methoxy triphenylmethyl, the Bian Ji of replacement, trialkylsilkl, as dimethyl-tert-butyl group silicyl) or diphenyl methyl silicyl.Aryl in the ester preferably comprises phenyl.In all cases, R and R ' can be identical or different substituent groups.
Term amino acid comprises natural and synthetic α, β, γ and δ aminoacid, include but not limited in the protein aminoacid, as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, agedoite acid, glutamine, acid, aspartic ester, glutamate, lysine, arginine and histidine.In preferred embodiment, aminoacid is the L-configuration.In another preferred embodiment, aminoacid is the L-valine.In addition, aminoacid also can be alanyl, valyl, leucyl, isoleucyl-, prolyl, the phenyl alanyl, tryptophanyl, methinyl, glycyl, seryl, threonyl, cysteinyl, tyrosyl, asparaginyl-, glutamy, the aspartoyl acyl, the glutaroyl acyl, lysyl, arginyl, histidyl-, β-alanyl, β-valyl, β-leucyl, β-isoleucyl-, β-prolyl, the beta-phenyl alanyl, β-tryptophanyl, β-methinyl, β-glycyl, β-seryl, β-threonyl, β-cysteinyl, β-tyrosyl, β-asparaginyl-, β-glutamy, β-aspartoyl acyl, β-glutaroyl acyl, β-lysyl, β-arginyl, the derivant of β-histidyl-.
Term " alpha-non-natural amino acid " refers to have amino end group but is not present in natural carboxylic acid.This term also comprises D-and L-aminoacid, and their any tautomerism or stereoisomeric forms in any ratio.
The term nucleoside base comprises purine or pyrimidine bases.The example of purine or pyrimidine bases includes but not limited to adenine, N 6-alkyl purine, N 6(wherein, acyl group is C (O) (alkyl, aryl, alkaryl or aralkyl), N to-acyl group purine 6-Bian Ji purine, N 6-halo purine, N 6-vinyl purine, N 6-vinyl purine, N 6-acetenyl purine, N 6-acyl group purine, N 6-hydroxy alkyl purine, N 6-alkylthio purine, N 2-alkyl purine, N 2-alkyl-6-thio-purine, thymus pyrimidine, cytosine, 5-flurocytosine, 5-methylcytosine, 6-nitrogen pyrimidine comprise 6-nitrogen cytosine, 2-and/or 4-mercaptopyrimidine, uracil, 5-halo uracil, comprise 5-fluorouracil, C 5-alkyl pyrimidine, C 5-Bian yl pyrimidines, C 5-halogenated pyrimidine, C 5-vinyl pyrimidine, C 5-acetenyl pyrimidine, C 5-acyl group pyrimidine, C 5-hydroxy alkyl purine, C 5-aminopyrimidine, C 5-cyanopyrimidine, C 5-nitro-pyrimidine, C 5-aminopyrimidine, N 2-alkyl purine, N 2-alkyl-6-thio-purine, U-18496 base, 5-AzU base, azido pyridine radicals, imidazole pyridyl, pyrroles's pyrimidine radicals and pyrazolyl-pyrimidine base.Purine bases include but not limited to: guanine, adenine, hypoxanthine, 2,6-diaminopurine and 6-chloropurine.Functional oxygen on the base and nitrogen groups if desired can be protected.Suitable protecting group is known for those skilled in the art; comprise trimethyl silyl, dimethyl hexyl silicyl, t-butyldimethylsilyl and t-butyldiphenylsilyl, triphenylmethyl, alkyl and acyl group; as acetyl group and propiono, methyl sulphonyl and p-methyl benzenesulfonic acid.In addition, purine or pyrimidine bases also can be chosen wantonly and be substituted, and form feasible prodrug, can divide in vivo.The example of suitable substituents comprises acyl moiety, amine or cyclopropyl (for example, 2-amino, 2,6-diaminourea or cyclopropyl guanosine).
Be used for method of the present invention or other reagent of the prior art are defined as follows: BSA (two (trimethyl silyl) acetamide), TMSCl is the chloro trimethyl silane; TFAA is a trifluoroacetic anhydride; TBDPSCl is a t-butyldiphenylsilyl chlorine; TBDMSCl is a tert-butyldimethylsilyl chloride; DCM is a dichloromethane.
Method of the present invention is not limited to use the amino-acid ester of nucleoside, protection and the reagent of example.Suitable optional reagent of the present invention be can be used for substituting above-mentioned reagent.For example, TEA (triethylamine) can be replaced by other amine that is fit to, and it includes but not limited to diisopropylethylamine, N-ethylmorpholine or other three grades of fatty amines; DME (1, the 2-dimethoxy-ethane) can be replaced by any suitable polar non-solute, for example THF (oxolane) or other ether; The toluene solution of Red-Al/EtOH (two (2-methoxy ethoxy) sodium aluminum hydride/ethanol) can be by NaHTe, SmI 2, H 2+ Pd-phosphine catalyst or LiAl (O tBu) 3H (three tert-butoxy lithium aluminium hydride reductions) replaces, and all these have all produced chemical selection and regional selective reduction, LiAlH only 4Produced the glycol of open chain.Adding MgSO 4Before and after wash the product serosity with THF and can be replaced by in acetone and wash.Really, when scale enlarged, acetone was a kind of preferred solvent.
In addition, although DMF is preferred handling easily and being easy to remove from reactant mixture, DMF (dimethyl formamide) can be replaced by any polar solvent, for example, and DMSO (dimethyl sulfoxine).EDC (1-[3-(dimethylamino) propyl group]-3-ethyl-carbon imidodicarbonic diamide hydrogen chloride); Be also referred to as DEC) can be carried out link coupled reagent replacement by any, it includes but not limited to: CDI (carbonyl dimidazoles), bop reagent (benzotriazole-1-base oxygen-three (dimethylamino)-phosphine hexafluorophosphate), or known similar coupling reagent.Although SnCl 4Be preferred, but available here lewis acid replace.Lewis acid includes but not limited to: SnCl 4, BF 3, AlCl 3, TiCl 2, TiCl 4, FeCl 3, SnCl 2And their mixture.In one embodiment, lewis acid is SnCl 4Alternative acetonitriles such as available any organic solvent such as toluene.Any activator all can be used for making nucleoside activation carrying out coupling as monosilane reagent.HMDS (hexamethyldisiloxane), TMSCl or TBDPSCl etc. all can be used for replacing BSA (two (trimethyl silyl) acetamide).Ammonia can be used for substituting the Feldalat NM that is dissolved in methanol, and any polar solvent DMSO can replace DMF.Multiple other silylation reagent all can replace TBDPSCl, and any villiaumite all can replace NH 4F, and other acid such as TFA can be used for replacing HCl.
The detailed description of preparation method step
The preparation of ribose acid lactone
Figure A20038010957600411
Ribose acid lactone can comprise the oxidation and the replacement technique preparation of standard by any deliver or method of not delivering.An embodiment of synthetic ribose acid lactone of the present invention is by D-fructose, carries out according to following step.
Ribose acid lactone can make by the reaction of D-fructose and calcium oxide (CaO).D-fructose can react with any molar ratio with CaO, condition is that reaction is carried out and the many by-products of no mistake in treatment with the speed that can accept, and to D-fructose, preferred molar ratio is 5: 1, preferred molar ratio is 3: 1, and most preferred molar ratio is 2.3: 1.3.CaO can be any speed add, condition is that reaction is carried out with the speed that can accept and can not produced excessive heat or too much by-product.In one embodiment, CaO increases progressively adding at room temperature 5 minutes.Reaction can be performed until D-fructose and almost be consumed, and for example carries out 6～22 hours, and therebetween, reaction process can be monitored, for example regularly takes out a certain amount of TLC that is used for and analyzes.
Reaction can be carried out under any temperature, and condition is that decomposition or excessive production of by-products are carried out and can not impelled in reaction with the speed that can accept.Preferred temperature is to about 23～40 ℃ from room temperature.
Can utilize precipitant to come from solution, to remove calcium.In one embodiment, in reactant mixture, add CO 2With the acid stronger than ribonic acid, one preferred embodiment in, adding be organic acid, form calcium carbonate.The organic acid that is fit to includes but not limited to: oxalic acid, malonic acid, succinic acid, 1,3-propanedicarboxylic acid, adipic acid, suberic acid, decanedioic acid, Azelaic Acid, maleic acid, acetic acid, propanoic acid, isopropylformic acid., acrylic acid, methacrylic acid, butanoic acid, valeric acid caproic acid or caproic acid.
Therefore, in an embodiment of the invention,, can in reasonable time, in mixture, blast CO in the reaction ending phase 2, for example, 2～3 hours, so that pH is reduced to neutral level from alkalescence.Any CaCO that in neutralization procedure, forms 3All can be removed, for example remove by vacuum filtration.
Then, the water layer usable acid is handled, and used acid for example be the organic acid stronger than nucleic acid, for example, oxalic acid, its molar ratio can be arbitrary proportion, condition is to react with the speed that can accept to carry out and can not produce too much by-product.In one embodiment, the acid of interpolation (for example oxalic acid) is 1: 2 with the molar ratio of D-fructose.
Reaction can proceed to nearly all calcium sedimentary degree all takes place, and is included in here under any temperature, deposits from solution with acceptable speed, and can not impel decomposition or form excessive by-product.For example, about 30 minutes of room temperature or 25 ℃ of left and right sides agitating solutions, to the oar liquid that white occurs.Stirring this oar liquid at about 45～50 ℃ then spends the night.
After finishing, but evaporating liquid for example under reduced pressure carry out, determine most water but still stay liquid mixture to remove.Product can be isolated from liquid mixture with known method.For example, can in oar liquid, add NaCl and organic solvent at room temperature about 30 minutes, as THF.Separate each layer that forms, in water layer, add fresh solvents such as THF, about 10 minutes of restir.The step that adds solvent, stirring and separating obtained water layer can repeat repeatedly as required, for example can repeat 3 times.At last, can merge organic solution, and add MgSO 4Deng stir about behind the desiccant 30 minutes, filter then, reuse solvent such as THF wash.Filtrate can be evaporated, and for example at 40 ℃ of following reduction vaporizations, can collect crude product then, and it is the semi-solid material of darkorange.
In addition, be purified product, also can add second kind of solvent such as acetone in crude product, mixture stirred 3 hours at 20 ℃.Can collect the ribose acid lactone product of white crystals by vacuum filtration, and wash with second kind of solvent as acetone and so on, and vacuum drying (seeing Fig. 1, chemical compound 1).
It is about 13.6% that the product yield of this reaction can reach, and improved 4% than the about of prior art.
The free hydroxyl of ribose acid lactone can continue optionally to be protected by suitable protecting group, and preferred protecting group is acyl group or silicyl, and guard method can be adopted method well known in the art, for example at Greene, and et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, the method for instruction in 1991.For example, chloro tert-butyl diphenyl silane and ribose acid lactone are reacted in anhydrous pyridine.Also can use acyl chlorides, preferably under the condition that in DME, refluxes in the presence of the alkali, react as Benzenecarbonyl chloride. and ribose acid lactone.
For example, the ribose acid lactone product can with mix with any molar ratio as alkali such as DMAP, condition is that reaction is carried out and can not produced too much by-product with the speed that can accept.In one embodiment, ribose acid lactone: the molar ratio of alkali (for example DMAP) is about 5: 1.Optional by using extra alkali to promote reaction, for example, use TEA with ratio arbitrarily, carry out with the rate that can receive speed and can not produce too much by-product but condition is reaction.In an embodiment of the invention, use excessive extra alkali (for example TEA).After the sufficiently long time,, make reaction carry out, and can not produce excessive by-product with acceptable speed with acyl chlorides such as the Benzenecarbonyl chloride .s of molar ratio adding arbitrarily.Be approximately 5: 1 with the molar ratio of ribose acid lactone.
Ribose acid lactone can prepare in the solvent arbitrarily, as long as the requirement of the dissolubility of this solvent satisfied temperature and reaction reagent.Solvent can be made of aprotic solvent arbitrarily, it includes but not limited to the alkyl solvent, as hexane and cyclohexane extraction, toluene, acetone, ethyl acetate, dithiane, THF, diox, acetonitrile, dichloromethane, dichloroethanes, diethyl ether, pyridine, dimethyl formamide (DMF), DME, dimethyl sulfoxide (DMSO), dimethyl acetylamide or its combination in any, but preferably use DME.
Reaction can be finished with the speed that can accept under arbitrary temp, but condition is can not produce too much by-product.Preferred temperature range is to about 5 ℃ from room temperature.
Then, can in reactant mixture, add frozen water, collect crude product thereafter, in suitable solvent such as t-butyl methyl ether, stir, filter, washing, and carry out drying by vacuum etc.
In order to compare, provided the method for preparing ribose acid lactone of Kiliani in the prior art among Fig. 2.The Adv.In Carbohydrate Chem.1957 that is described in detail in Sowden of Kiliani method provides among the 12:43, wherein instructs, and adds Ca (OH) 2The back added in 14 days once more, and mixture was placed 1～2 month then, during shake once in a while.Then, filtering mixt, filtrate is used CO 2Saturated.Then, by adding the oxalic acid of complete equivalent, make the calcium ion precipitation, filtering solution is condensed into oar liquid, treats this oar liquid crystallization several days again under cryogenic conditions.At last, mother solution is separated from crystalline solid, reuse water dissolution crystalline solid also carries out recrystallization.
The reduction of the ribose acid lactone of protection
Figure A20038010957600441
2-C-methyl D-ribonic acid-the gamma lactone of the optional protection that is obtained by abovementioned steps can reduce with any suitable Reducing agent; the molar ratio of used Reducing agent is optional, carries out with the speed that can accept and can not produce too much by-product but condition is reaction.Suitable Reducing agent includes but not limited to Red-Al/EtOH (two (2-methoxy ethoxy) sodium aluminum hydride/ethanol), NaHTe, SmI 2, H 2+ Pd-phosphine catalyst or LiAl (O tBu) 3H (three tert-butoxy lithium aluminium hydride reductions) replaces, and all these have all produced chemical selection and regional selective reduction.In an embodiment of the invention, Reducing agent is a Red-Al/ ethanol.For example, Red-Al solution can be added in the 2-C-methyl D-ribonic acid-gamma lactone solution of optional protection, wherein, Red-Al and 2,3, the molar ratio of 5-three-O-benzoyl-2-C-methyl D-ribonic acid-gamma lactone is about 2: 1.
What is interesting is, found that some reagent is unsatisfactory, produced mixture of products expectation and that do not expect in the process that is used for the inventive method.For example, when using LiAI (O t-Bu) 3When H replaces the alcoholic solution of Red-Al, LiAI (O t-Bu) 3H makes that reaction is slack-off, and has formed the multiple product of not expecting.Similarly, 9-boron bicyclo--[3.3.1]-nonane, 9-BBN and diisobutyl aluminium hydroxide, DIBALH do not react, and perhaps only produce the product of very small amount of expectation.
Reaction can be finished under any temperature, carries out and can not produce too much by-product with the speed that can accept but condition is reaction.Preferred temperature is from about 0 ℃～5 ℃.
Ribofuranose can prepare in the solvent of any satisfied temperature and agent dissolves requirement.Solvent can comprise organic solvent arbitrarily, include but not limited to the alkyl solvent, as pentane, hexane, toluene, acetone, ethyl acetate, dithiane, THF diox, acetonitrile, dichloromethane, dichloroethanes, diethyl ether, pyridine, dimethyl formamide (DMF), DME, dimethyl sulfoxide (DMSO), dimethyl acetylamide, alcohols solvent, as methanol, ethanol, propanol, isopropyl alcohol, butanols, amylalcohol and capryl alcohol, or their combination, but preferably dry toluene and ethanol solution.
Available suitable proton source finishes reaction as acetone, water and 1NHCl.Mixture can be used organic solvent such as ethyl acetate extraction, with salt washing, dry, remove down and desolvate for about 40 ℃ in decompression.
Then, the free hydroxyl of ribofuranose can be carried out selective protection with suitable protecting group, and preferred protecting group is acyl group or silicyl, and guard method can be adopted method well known in the art, for example at Greene, and et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, the method for instruction in 1991.Chloro tert-butyl diphenyl silane and ribofuranose are reacted in anhydrous pyridine.Also can use acyl chlorides, preferably under the condition that in DME, refluxes in the presence of the alkali, react as Benzenecarbonyl chloride. and ribofuranose.
Fig. 3 is Harry-O ' kuru et al, J.Org.Chem., (1997), 62 (6): the replaceable method that provides among the 1754-59.This method lacks the lactone intermediate, but is used to obtain the product identical with the present invention.The method of Harry-O ' kuru has been utilized equal protected D-arabinose or the D-ribose (Fig. 3) before forming ketone of all hydroxyls except that the C2 position.Protected sugar and Dess-Martin periodinane reagent are at CH 2Cl 2And MgSO 4Middle reaction obtains 2,4-two-O-benzoyl-5-methyl-O-benzoyl-dihydrofuran-3-ketone, and then use MeTiCl 3, MeMgBr/TiCl 4(or RCeCl 2, wherein, R will be the substituent group on the ribose C2) reduce.Chemical compound ( 3) and 4 ( 4a) and BzCl/DMAP/Et 3The reaction of N produced end product ( 4b), 1,2,3,5-four-O-benzoyl-2-R-ribose furanoside.It should be noted that in the first step hydrate of target product produces in a large number, need with excessive MgSO 4Reaction is to prepare the ketone product of bone dry basically.And, observe, in key intermediate 2-ketone and organic titanium reagent reacting; produced 1,3 of expectation, 5-benzoyl-protection-2-alkyl ribose furanoside and its ester exchange 2; 3, the α of the ribose furanoside of 5-benzoyl-protection-and the mixture of beta isomer.Because three kinds of products are all useful for this author, so this is unimportant for them.Like this synthetic just needs extra separating step to obtain the single isomer (J.Org.Chem., 1997,62 (6): 1754-9, at 1755) of deciding.No matter be the initiation material that D-arabinose or D-ribose all can be used as this purpose, but have to consider that its economy, its expense are approximately 250 times that use D-fructose when using the D-arabinose!
The method of the prior art among Fig. 3 and difference of the present invention are that except that the C2 position, all hydroxyls of D-arabinose or D-ribose are all protected before forming ketone.Then, by with Dess-Martinperiodinane (seeing Fig. 3, chemical compound 3) reagent reacting, on the C2 of initial compounds, formed ketone, then by MeTiCl 3Or RCeCl 2Reduction, R wherein is second substituent group that will replace on the ribose C2.(see Fig. 3, chemical compound 3With 4). End product 1,2,3, the productive rate of 5-four-O-benzoyl-2-alkyl-ribose furanoside is about 79%.
Comparatively speaking; method of the present invention is more effective; its lactone that provides is formed at the C1 of ribofuranose; to C2; C3 and C5 carry out hydroxyl protection; preferably in ethanol, reduce lactone, produce the regioselective isolating end group isomerized products that is easy to, then the independent residue free hydroxyl of the C1 that is positioned at ribofuranose is protected with Red-Al.
The condensation reaction of ribofuranose and activatory cytosine
Figure A20038010957600461
2-C-methyl D-ribonic acid the furanose of optional protection that obtained by the preceding step or that obtained by other known methods can be with any known method and nucleotide base coupling, and these methods comprise the standard coupling technology that adopts activation alkali.
An embodiment of the invention comprise the method for the synthetic β-D-2 '-C-methyl-cytidine that carries out according to following steps.
β-D-2 '-C-methyl-cytidine can obtain by making the reaction of 2-C-methyl D-ribonic acid furanose and activatory unprotected (as not benzoylated) cytosine; such cytosine can be as using the activatory cytosine of activator; silylation reagent for example; it includes but not limited to BSA ((N, O-two (trimethyl silyl) acetamide), HMDS, TMSCl or TBDPSCl.In one embodiment, silylation reagent is BSA.
Reaction can be chosen wantonly as SnCl 4Such lewis acid exists down and carries out, and used lewis acidic molar ratio is optional, but condition is to react with the speed that can accept to carry out and can not produce too much by-product.The suitable lewis acid of working as includes but not limited to SnCl 4, BF 3, AlCl 3, TiCl 2, TiCl 4, FeCl 3, SnCl 2And their mixture.In one embodiment, lewis acid is SnCl 4
β-D-2 '-C-methyl-cytidine can prepare in the solvent of any satisfied temperature and agent dissolves requirement.Solvent can comprise aprotic solvent arbitrarily, include but not limited to the alkyl solvent, as hexane and cyclohexane extraction, toluene, acetone, ethyl acetate, dithiane, THF diox, acetonitrile, dichloromethane, dichloroethanes, diethyl ether, pyridine, dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), dimethyl acetylamide or their combination, but acetonitrile preferably.
This reaction can be finished under any temperature, carries out and can not impel decomposition or too much by-product with the speed that can accept but condition is reaction.Preferred temperature is about 20 ℃～80 ℃.
Then, nucleoside can carry out deprotection with known road method, and the method for deprotection can adopt method well known in the art, for example at Greene, and et al., Protective Groups in Organic Synthesis, John Wiley andSons, Second Edition, the method for instruction in 1991.For example, the hydroxylic moiety of benzoyl protection can be sloughed protection with the MeOH solution of NaOMe under about room temperature.
The method of the prior art of representing among Fig. 5 comprises makes benzoyl cytosine, BSA and SnCl 4/ acetonitrile and 1,2,3,5-four-O-benzoyl-2-C-methyl-β-D-ribofuranose (4) reaction, formation 4-benzamido-1-(3,4-dibenzoyl oxygen base-5-benzoyloxy methyl-3-methyl-oxolane-2-yl)-lH-pyrimid-2-one ( 5a); Make ( 5a) react in methanol with NH3 and use the chromatographic isolation product, 4-amino-1-(3,4-dihydroxy-5-methylol-3-methyl-oxolane-2-yl)-lH-pyrimid-2-one ( 6), be also referred to as β-D-2 '-C-methyl-cytidine; Make ( 6) and Me 2NCH (OMe) 2In DMF, reacted 1.5 hours under the room temperature, form N[1-(3,4-dihydroxy-5-methylol-3-methyl-oxolane-2-yl)-2-oxygen-1,2-dihydro-pyrimidin-4-yl]-N, N-dimethyl-carbonamidine ( 7), make ( 7) with TBDPSCl and pyridine at room temperature react formed in 6 hours N '-1-[5-(tert-butyl group-diphenyl-silane oxygen ylmethyl)-3,4-dihydroxy-3-methyl-oxolane-2-yl]-2-oxygen-1,2-dihydro-pyrimidin-4-yl }-N, N-dimethyl-carbonamidine ( 8); Make ( 8) under room temperature, reacted 2 days in THF/DMF with N-Boc-L-valine, DEC and DMAP, and will obtain 2-tert-butoxycarbonyl amino-3-methyl-butanoic acid 2-(tert-butyl group-diphenyl-silane oxygen ylmethyl)-5-[4-(dimethylamino methene amido)-2-oxygen-2H-pyrimidine-1-yl by HPLC by the product that this reaction obtains]-4-hydroxy-4-methyl-oxolane-3-base ester ( 9a); Will ( 9a) and NH 4F refluxed in MeOH about 3 hours; to remove silicyl and amino protecting group; and product carried out chromatogram purification, obtain 2-tert-butoxycarbonyl amino-3-methyl-butanoic acid-5-(4-amino-2-oxygen-2H-pyrimidine-1-yl)-4-hydroxyl-2-hydroxymethyl-4-methyl-oxolane-3-base ester ( 10); Make at last, ( 10) with HCl in EtOAc under room temperature reaction obtain 2-amino-3-methyl-butanoic acid-5-(4-amino-2-oxygen-2H-pyrimidine-1-yl)-4-hydroxyl-2-hydroxymethyl-4-methyl-oxolane-3-base ester, dihydrochloride ( 11) as end product.÷
Fig. 6 here be as be used in the prior art to prepare β-D-2 '-C-methyl-cytidine ( 6) another for choosing method.The prior art method has utilized uracil as initiation material, and comprise make uracil and BSA in acetonitrile with 1,2,3,5-four-O-benzoyl-2-C-methyl-β-D-ribofuranose ( 4) reacted about 30 minutes, add lewis acid SnCl in the acetonitrile kind 4, gained solution was refluxed about 4 hours, chromatographic isolation product 1-(3,4-two Bian oxygen bases-5-Bian oxygen base-methyl-3-methyl-oxolane-2-yl)-1H-pyrimidine-2, the 4-diketone ( 12); Make ( 12) reacted about 4.5 hours in the methanol kind with NaOMe, to remove the benzoyl protecting group, separate then and crystallized product 1-(3,4-dihydroxy-5-methylol-3-methyl-oxolane-2-yl)-1H-pyrimidine-2, the 4-diketone ( 13), be also referred to as β-D-2 '-C-methyl-uridnine; Have most, make ( 13) in turn with TMSCl and N-crassitude at CH 3Reaction is about 3.5 hours among the CN, and cooling also added trifluoroacetic anhydride (TFAA) in about 30 minutes, add the 4-nitrophenol at 0 ℃, and stir about 3 hours adds NH 4OH De diox also is heated to 50 ℃, after spending the night, by chromatographic process and Crystallization Separation finally produce β-D-2 '-C-methyl-cytidine ( 6).
The esterification of β-D-2 '-C-methyl-cytidine
β-D-2 '-C-methyl-the cytidine of the optional protection that is obtained by abovementioned steps or any known method can carry out esterification by knowing any method.
An embodiment of the invention comprise 3 of synthetic β-D-2 '-C-methyl-cytidine '-method, the particularly Yi Xia β-D-2 '-C-methyl-cytidine of ester 3 '-method of L-valine ester.
3 of β-D-2 '-C-methyl-cytidine '-ester can make by the amino that adopts the optional protection of any known method β-D-2 '-C-methyl-cytidine.For example at Greene, et al., Protective Groups in Organic Synthesis, John Wiley and Sons, Second Edition, the method for instruction in 1991.In an embodiment of the invention, β-D-2 '-C-methyl-cytidine can with Me 2NCH (OMe) 2In DMF, react, produce N[1-(3,4-dihydroxy-5-methylol-3-methyl-oxolane-2-yl)-2-oxygen-1,2-dihydro-pyrimidine-4-yl]-N, N-dimethyl carbonamidine.
In a concrete embodiment; chemical compound can further be protected with TBDPSCl and imidazoles; provide 5 '-chemical compound of silicyl-protection; N '-{ 1-[5-(tert-butyl group-diphenyl-silane oxygen ylmethyl)-3; 4-dihydroxy-3-methyl-oxolane-2-yl]-2-oxygen-1,2-dihydro-pyrimidine-4-yl }-N, N-dimethyl carbonamidine; reaction can carried out in the solvent arbitrarily, and condition is the dissolubility fit of itself and temperature and reagent.Solvent can comprise any aprotic solvent, comprise but do not limit the alkyl solvent, as hexane and cyclohexane extraction, toluene, acetone, ethyl acetate, dithiane, THF diox, acetonitrile, dichloromethane (DCM), dichloroethanes, diethyl ether, pyridine, dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), dimethyl acetylamide or their combination in any, but preferred DCM.
Then, the β-D-2 '-C-methyl-cytidine of optional protection can with any suitable structure coupling obtain 3 of pharmaceutically acceptable β-D-2 '-C-methyl-cytidine '-prodrug, coupling can use known any method to carry out, and comprises standard condensation reaction.By link coupled part can be phosphate radical (comprise single-, two-or three-phosphate radical and stable phosphate radical); Straight chain, side chain or cyclic alkyl (comprising low alkyl group); Acyl group (comprising lower acyl); Aryl, sulphonic acid ester (comprise alkyl sulfonate esters or sulfonic acid aralkyl ester, comprise methyl sulphonyl) that CO-alkyl, CO-acyl group, CO-alkoxyalkyl, CO-alkoxyalkyl, CO-replace; Bian Ji, phenyl wherein is by the optional replacement of one or more substituent groups of describing in the aryl definition; Alkyl sulphonyl, aryl sulfonyl, aralkyl sulfonyl, lipid (comprising phospholipid); Aminoacid; Carbohydrate; Peptide; Cholesterol or pharmaceutically acceptable leaving group, when using in the body, it can provide free hydroxyl (or phosphate radical).
In an embodiment of the invention, 3 of expectation '-prodrug be 3 of β-D-2 '-C-methyl-cytidine '-L-valine ester, can prepare by following steps.
3 of β-D-2 '-C-methyl-cytidine '-β-D-2 '-C-methyl-cytidine that L-valine ester can be by making optional protection (for example 5 '-and the β-D-2 '-C-methyl-cytidine of N-protected) and N-Boc-L-valine be preferably at coupling agent; have reaction down as EDC and alkali such as DMAP, obtain 2-t-butoxycarbonyl amino-3-methyl-butanoic acid 2-(tert-butyl group-diphenyl-silicon alkoxyl-methyl)-5-[4-(dimethylamino-methene amido)-2-oxygen-2H-pyrimidine-1-yl]-4-hydroxy-4-methyl-oxolane-3-base ester and preparing.
3 of β-D-2 '-C-methyl-cytidine '-L-valine ester can prepare in the solvent of any satisfied temperature and agent dissolves requirement.Solvent can comprise aprotic solvent arbitrarily, include but not limited to the alkyl solvent, as hexane and cyclohexane extraction, toluene, acetone, ethyl acetate, dithiane, THF diox, acetonitrile, dichloromethane (DCM), dichloroethanes, diethyl ether, pyridine, dimethyl formamide (DMF), dimethyl sulfoxide (DMSO), dimethyl acetylamide or their combination in any, but DCM preferably.
This reaction can be finished under any temperature, carries out and can not impel decomposition or too much by-product with the speed that can accept but condition is reaction.Preferred temperature is near room temperature.
Then, 3 of β-D-2 '-C-methyl-cytidine '-L-valine ester can slough protection with known method, for example at Greene, and et al., Protective Groups in Organic Synthesis, John Wiley and Sons, SecondEdition, the method for instruction in 1991.In a specific embodiment of the present invention, 5 of t-butyldiphenylsilyl protection '-the L-valine of OH and N Boc-protection can use NH 4F is in MeOH; in the presence of the ethyl acetate of about 10 molar equivalents (for prevent the ammonia of separating out cut off '-the O-L-valine ester); the backflow mixture obtains 2-t-butoxycarbonyl amino-3-methyl-butanoic acid 5-(4-amino-2-oxygen-2H-pyrimidine-1-yl)-4-hydroxyl-2-methylol-4-methyl-oxolane-3-base ester, thereby sloughs protection.
3 '-L-valine ester can be converted into salt with known method, these methods comprise make 3 of β-D-2 '-C-methyl-cytidine '-L-valine ester and HCl react in EtOH, obtain 2-amino-3-methyl-butanoic acid 5-(4-amino-2-oxygen-2H-pyrimidine-1-yl)-4-hydroxyl-2-methylol-4-methyl-oxolane-3-base ester, dihydrochloride is as end product.
Preferred implementation
One of the present invention preferably to decide embodiment shown in Figure 1, and it comprises: D-fructose was reacted 6-22 hour in 23-40 ℃ in the presence of CaO/ water, add CO then in reactant mixture 2And oxalic acid, reaction was carried out 8-12 hour, formation 2-C-methyl D-ribonic acid-gamma lactone ( 1); Make 2-C-methyl D-ribonic acid-gamma lactone ( 1) with 4-dimethylaminopyridine (DMAP) and triethylamine (TEA) 1, in about 30 minutes of 5-25 ℃ of reaction, cooling mixture was to about 5 ℃ in the 2-dimethoxy-ethane (DME).Add Benzenecarbonyl chloride., obtain 2,3,5-three-O-benzoyl-2-C-methyl D-ribonic acid-gamma lactone ( 2); Make 2,3,5-three-O-benzoyl-2-C-methyl D-ribonic acid-gamma lactone ( 2) reacted about 40 minutes down in-5～0 ℃ in toluene with Red-Al/ ethanol, obtain 2,3,5-three-O-benzoyl-2-C-methyl-β-D-ribofuranose ( 3), at last in the presence of DMAP and DME, to 2,3,5-three-O-benzoyl-2-C-methyl-β-D-ribofuranose ( 3) cold soln in, add Benzenecarbonyl chloride ./TEA, reaction was carried out about 4 hours to about 12 hours, obtain end product ( 4), 2,3,5-three-O-benzoyl-2-C-methyl-β-D-ribofuranose.
Particularly, the aqueous solution of D-fructose is at room temperature modulated, and the preferred molar ratio rate that joins CaO wherein is 5: 1, and more preferably 3: 1, most preferably be 2.3: 1.3, the adding mode is cumulative adding in 5 minutes.React on and carried out under 23～40 ℃ 6～22 hours, regularly take out reactant liquor and carry out the TLC analysis.
When finish reaction time, can in reasonable time, in mixture, blast CO 2About 2～3 hours, so that pH is reduced to neutral level from alkalescence.Any CaCO that in neutralization procedure, forms 3All can be removed, for example remove by vacuum filtration.Combining water layer, and handle with 1: 2 molar ratio with oxalic acid (or other organic acid) and D-fructose, 25 ℃ of stir abouts 30 minutes up to white serosity occurring.Then, stir this serosity in 45-50 ℃ and spend the night, the water of the overwhelming majority is removed in decompression distillation down, obtains still being the mixture of liquid condition.In serosity, add NaCl and THF, stir about 30 minutes under the room temperature.Separating obtained layer joins water layer among the fresh THF, and other stirred 10 minutes.The process that adds THF, stirring and separating obtained liquid level repeats 3 times.At last, merge all THF solution, with anhydrous MgSO 4Together stirred 30 minutes, filtering mixt is washed MgSO with THF 4Filter cake.Filtrate is collected crude product in about 40 ℃ of following distilling under reduced pressure, is the darkorange semi-solid.
Then, add acetone in crude product, mixture stirred 3 hours down at 20 ℃.Vacuum filtration is collected the ribose acid lactone product of white crystals, washes and vacuum drying (seeing Fig. 1, chemical compound 1, flow process 1) with acetone.The product productive rate of this reaction is about 13.6%, compared with prior art, has improved nearly 4%.
Then, (ribose acid lactone: DMAP), excessive TEA and DME mix, stir about was 30 minutes under the room temperature with about 5: 1 of molar ratio for the ribose acid lactone product of acquisition and DMAP.The suspension of gained is cooled to 5 ℃, with the ratio adding Benzenecarbonyl chloride. about 5: 1 with ribose acid lactone.Stirred the mixture under the room temperature about 4 hours, up to confirming that by TLC initiation material runs out of.Then, in reactant mixture, added frozen water and stir about 30 minutes, then, collect crude product, with t-butyl methyl ether stirring, filtration, washing and vacuum drying.The white solid of collecting is 2,3, and 5-three-O-benzoyl-2-C-methyl D-ribonic acid-gamma lactone, productive rate are 83.4%, and purity (is seen Fig. 1, chemical compound near 98% 2).
By 2,3 of previous steps acquisition, 5-three-O-benzoyl-2-C-methyl D-ribonic acid-gamma lactone is cooled to-5 ℃ approximately, to wherein adding in advance at the dry toluene of 0 ℃ of blended Red-Al and the solution of dehydrated alcohol.Red-Al and 2,3, the molar ratio of 5-three-O-benzoyl-2-C-methyl D-ribonic acid-gamma lactone is about 2: 1.Mixture remained on-5 ℃ of stir abouts 40 minutes.Take out sample liquid from mixture and be used for TLC and/or HPLC analysis,, with acetone, water and 1N HCl reaction is finished then, return to room temperature to confirm whether initiation material runs out of.At last, the mixture ethyl acetate extraction is with salt washing, drying, in about 40 ℃ of following removal of solvent under reduced pressure.Products therefrom 2,3, the productive rate of 5-three-O-benzoyl-2-C-methyl D-ribofuranose are that the amount of 5-three-O-benzoyl-2-C-methyl D-ribonic acid-gamma lactone is the quantitative yield of benchmark with 2,3 of use when this step begins.(see Fig. 1, chemical compound 3).
The protecting group of ribofuranose C-1 is and then in next step formation.Benzenecarbonyl chloride. with about 2: 1 molar ratio join 5 ℃ 2,3, in the solution of 5-three-O-benzoyl-2-C-methyl D-ribofuranose, the DMAP that is dissolved in anhydrous DME in addition and the TEA of Jia Ruing simultaneously.Stirring reaction also spends the night, and then, with frozen water and aqueous sodium carbonate reaction is finished.Then, remove THF, the mixture ethyl acetate extraction.Produce condensed oily product behind washing, drying and the removal solvent.To wherein adding t-butyl methyl ether, heptane and water, about 20 ℃ of stir abouts 2 hours.Behind washing and the vacuum drying, end product 1,2,3, the productive rate of 5-four-O-benzoyl-2-C-methyl-β-D-ribofuranose is 52%, purity (is seen Fig. 1, chemical compound greater than 98% 4, flow process 1).
Another preferred implementation of the present invention is shown in Fig. 4, comprises making cytosine, BSA and SnCl 4In/acetonitrile and the first embodiment of the invention 1,2,3,5-four-O-benzoyl-2-C-methyl-β-D-ribofuranose ( 4) reaction, obtain 4-amino-1-(3,4-dibenzoyl oxygen base-5-benzoyloxy methyl-3-methyl-oxolane-2-yl)-1H-pyrimid-2-one ( 5); Make ( 5) react with NaOMe/MeOH, obtain (4-amino-1-(3,4-dihydroxy-5-methylol-3-C-methyl-oxolane-2-yl)-1H-pyrimid-2-one ( 6), be also referred to as β-D-2 '-C-methyl-cytidine.Use cytosine as initiation material rather than benzoyl-cytosine, improved " the atom benefit " of process, and simplified the purification in the subsequent step.
Synthetic can ( 6) formation and terminate, product separates by known method.In addition, also can further synthesize, 3 of preparation β-D-2 '-C-methyl-cytidine (2-amino-3-methyl-butanoic acid 5-(4-amino-2-oxygen-2H-pyrimidine-1-yl)-4-hydroxyl-4-C-methyl-2-methylol-oxolane-3-base ester) '-the O-L-valine ester, perhaps its preferred hydrochloride form, the 4th preferred implementation of the present invention.
In another preferred implementation of the present invention, chemical compound ( 6) and Me 2NCH (OMe) 2In DMF, react, form ( 7), N-[1-(3,4-dihydroxy-5-methylol-3-methyl-oxolane-2-yl)-2-oxygen-1,2-dihydro-pyrimidine-4-yl]-N, N-dimethyl carbonamidine, its be ( 6) aminoacid-protection form; Make ( 7) react in DCM with TBDPSCl and imidazoles, obtain ( 7) 5 '-form of silicyl-protection, promptly N '-1-[5-(tert-butyl group-diphenyl-silane oxygen ylmethyl)-3,4-dihydroxy-3-methyl-oxolane-2-yl]-2-oxygen-1,2-dihydro-pyrimidine-4-yl }-N, N-dimethyl-carbonamidine ( 8), wherein, using the benefit of DCM is the control better to being formed with of dimethylamino silane by-product; Make ( 8) under room temperature, react in DCM with N-Boc-L-valine, EDC and DMAP, form 2-t-butoxycarbonyl amino-3-methyl-butanoic acid 2-(tert-butyl group-diphenyl-silicon alkoxyl-methyl)-5-[4-(dimethylamino-methene amido)-2-oxygen-2H-pyrimidine-1-yl]-4-hydroxy-4-methyl-oxolane-3-base ester ( 9); By make ( 9) and NH 4F reacts in the presence of the ethyl acetate of about 10 molar equivalents (cut off for the ammonia that prevents to separate out 3 '-O-L-valine ester) in MeOH; remove silicyl and amino-protecting group; and the backflow mixture, obtain 2-t-butoxycarbonyl amino-3-methyl-butanoic acid 5-(4-amino-2-oxygen-2H-pyrimidine-1-yl)-4-hydroxyl-2-methylol-4-methyl-oxolane-3-base ester ( 10); Make at last, ( 10) react in EtOH with HCl, obtain 2-amino-3-methyl-butanoic acid 5-(4-amino-2-oxygen-2H-pyrimidine-1-yl)-4-hydroxyl-2-methylol-4-methyl-oxolane-3-base ester, dihydrochloride ( 11) as final products.
The present invention further is illustrated in the following non-limiting example.These examples given here are in order to help the understanding to invention.They should not be construed as the description of the inventive method and product the present invention who limits in the claim are constituted any restriction.Under the prerequisite that does not break away from spirit of the present invention, equivalent, similar or suitable solvent, reagent or reaction condition can be replaced by those specific solvents described herein, reagent and/or reaction condition.
Embodiment
Embodiment 1
2-C-methyl D-ribonic acid-gamma lactone
In 3 mouthfuls of round-bottomed flasks of the 250mL that has been equipped with suspension type agitator, shaft, digital temperature sensing device and ar gas passage, agitating deionized water (100mL).Blasted argon 30 minutes, (20.0g, 0.111mol), it is limpid that solution became in a few minutes to add D-fructose.In 5 minutes, add in batches calcium oxide (12.5g, 0.223mol), the vigorous stirring mixture.Observe heat release, rise to 39.6 ℃ from adding 10 minutes afterreaction temperature of calcium oxide.After about 15 minutes, reactant mixture becomes yellow, and color is deepened in time.After 3 hours, take out the partial reaction mixture and carry out the TLC analysis.The part of taking out uses the saturated oxalic acid acidified aqueous solution to pH 2.Resultant white suspension is dephlegmate under reduced pressure.Add toluene (2mL) in residual liquid, mixture under reduced pressure distills (at 45-50 ℃), removes all residual water.Residual solid joins 1: 1 the oxolane of 2mL once more: in the carbinol mixture.After fully mixing, suspension is left standstill, (silica gel plate launches with the ethyl acetate that contains 2% methanol, is immersed in 1% alkalinity potassium permanganate and dyes to put TLC with supernatant.Then, use hot rifle hot plate, up to xanchromatic point in the pink colour background, occurring).Under these conditions, the lactone of expectation appears at R usually fValue is 0.33 place.More polarity by-product and unreacted raw material are at R fValue is that 0.0～0.2 place detects.
Although observe the formation of product after 3 hours, reaction is still proceeded 22 hours.Therebetween, reactant mixture is 25 ℃ of stirrings.Last during this period, the pH of mixture is 13.06.In reactant mixture, blast carbon dioxide about 2.5 hours (pH is 7.25).The calcium carbonate solid that forms is removed by vacuum filtration, and filter cake is washed with the 50mL deionization.Combining water layer and with oxalic acid (5.0g 0.056mol) handles, under 25 ℃ to mixture vigorous stirring 30 minutes (initial dark color disappears basically, and mixture becomes the milky serosity).The pH of the mixture in this stage is generally 2-3.Slurry mixture stirs down at 45-50 ℃ and spends the night.Then, at 45-50 ℃ of following distilling under reduced pressure mixture, remove 75mL water.Add sodium chloride (30g) and oxolane (100mL) in hydrotropism's serosity (about 75mL), in 25 ℃ of vigorous stirring mixture 30 minutes.Separate each layer, use the fresh oxolane of 75mL to stir water layer 10 minutes.This process repeats 3 times, merges tetrahydrofuran solution and stirs 30 minutes with the anhydrous magnesium sulfate of 10g.Filtering mixt, the magnesium sulfate filter cake is washed with the 60mL oxolane.Filtrate obtains the 10.86g crude product 40 ℃ of following distilling under reduced pressure, and it is the darkorange semisolid.(, replacing oxolane to replace the distillation crude product) to dry with acetone for more massive synthetic.Under 20 ℃, crude product was stirred 3 hours in acetone (20mL).Vacuum filtration is collected product, with 12mL acetone filter wash cake, obtains the expectation product 1 of white crystalline solid.Vacuum drying obtains 2.45g (13.6% productive rate).The fusing point of chemical compound 1: 158-162 ℃ (bibliographical information fusing point: 160-161 ℃). 1H NMR (DMSO-d 6) (s, 1H use D to δ ppm 5.69 2O exchange), 5.41 (d, 1H use D 2O exchange), 5.00 (t, 1H use D 2The O exchange), 4.15 (m, 1H), 3.73 (m, 2H), 3.52 (m, 1H), 1.22 (s, 3H). 13C NMR(DMSO-d 6)δppm 176.44，82.95，72.17，72.02，59.63，20.95。(C 6H 10O 5: value of calculation C, 44.45; H, 6.22.Measured value: C, 44.34; H, 6.30).
Embodiment 2
2,3,5-three-O-benzoyl-2-C-methyl D-ribonic acid-lactone
In 25 ℃ of argon atmospheres, stir acetone 1(3.0g, 18.50mmol.), 4-dimethylaminopyridine (0.45g, 3.72mmol.) and triethylamine (25.27g, 249.72mmol.) 1, the mixture in the 2-dimethoxy-ethane (50mL) 30 minutes.This white suspension is cooled to 5 ℃, in 15 minutes, add Benzenecarbonyl chloride. (11.7g, 83.23mmol.).Stirred the mixture two hours at 25 ℃.TLC analyzes (silica gel, the ethyl acetate solution of 2% methanol) and shows the initiation material full consumption.In reactant mixture, add frozen water (100g), continue to stir 30 minutes.The white solid that forms is collected by vacuum filtration, with cold water (50mL) filter wash cake.At 20 ℃ this crude product and t-butyl methyl ether (60mL) are together stirred, filter then, filter cake is washed with t-butyl methyl ether (25mL), and vacuum drying, obtains 7.33g chemical compound 2 (83.4% productive rate), and it is a white solid, and purity is 97.74% (HPLC/AUC).Chemical compound 2Fusing point: 137-140 ℃ (literature value: 141-142 ℃). 1H NMR(CDCl 3)δppm 8.04(d，2H)，7.92(d，2H)，7.73(d，2H)，7.59(t，1H)，7.45(m，4H)，7.32(t，2H)，7.17(t，2H)，5.51(d，1H)，5.17(m，1H)，4.82-4.66(d of an ABquartet，2H)1.95，(s，3H)。 13C NMR(CDCl 3)δppm 172.87，166.17，166.08，165.58，134.06，133.91，133.72，130.09，129.85，129.80，129.37，128.78，128.60，128.49，127.96，127.89，79.67，75.49，72.60，63.29，23.80。TOF MS ES+(M+1：475)。
Embodiment 3
2,3,5-three-O-benzoyl-2-C-methyl-β-D-ribofuranose
To Red-Al (65wt.% is dissolved in toluene, 2.0mL, 6.56mmol) solution in dry toluene (2.0mL) stirs under 0 ℃ of argon atmosphere.(0.38mL, 6.56mmol) solution in dry toluene (1.6mL) was added in the above-mentioned toluene solution in 5 minutes dehydrated alcohol.The gained mixture stirred 15 minutes at 0 ℃, the 2mL (2.18mmol) in this Red-Al/ ethanol reagent was joined 2,3 in 10 minutes, 5-three-O-benzoyl-2-C-methyl D-ribose acid lactone 2(475mg is in the cold soln of dry toluene 1.0mmol) (10mL) (5 ℃).TLC analyzes (silica gel plate, the n-heptane solution of 35% ethyl acetate) and shows the initiation material full consumption.HPLC the analysis showed that only remaining 0.1% initiation material.With acetone (0.2mL), water (15mL) and 1NHCl (15mL) finish reaction at 0 ℃, and are heated to room temperature.Add 1N HCl (5mL) dissolve inorganic salts (pH:2-3).(3 * 25mL) extract mixture, and (25mL) washes organic solution with saline, and dry (anhydrous sodium sulfate, 10g), 40 ℃ of decompressions remove down desolvates the product that obtains expecting with ethyl acetate 3, quantitative yield (480mg).This material is used to following step.
Embodiment 4
1,2,3,5-four-O-benzoyl-2-C-methyl-β-D-ribofuranose
To cold chemical compound 3 (480mg, 1.0mmol), 3,4-dimethylaminopyridine (12.3mg, 0.1mmol) and triethylamine (506mg, in anhydrous tetrahydro furan 5.0mmol) (5mL) solution, in 5 minutes, add Benzenecarbonyl chloride. (283mg, 2.0mmol).Under the room temperature in the argon atmosphere stirred reaction mixture spend the night.HPLC the analysis showed that 0.25% unreacted initial substance.Reaction finishes by adding frozen water (10g) and saturated solution of sodium bicarbonate.Remove oxolane under the decompression, with ethyl acetate (50mL) extraction mixture.Water (25mL), saline (25mL) are washed organic solution, and dry (anhydrous sodium sulfate, 12g), decompression removes down and desolvates, and obtains the stiff grease of 650mg.The t-butyl methyl ether of this crude product and 5mL together stirred 5 minutes, added heptane (5mL) and water (0.1mL), continued to stir 2 hours at 20 ℃.Vacuum filtration is collected solid, and filter cake was with 1: 1 heptane: t-butyl methyl ether solution (6mL) and t-butyl methyl ether (2mL) are washed.The vacuum drying solid obtains 300mg (52%) expectation product 4 (purity 98.43% is by the HPLC/AUC purification), and it is a white solid, fusing point: 154-156.3 ℃ (literature value: 155-156 ℃). 1H NMR(CDCl 3)δppm 8.13(m，4H)，8.07(d，2H)，7.89(d，2H)，7.63(m，3H)，7.48(m，6H)，7.15(m，3H)，7.06(s，1H)，5.86(dd，1H)，4.79(m，1H)，4.70-4.52(d of an AB quartet，2H)，1.95，(s，3H)。 13CNMR(CDCl 3)8ppm 166.31，165.83，165.01，164.77，134.01，133.86，133.70，133.17，130.44，130.13，129.97，129.81，129.59，129.39，129.07，128.84，128.76，128.37，98.01，86.87，78.77，76.35，64.05，17.07。(C 34H 28O 9: value of calculation C, 70.34; H, 4.86.Measured value: C, 70.20; H, 4.95).
Embodiment 5
4-amino-1-(3,4-dibenzoyl oxygen base-5-benzoyloxy methyl-3-methyl-oxolane-2-yl)-1H-pyrimid-2-one: (chemical compound 2, Fig. 4)
In the 12L round-bottomed flask that has been equipped with reflux condenser, suspension type agitator and argon inlet adapter, (89g 0.80mol) is suspended in the acetonitrile (900ml) with cytosine.Under 20 ℃ argon atmosphere, stir the mixture, add a part of N, and O-two (trimethyl silyl) acetamide (537ml, 2.2mol).Gained solution is heated to 80 ℃ and stirred 1 hour under uniform temp.1,2,3, (425.0g 0.73mol) is suspended in the acetonitrile (4000ml), and joins in the reactant mixture 5-four-O-benzoyl-2-C-methyl-β-D-ribofuranose.After a few minutes, it is limpid that reactant mixture becomes, and temperature drops to ca.50 ℃.(154ml 1.31mol), continues to stir at 80 ℃ to add stannic chloride (IV) in 15 minutes.After 1 hour, add sodium bicarbonate solution and make the partial reaction mixture finish reaction, use the acetic acid ethyl acetate extract layer.With TLC (silica gel, the n-heptane solution of 20% ethyl acetate, the R of sugar derivatives f: 0.40) detect ethyl acetate layer.TLC the analysis showed that the sugar derivatives full consumption.Use the dichloromethane solution of 10% methanol, detect the product (R of expectation by TLC f: 0.37).Reaction is also monitored (method #2) by HPLC.Reactant mixture is cooled to 20 ℃, adds saturated sodium bicarbonate solution (3000mL) in 30 minutes and finishes reaction (observing heat release when adding several milliliters of initial sodium bicarbonate solutions).Gradation adds solid sodium bicarbonate (1350g) to avoid foaming.Detect mixture to guarantee its pH 〉=7.Stop to stir, made each layer separation 20 minutes.Get rid of water layer, together stir, make mixture separation (30 minutes) with ethyl acetate (1500ml).Separate organic layer, merge with acetonitrile solution.Organic solution is washed with saline (500ml), removes then and desolvates, and volume reaches ca.750ml.Product can be used for subsequent reaction.Also can further remove to desolvate and obtain the white foam shape solid of quantitative yield.Chemical compound ( 2) structure by 1H NMR analysis confirmation.
Embodiment 6
4-amino-1-(3,4-dihydroxy-5-methylol-3-methyl-oxolane-2-yl)-lH-pyrimid-2-one: (chemical compound 3)
To chemical compound ( 2) (416g, in methanol 0.73mol) (2000ml) solution, the adding Feldalat NM (13.8g, 0.26mol).Reactant mixture at room temperature stirs, and by TLC (silica gel, the dichloromethane solution of 10% methanol, chemical compound 1R f: 0.53) and (silica gel, the dichloromethane solution of 30% methanol, chemical compound 3R f: 0.21) monitoring.After 30 minutes, product begins precipitation, and TLC shows that two hours afterreactions finish.Reaction is also monitored (method #2) by HPLC.The decompression under remove methanol to volume ca.500ml, re-use ethanol drive (2 * 500ml), make volume reach ca.500ml.Remaining stiff serosity 750ml ethanol dilution, mixture stirred 1 hour at 20 ℃.Filter to collect product, filter cake is washed with ethanol (100ml) and t-butyl methyl ether (100ml), obtain after the drying 168g (two productive rates that go on foot are 90%) product ( 3), pure＞97% (HPLC/AUC).Product has also passed through 1H and 13The analysis of C NMR.
Embodiment 7
N-[1-(3,4-dihydroxy-5-methylol-3-methyl-oxolane-2-yl)-2-oxygen-1,2-dihydro-pyrimidine-4-yl]-N, N-dimethyl-carbonamidine: (chemical compound 4)
(19g 0.0738mol) at anhydrous N, in the suspension of dinethylformamide (150ml), adds N, and (98ml's dinethylformamide dimethyl-acetal 0.7385mol), stirs the mixture at 20-22 ℃ to chemical compound 3.After one hour, TLC (silica gel, the dichloromethane solution of 30% methanol, the R of chemical compound 3 fBe 0.21, the R of product 4 fBe 0.55) show to react and finish.Decompression removes down and desolvates and reagent (maintaining the temperature at below 40 ℃).Add ethanol (50ml) in the oily residue that obtains, decompression removes down and desolvates.This process repeats twice, and crude product is solidified.Crude product and 190ml ethanol together stirred under 20 ℃ 1 hour, kept 12 hours at 5 ℃.Solid collected by filtration, filter cake is washed with the cold ethanol of 30ml and the cold t-butyl methyl ether of 30ml.The drying under reduced pressure solid obtains 14.7g (64%) chemical compound (4), as first product.TLC (silica gel, the dichloromethane solution of 30% methanol, the R of product (4) fBe 0.55) and (silica gel, the dichloromethane solution of 10% methanol, the R of product (4) fBe 0.1) only demonstrate a point of chemical compound (4).Mother solution after ethanol is purified is evaporated drying, and residue and ethanol (80ml) stirred 1 hour down at 20 ℃, and kept 12 hours at 5 ℃.Solid collected by filtration, filter cake is washed with the cold ethanol of 15ml and the cold t-butyl methyl ether of 15ml.Behind the vacuum drying solid, obtain 3.5g (15%) solid, as second product.TLC (silica gel, the dichloromethane solution of 30% methanol, the R of product (4) fBe 0.55) and (silica gel, the dichloromethane solution of 10% methanol, the R of product (4) fBe 0.1) only demonstrate a point of chemical compound (4); M.p.201-209 ℃; 1H NMR (DMSO-d 6) δ ppm 8.62 (s, 1H, N=CH), 8.17 (d, 1H, H-6, J 5-6=7.3Hz), 5.91 (m, 2H, H-1 ', H-5), 5.16 (t, 1H, OH-5 ', D 2O is commutative), 5.06 (s, 1H, OH-2 ', D 2O is commutative), 3.8-3.5 (m, 4H, H-3 ', H-4 ', H-5 ' and H-5 "), 3.15 and 3.02 (2s, 6H, N (CH 3) 2), 0.92 (s, 3H, CH 3); FAB＞0 (GT) 625 (2M+H) +, 313 (M+H) +, 167 (B+2H) +FAB＜0, (GT) m/z 419 (M+T-H) -, 403 (M+G-H) -, 311 (M-H) -, 165 (B) -
Embodiment 8
N '-and 1-[5-(tert-butyl group-diphenyl-silane oxygen ylmethyl)-3,4-dihydroxy-3-methyl-oxolane-2-yl] 2-oxygen-1,2-dihydro-pyrimidine-4-yl }-N, N-dimethyl-carbonamidine: (chemical compound 5)
Chemical compound ( 4) (42.9g 0.137mol) was distributed in the anhydrous methylene chloride (200ml) by the stirring of suspension type agitator in 30 minutes.Then, use rotary evaporator that mixture is evaporated to drying, at ca.30 ℃).Should exsiccant chemical compound ( 4), imidazoles (37.4g, 0.55mol) and anhydrous methylene chloride (800ml) under argon atmosphere, join in the 4-mouth round-bottomed flask of 2L, tert-butyl diphenyl chlorosilane (43.1g, 0.156mol are divided into several times and add) is transferred in the additional channel that is connected in reaction flask.Reactant mixture is cooled to 10 ℃, and (13.74g 0.05mol), keeps reaction temperature while stirring at 10～12 ℃ to add tert-butyl diphenyl chlorosilane from additional channel in 20 minutes.Reaction is by HPLC monitoring (method #2).1.5 after hour, (14.76g 0.053mol), keeps reaction temperature at 10～12 ℃ to the tert-butyl diphenyl chlorosilane of adding second portion while stirring in 20 minutes.After 1 as a child, (14.8g 0.053mol), keeps reaction temperature while stirring at 10～12 ℃ to add remaining tert-butyl diphenyl chlorosilane in 20 minutes.Then, 12-15 ℃ of restir 1.5 hours.HPLC shows 95.40% product, 3.00% two-silicyl derivant and unreacted raw material.Add saturated sodium bicarbonate solution (150ml) and finish reaction, and stir near 15 minutes (pH is 8) at 15 ℃.Separate water layer and dichloromethane layer.Water (2 * 150ml) and saline (1 * 200ml) washes dichloromethane layer, with anhydrous sodium sulfate drying (60.0g, 30 minutes).Then, remove by filter solvent under the decompression.Remaining foam shape solid is used for the reaction in step down.
Embodiment 9
2-tert-butoxycarbonyl amino-3-methyl-butanoic acid 2-(tert-butyl group-diphenyl-silane oxygen ylmethyl)-5[-4 (dimethylamino-methene amido)-2-oxygen-2H-pyrimidine-1-yl]-4-hydroxy-4-methyl-oxolane-3-base ester: (chemical compound 6)
Agitate compounds in 25 ℃ of following argon atmospheres ( 5) (58g, dichloromethane 0.1053mol) (500ml) solution.Add N-(tert-butoxycarbonyl)-L-valine (29.7g, 0.1367mol), 1-[3-(dimethylamino) propyl group]-3-ethyl carbon imidodicarbonic diamide hydrogen chloride (26.2g, 0.1367mol) and 4-(dimethylamino) pyridine (1.3g, 0.0106mol), 25 ℃ of following stirred reaction mixtures are by HPLC monitoring (method #2).After 4 hours, HPLC shows and contains 7.9% initiation material.Add N-(tert-butoxycarbonyl)-L-valine (4.57g, 0.0210mol) and 1-[3-(dimethylamino) propyl group]-(4.03g 0.0210mol), continues 25 ℃ of restir 2 hours 3-ethyl carbon imidodicarbonic diamide hydrogen chloride.Then, detect 0.7% initiation material through HPLC.In reactant mixture, add methanol (60ml), solvent removed by evaporation at reduced pressure (temperature remains on below 40 ℃), obtain chemical compound ( 6), be stiff oil.This material (93% purity, HPLC/AUC) be used to down the step reaction.
Embodiment 10
Tert-butoxycarbonyl amino-3-methyl-butanoic acid 5-(4-amino-2-oxygen-2H pyrimidine-1-yl)-4-hydroxyl-2-methylol-4-methyl-oxolane-3-base ester: (chemical compound 7)
With chemical compound ( 6) (0.3mol), MeOH (1650ml) and EtOAc (265g 3.0mol) joins in the 5-mouth round-bottomed flask of 3L, stir the mixture make chemical compound ( 6) dissolving.(45.0g 1.21mol), makes temperature reach 64.5 ℃ under refluxing, and stirs 4 hours to add amine fluoride.HPLC (method #2) shows and finishes after being reflected at 4 hours.Then, decompression removes in 40-45 ℃ down and desolvates, and drives with EtOAc (300ml).Remaining foam and EtOAc (400ml), water (600ml) and t-butyl methyl ether (300ml) merge, and crushed mixture is 2.5 hours under the room temperature.Filter to collect isolating white solid, water (200ml), 1: 1 EtOAc/ t-butyl methyl ether (120ml) and t-butyl methyl ether (120ml) are washed.Then, the vacuum drying solid surpasses 20 hours, obtain chemical compound ( 7), be white solid.Productive rate is 71.54g, and three steps were 52%.Chemical compound ( 7) purity be 99.08% (HPLC, method #3). 1H NMR (DMSO-d 6) δ ppm 7.99 (d, 1H, H-6, J 6-5=7.42Hz), 7.3-7.1 (m, 3H, CH and NH 2, D 2O is commutative), 5.9 (s, 1H, H-1 '), 5.75 (d, 1H, H-5, J 6-5=7.43Hz), 5.43 (s, 1H, OH-2 ', D 2O is commutative), 5.24 (t, 1H, OH-5 '), 5.04 (d, 1H, H-3 ', J 3 '-4 '=9.1Hz), 4.1-4.0 (m, 2H, H-4 ', CH), 3.8-3.4 (2m, 2H, H-5 ', H-5 "), and 2.2-2.0 (m, 1H, CB), 1.40 (s, 9H, (CH 3) 3C), 1.0 (s, 3H, CH 3), 0.9-0.8 (m, 6H, (CH 3) 2CH); FAB＜0, (GT) m/e 911 (2M-H) -, 455 (M-H) -, 256 (M-BocVal) -, 216 (BocValOH) -, 110 (B) -FAB＞0 (GT) 913 (2M+H) +, 457 (M+H) +, 112 (B+2H) +, 57 (CH 3) 3C) +FAB＜0 (GT) 911 (2M-H) -, 455 (M-H) -, 256 (M-BocVal) -, 216 (BocVal) -, 110 (B) -
Embodiment 11
2-amino-3-methyl-butanoic acid 5-(4-amino-2-oxygen-2H-pyrimidine-1-yl)-4-hydroxyl-2-methylol-4-methyl-oxolane-3-base ester (dihydrochloride): (chemical compound 8)
In the round-bottomed flask that has been equipped with suspension type agitator, temperature probe, ar gas passage and hydrogen chloride bubbler, agitate compounds (7) (21.0g, ethanol 0.046mol) (168ml) solution.In clear solutions, blasted hydrogen chloride gas (22g) 1 hour.With ice-water bath reaction temperature is remained on below 30 ℃.A few minutes begin to have solid to form after feeding hydrogen chloride gas.After 4 hours, HPLC (method #3) shows to have only 0.8% initiation material.Solid collected by filtration, filter cake is washed with ethanol (20ml) and diethyl ether (100ml).Behind the drying under reduced pressure product 16 hours, obtain 19.06g (96.5%) product (8), purity is 97.26% (HPLC, method #3); M.p.210 ℃ (becoming brown), 248-250 ℃ (fusion); 1H NMR (DMSO-d 6) δ ppm 10.0 (s, 1H, 1/2NH 2, D 2O is commutative), 8.9-8.6 (2 br s, 4H, 1/2NH 2, NH 3, D 2O is commutative), 8.42 (d, 1H, H-6, J 5-6=7.9Hz), 6.24 (d, 1H, H-5, J 5-6=7.9Hz), 5.84 (s, 1H, H-1 '), 5.12 (d, 1H, H-3 ', J 3 '-4 '=8.8Hz), 4.22 (d, 1H, H-4, J 3 '-4 '=8.7Hz), 4.0-3.9 (m, 1H, CH), 3.8-3.5 (m, 2H, H-5 ', H-5 "), and 2.3-2.1 (m, 1H, CH), 1.16 (s, 3H, CH 3), 1.0 (m, 6H, (CH 3) 2CH); FAB＞0 (GT) 713 (2M+H) +, 449 (M+G+H) +, 357 (M+H) +, 246 (S) +, 112 (B+2H) +FAB＜0 (GT) 747 (2M+Cl) -, 483 (M+G+Cl) -, 391 (M+Cl) -, 355 (M-H) -, 116 (Val) -, 110 (B) -, 35 (Cl) -
Embodiment 12
The HPLC method of testing
Above-mentioned all methods are all used the reverse chromatograms post; Waters Numbering of part #WAT086344; Nova-Pak C18,60 ' hole size, 4 μ m granular sizes, 3.9 * 150mm.All chromatographs are all used Waters 2695 HPLC and 996 PDA monitors obtain.
Mobile phase: HPLC level acetonitrile and water are buied from JT Baker, and the acetic acid triethylamine solution of 1M is from Fluka Buy.
Method #1: be used for test compounds 4, Fig. 4:
Flow velocity: 1.00ml/min.The linear gradient of acetonitrile/water is as follows.
System's balance 5 minutes all before each test.
Wavelength: 254nm.
Chemical compound 4Retention time=12.8 minute.
Time The % acetonitrile % water
0.00 40.0 60.0
1.00 40.0 60.0
13.0 95.0 5.0
15.0 95.0 5.0
Method #2: be used for test compounds 2,4,5,6 and 7, Fig. 4:
Flow velocity: 1.00ml/min.The linear gradient of acetonitrile/20mM acetic acid triethylamine buffer solution is as follows.
System's balance 5 minutes all before each test.
Wavelength: 320 and 272nm.
Time The % acetonitrile The % buffer
0.00 0.00 100.0
15.00 80.0 20.0
30.00 80.0 20.0
Contrast table: chemical compound and retention time, method #2:
Chemical compound Retention time (minute) wavelength
1 18.2，272nm
2 13.4，272nm
3 2.9，272nm
Essence of Niobe 11，272nm
The chemical compound of part protection 3 72，272nm
The chemical compound of part protection 3 10.0，272nm
4 4.0，320nm
5 13.2，320nm
Two-silylated chemical compound 5 16.6，320nm
6 17.8，320nm
DMAP (3.7 broad peak), 272nm
7 8.3，272nm
6 of part protection 16.3，272nm
Method #3: be used for test compounds 3,7 and 8, Fig. 4:
Flow velocity: 1.00ml/min.The linear gradient of acetonitrile/20mM acetic acid triethylamine buffer solution is as follows.
System's balance 5 minutes all before each test.
Wavelength: 272nm.
Time The % acetonitrile The % buffer
0.00 0.00 100.0
30.0 50.0 50.0
Contrast table: chemical compound and retention time, method #3:
Chemical compound Retention time (minute)
7 18.4
8 8.5
3 3.6
Art methods
Embodiment 13
The N4-[(dimethylamino) methylene]-preparation of β-D 2 '-C-methyl-cytidine (4):
Use N, the dinethylformamide dimethyl-acetal (8.2ml, 61.73mmol) handle β-D-2 '-C-methyl-cytidine ( 3) (stir about is 1.5 hours under the room temperature for 1.65g, the 6.43mmol) solution in DMF (32ml).Solution under reduced pressure with the ethanol coevaporation.Crystallization from ethanol/ether, produce unknown so far chemical compound ( 4) (first product, 1.21g, 60% productive rate, second product, slightly impure, 0.46g, 23% productive rate), be crystalloid.
Following physical-chemical characteristic is the crystalline solid acquisition by first product.F=201-209 ℃; 1H NMR (DMSO-d 6) δ ppm 8.62 (s, 1H, N=CH), 8.17 (d, 1H, H-6, J 5-6=7.3Hz), 5.91 (m, 2H, H-1 ', H-5), 5.16 (t, 1H, OH-5 ', D 2O is commutative), 5.06 (s, 1H, OH-2 ', D 2O is commutative), 3.8-3.5 (m, 4H, H-3 ', H-4 ', H-5 ' and H-5 "), 3.15 and 3.02 (2s, 6H, N (CH 3) 2), 0.92 (s, 3H, CH 3);
FAB＞0 (GT) 625 (2M+H) +, 313 (M+H) +, 167 (B+2H) +FAB＜0, (GT) m/z419 (M+T-H) -, 403 (M+G-H) -, 311 (M-H) -, 165 (B) -Carry out 5.96 minutes (CH of from 0 to 50% of HPLC under the room temperature 3The buffer of N in 20mM acetic acid triethylamine, program is 30 minutes, flow velocity is 1ml/min), λ max=316.1nm.
Embodiment 14
N 4-[(dimethylamino) methylene]-5 '-preparation of O-t-butyldiphenylsilyl-β-D 2 '-C-methyl-cytidine (5):
To chemical compound ( 4) (1.167g, 3.73mmol) in the solution of dried pyridine (15ml), continue to add imidazoles (760mg, 11.19mmol) and tert-butyl diphenyl chlorosilane (0.66ml, 2.53mmol).Agitating solution under the room temperature.After 4 hours, (0.40ml 2.28mmol) also at room temperature stirred 2 hours to mend tert-butyl diphenyl chlorosilane in the reactant mixture.Then with the sodium bicarbonate extraction, washing has basic unit, dried over sodium sulfate, decompression evaporation down.Crude mixture is mixed with dry acetonitrile (30ml) and dried dimethyl formamide (15ml).
Embodiment 15
N 4-[(dimethylamino) methylene]-5 '-O-t-butyldiphenylsilyl-β-D 2 '-C-methyl-cytidine 3 '-preparation of O-L-N-(tertbutyloxycarbonyl) L-valine ester (6):
Forward the step obtain chemical compound ( 5) in continue to add N-(tertbutyloxycarbonyl)-L-valine (Boc-Val-OH, 400mg, 1.87mmol), N '-(3-dimethylaminopropyl)-N-ethyl carbon imidodicarbonic diamide hydrogen chloride (DEC, 715mg, 3.73mmol) and 4-dimethylaminopyridine (DMAP, 68mg, 0.56mmol), stir the gained mixture under the room temperature.Course of reaction is monitored by HPLC.Mend 3 Boc-Val-OH (400mg * 3) in the reactant mixture, DEC (715mg * 3) and DMAP (68mg * 3) mend a Boc-Val-OH (200mg), DEC (357mg) and DMAP (34mg) at last again.Two days later, initial substance all consumes, and removes DMF under the decompression.The residue chemical compound ( 6) join in the dried ethanol (70ml).
Embodiment 16
3 of β-D-2 '-C-methyl-cytidine (7) '-preparation of O-L-N-(tertbutyloxycarbonyl) L-valine ester (6):
To contain the residue chemical compound ( 6) dried ethanol (70ml) in add amine fluoride (1.38g, 37.30mmol), backflow mixture 3 hours.Filtering mixt and removal of solvent under reduced pressure.Residue is added the extraction of ethyl acetate and water several times.The vacuum evaporation organic facies, purification on silica gel chromatographic column (eluant: MeOH (20%): EtOAc (80%)).Isolate desired compounds ( 7) (1.37g, the productive rate in 3 steps is 78%), be white foam.
The physical-chemical data comprise: 1H NMR (DMSO-d 6) δ ppm 7.99 (d, 1H, H-6, J 6-5=7.42Hz), 7.3-7.1 (m, 3H, CH and NH 2, D 2O is commutative), 5.9 (s, 1H, H-1 '), 5.75 (d, 1H, H-5, J 6-5=7.43Hz), 5.43 (s, 1H, OH-2 ', D 2O is commutative), 5.24 (t, 1H, OH-5 '), 5.04 (d, 1H, H-3 ', J 3 '-4 '=9.1Hz), 4.1-4.0 (m, 2H, H-4 ', CH), 3.8-3.4 (2m, 2H, H-5 ', H-5 "), and 2.2-2.0 (m, 1H, CH), 1.40 (s, 9H, (CH 3) 3C), 1.0 (s, 3H, CH 3), 0.9-0.8 (m, 6H, (CH 3) 2CH); FAB＜0, (GT) m/e 911 (2M-H) -, 455 (M-H) -, 256 (M-BocVal) -, 216 (BocValOH) -, 110 (B -FAB＞0 (GT) 913 (2M+H) +, 457 (M+H) +, 112 (B+2H) +, 57 (CH 3) 3C) +FAB＜0 (GT) 911 (2M-H) -, 455 (M-H) -, 256 (M-BocVal) -, 216 (BocValOH) -, 110 (B) -
Embodiment 17
β-D 2 '-C-methyl-cytidine (dihydrochloride, (8) 3 '-preparation of O-L-L-valine ester:
With 20%HCl/ ethyl acetate solution (75ml) handle chemical compound ( 7) (1.32g, 2.9mmol) solution in dry ethyl acetate (75ml).Stirred reaction mixture is 2 hours under the room temperature.Title compound ( 8) from reactant mixture, be settled out, filter and use Et 2O (1.01g, 81% productive rate) washes.The physical-chemical data comprise: F=210 ℃ (sending out brown), 234-241 ℃ (fusion); 1H NMR (DMSO-d 6) δ ppm 10.0 (s, 1H, 1/2NH 2, D 2O is commutative), 8.9-8.6 (2 br s, 4H, 1/2NH 2, NH 3, D 2O is commutative), 8.42 (d, 1H, H-6, J 5-6=7.9Hz), 6.24 (d, 1H, H-5, J 5-6=7.9Hz), 5.84 (s, 1H, H-1 '), 5.12 (d, 1H, H-3 ', J 3 '-4 '=8.8Hz), 4.22 (d, 1H, H-4, J 3 '-4 '=8.7Hz), 4.0-3.9 (m, 1H, CH), 3.8-3.5 (m, 2H, H-5 ', H-5 "), and 2.3-2.1 (m, 1H, CH), 1.16 (s, 3H, CH 3), 1.0 (m, 6H, (CH 3) 2CH); FAB＞0 (GT) 713 (2M+H) +, 449 (M+G+H) +, 357 (M+H) +, 246 (S) +, 112 (B+2H) +FAB＜0 (GT) 747 (2M+Cl) -, 483 (M+G+Gl) -, 391 (M+Cl) -, 355 (M-H) -, 116 (Val) -, 110 (B) -, 35 (Cl) -HPLC rt=7.26min (from 0 to 50% CH 3The buffer of N in 20mM acetic acid triethylamine, program is 30 minutes, flow velocity is 1ml/min), λ max=273.5nm; UV (H 2O): λ max=271nm (ε 7500), λ min=249nm (ε 5200), λ s=234nm (ε 6200).
Embodiment 18
Synthetic (Fig. 6) of β-D-2 '-C-methyl-cytidine:
Another synthetic route of preparation β-D-2 '-C-methyl-cytidine is seen Fig. 3.In the method, heating uracil (2.1eq.) and the mixture of BSA (1.1mL/mmol) in acetonitrile (7mL/mmol) are to refluxing about 30 minutes.Gained solution is with 1,2,3, acetonitrile (7mL/mmol) solution and the SnCl of 5-four-O-benzoyl-2-C-methyl-β-D-ribofuranose (1) 4Solution-treated (3.5eq.).Gained solution is heated to and refluxed about 4 hours.The dark mixture of reactant is diluted with ethyl acetate (2.5 volume of toluene), and uses and the isopyknic cold NaHCO of ethyl acetate 3Saturated solution is at room temperature handled.Filter whole mixture by celite (celite), wash solid material with ethyl acetate.Isolate organic layer from filtrate, Na is used in water and salt washing 2SO 4Drying, decompression evaporation down.The silica gel column chromatography of the hexane solution by using 50% ethyl acetate obtain productive rate be 65% chemical compound ( 9), β-D-2 '-, 3 '-, 5 '-benzoyl-2 '-C-methyl-uridnine, be white solid.
By make ( 9) be dissolved in the methanol of handling with MeONa (3.3eq.) (12.5ml/mmol) and at room temperature stirred the gained yellow solution about 4.5 hours, with the benzoyl protecting group from β-D-2 '-, 3 '-, 5 '-benzoyl-2 '-C-methyl-uridnine ( 9) on remove.Add the Dowex H that washed with methanol in advance +50wX4 makes the solution neutralization.Filtering mixt, with hot methanol extraction resin several times.Merging filtrate and vapourisation under reduced pressure.Residue adds in the entry, washes 3 times with dichloromethane.Water layer decompression evaporation down.Crystallization generation from water ( 10), β-D-2 '-C-methyl-uridnine, productive rate 87%.
Then, by β-D-2 '-C-methyl-uridnine ( 10), the solution of acetonitrile (10ml/mmol) solution composition of (3eq.) of 1-crassitude (1ml/mmol), chloro trimethyl silane stir about 3.5 hours at room temperature.Solution is cooled to 0 ℃, handles, under uniform temp, stirred 30 minutes with trifluoroacetic anhydride (3eq.).Add 4-nitrophenol (3eq.), about 3 hours of agitating solution.Add entry and finish reaction in solution, decompression is evaporating solvent down.Residue is added dichloromethane, use saturated NaHCO 3Solution and washing.Decompression is the evaporation organic layer down.Thick residue is added diox (25ml/mmol), and use 28%NH 4OH (5ml/mmol) aqueous solution is handled.Solution is heated to 50 ℃ and spends the night.Then, solvent evaporated under reduced pressure, the chromatographic isolation of use gradient methanol (5-20% is in dichloromethane).Produced β-D-2 '-C-methyl-cytidine ( 11) as the product of expecting, productive rate 75%.Further crystallization in EtOH of product.
Reagent source among the embodiment 5 and 6 comprises: N, the dinethylformamide dimethyl-acetal is available from Fluka , numbering No.40271;
N, dinethylformamide is crossed molecular sieve, available from Fluka , numbering No.40248;
Straight alcohol available from Carlo Erba ACS, is analyzed usefulness, numbering No.414607;
Diethyl ether is available from Merck , numbering No.1.00921.5000;
Tert-butyl diphenyl chlorosilane is available from Avocado , numbering No.12721;
Imidazoles is available from Fluka , numbering No.56750;
Pyridine is crossed molecular sieve, available from Fluka , numbering 82704;
Sodium bicarbonate is available from Fluka , numbering No.71628;
Anhydrous sodium sulfate is available from Fluka , numbering No.71960;
Acetonitrile is crossed molecular sieve, available from Fluka , numbering No.00695;
N, dinethylformamide is crossed molecular sieve, available from Fluka , numbering No.40248;
N-(tertbutyloxycarbonyl)-L-figured silk fabrics ammonia is available from Aldrich , numbering No.35,972-6;
4-dimethylaminopyridine is available from Aldrich , numbering No.10,770-0;
N '-(3-dimethylaminopropyl)-N-ethyl carbon imidodicarbonic diamide hydrogen chloride is available from Aldrich , numbering No.16,146-2;
Amine fluoride is available from Fluka , numbering No.09742;
Through the distillatory methanol of sodium; Through the distillatory ethyl acetate of phosphorus pentoxide anhydrous hydrogen chloride, available from Praxair, numbering No.1741100; And
Diethyl ether is available from Merok , numbering No.1.00921.5000.
Below only be that the present invention has been carried out exemplary illustration, be not limited to disclosed method and reaction condition.Being apparent to various variations to those skilled in the art includes within the defined scope and spirit of claim of the present invention.

Claims (88)

1. prepare 3 '-method of O-amino-acid ester nucleoside, may further comprise the steps:
(a) make optional protection, optional ribofuranose and unprotected nucleoside base and the coupling in the presence of lewis acid of silylation reagent that replaces, form the nucleoside of optional protection;
(b) as needs, optional nucleoside and the deprotecting regent reaction that makes the protection in the step (a) provides unprotected nucleoside;
(c) if nucleoside has amido, optional amido protection or unprotected nucleoside and the amine protection reagent reacting of making;
(d) optional that make protection or unprotected nucleoside and silylation reagent reacting, obtain 5 '-nucleoside that the O-silicyl is protected;
(e) optional amino acid derivativges protection or unprotected nucleoside and protection is reacted under one or more coupling agents, form protect 3 '-the O-amino-acid ester;
(f) if desired, optional make in the step (e) product with from 5 '-C removes the silicyl protecting group and removes the reagent reacting of carbonamidine protecting group from nucleoside amine; With
(g) optional make in the step (f) product with from 3 '-the O-amino-acid ester removes the reagent reacting of protecting group, produce that replace or unsubstituted 3 '-nucleoside of O-ester-replacement.
2. the process of claim 1 wherein, the ribofuranose of the optional protection in the step (a) 2 '-the C position comprises methyl.
3. the process of claim 1 wherein that the lewis acid in the step (a) is selected from by SnCl 4, BF 3, AlCl 3, TiCl 4, FeCl 3And SnCl 2The group of forming.
4. the method for claim 3, wherein, lewis acid is SnCl 4
5. the process of claim 1 wherein that the silylation reagent in the step (a) is selected from the group of being made up of BSA, HMDS, TMSC1 and TBDPSC1.
6. the method for claim 5, wherein, silylation reagent is BSA.
7. the process of claim 1 wherein that the coupling reaction in the step (a) is carried out in acetonitrile solvent.
8. the process of claim 1 wherein that the deprotecting regent in the step (b) is NaOMe or NH 3
9. the process of claim 1 wherein that the amine protection reagent in the step (c) is selected from by N dinethylformamide dimethyl-acetal and N-1, the group that the 1-dimethyl disulfide is formed for benzylidene amino.
10. the method for claim 9, wherein, amine protection reagent is N, the dinethylformamide dimethyl-acetal.
11. the process of claim 1 wherein that the silylation reagent in the step (d) is selected from the group of being made up of TBDPSC1, TMSC1 and TBDMSC1.
12. the method for claim 11, wherein, silylation reagent is TBDPSC1.
13. the process of claim 1 wherein that the amino acid derivativges of step (e) is a valine derivative.
14. the process of claim 1 wherein, the aminoacid protecting group of step (e) be selected from BOC ,-(C=O)-aralkyl ,-(C=O)-alkyl or-(C=O)-aryl.
15. the method for claim 14, wherein, the aminoacid protecting group is BOC.
16. the process of claim 1 wherein that one of coupling agent in the step (e) is EDC.
17. the process of claim 1 wherein that the silicyl in the step (f)-remove reagent is NH 4F.
18. the process of claim 1 wherein, 3 in the step (g) '-to remove reagent be HCl to O-amino-acid ester protecting group.
19. prepare the method for nucleoside, may further comprise the steps:
(a) make 1,2,3,5-four-O-benzoyl-2-C-methyl-β-D-ribofuranose and cytosine are at BSA and SnCl 4There is coupling down, forms 4-amino-1-(3,4-dibenzoyl oxygen ylmethyl-5-benzoyloxy methyl-3-methyl-oxolane-2-yl)-1H-pyrimid-2-one;
(b) make by obtaining 4-amino-1-(3 in the step (a), 4-dibenzoyl oxygen ylmethyl-5-benzoyloxy methyl-3-methyl-oxolane-2-yl)-1H-pyrimid-2-one and Feldalat NM reaction, remove the benzoyl protecting group, obtain 4-amino-1-(3,4-dihydroxy-5-methylol-3-methyl-oxolane-2-yl)-1H-pyrimid-2-one;
(c) make by the 4-amino-1-that obtains in the step (b) (3,4-dihydroxy-5-methylol-3-methyl-oxolane-2-yl)-1H-pyrimid-2-one and N the reaction of dinethylformamide dimethyl-acetal, protection N 4-amino produces N-[1-(3,4-dihydroxy-5-methylol-3-methyl-oxolane-2-yl)-2-oxygen-1,2-dihydro-pyrimidine-4-yl]-N, N-dimethyl carbonamidine;
(d) make by step b) or c) in the nucleoside that obtains and silylation reagent TBDPSC1 reaction, obtain N '-{ 1-[5-(tert-butyl group-diphenyl-silane oxygen ylmethyl)-3,4-dihydroxy-3-methyl-oxolane-2-yl]-2-oxygen-1,2-dihydro-pyrimidine-4-yl }-N, N-dimethyl carbonamidine;
(e) make N '-{ 1-[5-(tert-butyl group-diphenyl-silane oxygen ylmethyl)-3,4-dihydroxy-3-methyl-oxolane-2-yl]-2-oxygen-1,2-dihydro-pyrimidine-4-yl }-N, N-dimethyl-carbonamidine and N-BOC-L-valine and EDC react in dichloromethane, obtain 2-t-butoxycarbonyl amino-3-methyl-butanoic acid 2-(tert-butyl group-diphenyl-silane oxygen ylmethyl)-5-[4-(dimethylamino-methene amido)-2-oxygen-2H-pyrimidine-1-yl]-4-hydroxy-4-methyl-oxolane-3-base ester;
(f) by backflow chemical compound and NH 4F removes 2-t-butoxycarbonyl amino-3-methyl-butanoic acid 2-(tert-butyl group-diphenyl-silane oxygen ylmethyl)-5-[4-(dimethylamino-methene amido)-2-oxygen-2H-pyrimidine-1-yl in the step (e)]-the silicyl protecting group and the amidino of 4-hydroxy-4-methyl-oxolane-3-base ester; Form 2-t-butoxycarbonyl amino-3-methyl-butanoic acid 5-(4-amino-2-oxygen-2H-pyrimidine-1-yl)-4-hydroxyl-2-methylol-4-methyl-oxolane-3-base ester, and
(g) by making 2-t-butoxycarbonyl amino-3-methyl-butanoic acid 5-(4-amino-2-oxygen-2H-pyrimidine-1-yl)-4-hydroxyl-2-methylol-4-methyl-oxolane-3-base ester and the HCl reaction in the step (f); from 3 '-O-L-valine ester substituent group removes the BOC-protecting group, obtains 2-amino-3-methyl-butanoic acid 5-(4-amino-2-oxygen-2H-pyrimidine-1-yl)-4-hydroxyl-2-methylol-4-methyl-oxolane-3-base ester (dihydrochloride).
20. prepare the method for furanose, may further comprise the steps:
(a) make the reaction of CaO solution and cyclic ethers, this cyclic ethers with ring on have hydroxyl and CH on the carbon of oxygen atom adjacency 2OH, thus the furyl lactone formed;
(b) if desired, with the optional protection of protecting group furyl lactone;
(c) the furyl lactone of optional protection and Reducing agent are reacted, lactone is reduced to hydroxyl, produce furanose product chemical compound; And
(d) optional furanose product chemical compound and the protecting group of making reacted.
21. the method for claim 20, wherein, the cyclic ethers that reacts with CaO is a D-fructose.
22. the method for claim 20, wherein, the furyl lactone is 2-C-methyl D-ribonic acid-lactone.
23. the method for claim 20, wherein, the furyl lactone of protection is 2,3,5-three-O-benzoyl-2-C-methyl D-ribonic acid-lactone.
24. the method for claim 20, wherein, furanose is 2,3,5-three-O-benzoyl-2-C-methyl-β-D-ribofuranose.
25. the method for claim 20, wherein, the furanose of protection is 1,2,3,5-four-O-benzoyl-2-C-methyl-β-D-ribofuranose.
26. the method for claim 20; wherein; protecting group be selected from by silicyl, benzoyl, p-toluyl, p-nitro benzoyl, p-chlorobenzene formacyl, acyl group, acetyl group ,-(C=O)-alkyl and-(C=O)-group that aryl is formed, optional by one or more groups replacements that are not subjected to Reducing agent influence in the step (c).
27. the method for claim 26, wherein, protecting group is a benzoyl.
28. the method for claim 26, wherein, protecting group is-(C=O)-alkyl.
29. the method for claim 20, wherein, Reducing agent is selected from by Red-Al/ ethanol, NaHTe, SmI 2, H 2+ Pd-phosphine catalyst and LiAI (O tBu) 3The group that H forms.
30. the method for claim 29, wherein, Reducing agent is a Red-Al/ ethanol.
31. the method for claim 20 wherein, is reflected in the solvent and carries out, this solvent is selected from the group of being made up of TEA, DMAP, DME, toluene and ethanol.
32. the method for claim 20, wherein, for the first product chemical compound lactone, reaction temperature is in change between-5 ℃ to about 50 ℃ approximately.
33. the method for claim 20, wherein, total generated time is from about 5 days to about 14 days.
34. the method for claim 33, wherein, total generated time is from about 5 days to about 10 days.
35. the method for claim 33, wherein, total generated time is about 60 hours.
36. a method may further comprise the steps:
(a) make CaO solution and D-fructose arrive about 40 ℃ of following reactions about 5 hours to about 25 hours for about 23 ℃ in temperature;
(b) make product and CO in the step (a) 2Reacted about 8 hours to about 12 hours with oxalic acid, form 2-C-methyl D-ribose acid lactone;
(c) 2-C-methyl D-ribose acid lactone and Benzenecarbonyl chloride. were reacted about 3 hours to about 6 hours, obtain 2,3,5-three-O-benzoyl-2-C-methyl D-ribose acid lactone;
(d) under about 5 ℃ to about 0 ℃, with Red-Al/ ethanol reductase 12,3,5-three-O-benzoyl-2-C-methyl D-ribose acid lactone about 30 obtained 2,3,5-three-O-benzoyl-2-C-methyl-β-D-ribofuranose by about 60 minutes;
(e) under about 0 ℃ to about 50 ℃, benzoylation 2,3 in solvent, and 5-three-O-benzoyl-2-C-methyl-β-D-ribofuranose about 4 hours to about 14 hours forms 1,2,3,5-four-O-benzoyl-2-C-methyl-β-D-ribofuranose; And
(f) optional separated 1,2,3,5-four-O-benzoyl-2-C-methyl-β-D-ribofuranose.
37. the method for claim 36, wherein, the response time in the step (a) is from about 6 to about 22 hours.
38. the method for claim 36, wherein, the reaction temperature in the step (a) is from about 23 to about 40 ℃.
39. the method for claim 36, wherein, the solvent in the step (c) is DME.
40. the method for claim 36, wherein, the reaction in the step (c) was carried out about 4 hours.
41. the method for claim 36, wherein, the reduction in the step (d) was carried out about 40 minutes.
42. the method for claim 36, wherein, the solvent in the step (d) comprises toluene.
43. the method for claim 36, wherein, the solvent in the step (e) comprises DME.
44. the method for claim 36, wherein, the temperature in the step (e) is from about 5 to about 50 ℃, and reaction was carried out about 4 to about 12 hours.
45. the method for claim 36, wherein, the separation in the step (f) is to be undertaken by method well known in the prior art.
46. the process of claim 1 wherein that the ribofuranose that replaces benzoyl protection, optional is by the method preparation of claim 20.
47. the process of claim 1 wherein that the ribofuranose that replaces benzoyl protection, optional is by the method preparation of claim 36.
48. prepare the method for furyl lactone, comprising:
(a) make the reaction of CaO solution and cyclic ethers, this cyclic ethers with ring on have hydroxyl and CH on the carbon of oxygen atom adjacency 2OH forms the furyl lactone.
49. prepare the method for 2-C-methyl D-ribose acid lactone, may further comprise the steps:
(a) be under about 23 ℃ to about 40 ℃ in temperature, CaO solution and D-fructose were reacted about 5 hours to about 25 hours;
(b) make the product and the CO of step (a) 2Reacted about 8 hours to about 12 hours with oxalic acid, form 2-C-methyl D-ribose acid lactone.
50. the method for the 2-C-methyl-β-D-ribofuranose chemical compound of the optional protection of preparation comprises:
(a), obtain the 2-C-methyl-β-D-ribofuranose of optional protection with the 2-C-methyl D-ribose acid lactone of the optional protection of Red-Al/ ethanol reduction.
51. the method for the nucleoside of the optional protection of preparation comprises:
(a) make optional protection, optional ribofuranose and unprotected nucleoside base and the coupling in the presence of lewis acid of silylation reagent that replaces, form the nucleoside of optional protection.
52. the method for the β-D-2 '-C-methyl-cytidine of the optional protection of preparation may further comprise the steps:
(a) make the optional 2-C-methyl-β-D-ribofuranose protected and cytosine at BSA and SnCl 4There is coupling down, forms the β-D-2 '-C-methyl-cytidine of optional protection.
53. prepare 2 '-method of C-methyl-cytidine, may further comprise the steps:
(a) make cytosine and activator, choose in the presence of lewis acid the 2-C-methyl-β-D-ribofuranose reaction with optional protection wantonly, form 2 of optional protection '-C-methyl-cytidine
Wherein, each P 1, P 2, P 3And P 4Be hydrogen or suitable oxygen protecting group independently; Then,
(b) as needs, before optional the sloughing in the step reaction optional protection 2 '-protection of C-methyl-cytidine, form 2 '-C-methyl-cytidine ( VI).
Figure A2003801095760007C2
54. the method for claim 53, wherein, each P 1, P 2, P 3And P 4Be hydrogen or acyl group independently.
55. the method for claim 53, wherein, each P 1, P 2, P 3And P 4Be hydrogen or benzoyl independently.
56. the method for claim 53, wherein, activator is a silylation reagent.
57. the method for claim 56, wherein, silylation reagent is BSA, HMDS, TMSC1 or TBDPSC1.
58. the method for claim 56, wherein, silylation reagent is BSA.
59. the method for claim 53, wherein, reactions steps (a) is finished under lewis acid, and lewis acid is selected from by SnCl 4, BF 3, AlCl 3, TiCl 2, TiCl 4, FeCl 3And SnCl 2The group of forming, or their any mixture.
60. the method for claim 53, wherein, lewis acid is SnCl 4
61. the method for claim 53, wherein, this method further comprise with ester moiety to 2 '-C-methyl-cytidine 3 '-step that esterification is carried out in the position.
62. the method for claim 61, wherein, ester moiety is an aminoacid.
63. the method for claim 62, wherein, aminoacid is the L-valine.
64. the method for the 2-C-methyl-β-D-ribofuranose of the optional protection of preparation may further comprise the steps:
(a) the 2-C-methyl D-ribose acid lactone of optional protection and Reducing agent are reacted
Figure A2003801095760008C1
Wherein, each P 1, P 2And P 3Be hydrogen or suitable oxygen protecting group independently; Then,
(b) the ribofuranose derivative compound in the step reaction before the protection optionally, form optional protection-2-C-methyl-β-D-ribofuranose,
Wherein, P 4Be hydrogen or suitable oxygen protecting group independently.
65. the method for claim 64, wherein, each P 1, P 2, P 3And P 4Be hydrogen or acyl group independently.
66. the method for claim 64, wherein, each P 1, P 2, P 3And P 4Be hydrogen or benzoyl independently.
67. the method for claim 64, wherein, Reducing agent is two (2-methoxy ethoxy) sodium aluminum hydrides (Red-Al), chooses wantonly in solvent.
68. the method for claim 67, wherein, solvent is an ethanol.
69. the method for the 2-C-methyl D-ribose acid lactone of the optional protection of preparation may further comprise the steps:
(a) make D-fructose and CaO reaction;
Then,
(b) optional protection lactone forms optional 2-C-methyl D-ribose acid lactone of protecting
Figure A2003801095760009C2
Wherein, each P 1, P 2And P 3Be hydrogen or suitable oxygen protecting group independently.
70. the method for claim 69, wherein, each P 1, P 2And P 3Be hydrogen or acyl group independently.
71. the method for claim 69, wherein, each P 1, P 2And P 3Be hydrogen or benzoyl independently.
72. the method for claim 69 wherein, utilizes precipitant to remove the calcium that produces in step (a).
73. the method for claim 72, wherein, precipitant is the organic acid stronger than ribonic acid.
74. the method for claim 73, wherein, organic acid is selected from the group of being made up of oxalic acid, malonic acid, succinic acid, 1,3-propanedicarboxylic acid, adipic acid, suberic acid, decanedioic acid, Azelaic Acid, maleic acid, acetic acid, propanoic acid, isopropylformic acid., acrylic acid, methacrylic acid, butanoic acid, valeric acid caproic acid or caproic acid.
75. the method for claim 73, wherein, organic acid is an oxalic acid.
76. from 2 of the optional protection of D-fructose preparation '-method of C-methyl D-cytidine, may further comprise the steps:
(a) make D-fructose and CaO reaction obtain 2-C-methyl D-ribonic acid-gamma lactone;
Figure A2003801095760010C1
(b) if desired, optional protection lactone forms optional 2-C-methyl D-ribose acid lactone of protecting;
Figure A2003801095760010C2
Wherein, each P 1, P 2And P 3Be hydrogen or suitable oxygen protecting group independently;
(c) the 2-C-methyl D-ribose acid lactone of optional protection and Reducing agent are reacted,
Figure A2003801095760010C3
Wherein, each P 1, P 2And P 3Be hydrogen or suitable oxygen protecting group independently;
(d) if desired, go on foot the ribofuranose derivative compound in the reaction before the optional protection, form the 2-C-methyl-β-D-ribofuranose of optional protection,
Figure A2003801095760011C1
Wherein, P 4Be hydrogen or suitable oxygen protecting group independently;
(e) make the optional 2-C-methyl-β-D-ribofuranose protected and cytosine and activator choose reaction in the presence of lewis acid wantonly, 2 of the optional protection of formation '-the C-methylcytidine
Figure A2003801095760011C2
Wherein, each P 1, P 2, P 3And P 4Be hydrogen or suitable oxygen protecting group independently;
Then,
(f) if desired, optional slough 2 of optional protection '-protection of C-methylcytidine, form 2 of optional protection '-the C-methylcytidine
Figure A2003801095760011C3
77. the method for claim 76, wherein, each P 1, P 2, P 3And P 4Be hydrogen or acyl group independently.
78. the method for claim 76, wherein, each P 1, P 2, P 3And P 4Be hydrogen or benzoyl independently.
79. the method for claim 76, wherein, activator is a silylation reagent.
80. the method for claim 79, wherein, silylation reagent is BSA, HMDS, TMSC1 or TBDPSC1.
81. the method for claim 79, wherein, silylation reagent is BSA.
82. the method for claim 76, wherein, reactions steps (e) is finished under lewis acid, and lewis acid is selected from by SnCl 4, BF 3, AlCl 3, TiCl 2, TiCl 4, FeCl 3And SnCl 2The group of forming, or their any mixture.
83. the method for claim 82, wherein, lewis acid is SnCl 4
84. the method for claim 76, wherein, Reducing agent is two (2-methoxy ethoxy) sodium aluminum hydrides (Red-Al), chooses wantonly in solvent.
85. the method for claim 84, wherein, solvent is an ethanol.
86. the method for claim 76, wherein, this method further comprise with ester moiety to 2 '-C-methyl-cytidine 3 '-step that esterification is carried out in the position.
87. the method for claim 86, wherein, ester moiety is an aminoacid.
88. the method for claim 87, wherein, aminoacid L-valine.
CNA2003801095761A 2002-12-12 2003-12-12 Process for the production of 2'-branched nucleosides Pending CN1744903A (en)

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CN103992360A (en) * 2014-04-21 2014-08-20 中美华世通生物医药科技（武汉）有限公司 Reduction method suitable for industrialized production
US10739908B2 (en) 2011-09-30 2020-08-11 Apple Inc. Flexible electronic devices

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US10739908B2 (en) 2011-09-30 2020-08-11 Apple Inc. Flexible electronic devices
US11675390B2 (en) 2011-09-30 2023-06-13 Apple Inc. Flexible electronic devices
US11994906B2 (en) 2011-09-30 2024-05-28 Apple Inc. Flexible electronic devices
CN103539826A (en) * 2013-10-23 2014-01-29 南京理工大学 Synthesis method of 1,2,3,5-tetra-O-benzoyl-2-C-methyl-beta-D-ribofuranose
CN103992360A (en) * 2014-04-21 2014-08-20 中美华世通生物医药科技（武汉）有限公司 Reduction method suitable for industrialized production

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