Abstract:
A method for the preparation of acyclic nucleosides such as valomaciclovir stearate comprising the acid hydrolysis of an intermediate of the formula II: where X is a leaving group or an optionally protected guanine moiety, R1 is a hydroxy protecting group or a —C(═O)C1-C22 alkyl ester group; R2 and R3 are independently lower alkyl or benzyl, or R2 and R3 taken together are —CH2CH2— or —CH2CH2CH2— or —CH2CH2CH2CH2—; to the corresponding aldehyde of the formula III: and the reduction of the aldehyde to the corresponding alcohol of the formula IV: by the addition of a borohydride or borane aldehyde reducing agent, characterised in that the borohydride or borane aldehyde reducing agent is introduced under acid conditions.

Description:
TECHNICAL FIELD  
         [0001]    This invention relates to improvements in the synthesis of branched acyclic nucleosides such as valomaciclovir stearate. In particular the invention provides for an improved acetal hydrolysis and concomitant reduction of the aldehyde to an alcohol in respect of key intermediates in the synthesis of such branched acyclic nucleosides.  
         BACKGROUND ART  
         [0002]    Valomaciclovir stearate of the formula XX below is an acyclic nucleoside analogue, useful in the treatment of VZV and other herpesviruses and HIV.  
                         
 
           [0003]    Our earlier patent applications WO98/34917, U.S. Pat. No. 6,184,376 and WO00/08025 describe various synthesis routes, of which several go through a key acetal intermediate which can be generalised to the following formula I/II:  
                         
 
           [0004]    where X is a leaving group or an optionally protected guanine moiety,  
           [0005]    R1 is hydrogen, an hydroxy protecting group or a —C(═O)C1-C22 alkyl ester R2 and R3 are independently lower alkyl or benzyl, or R2 and R3 taken together are —CH 2 CH 2 — or —CH2CH2CH2— or —CH2CH2CH2CH2—.  
           [0006]    In the prior art processes, (see for example page 30 lines 1-24 of WO98/34917) these acetals are hydrolysed by the addition of an acid such as triflic acid, HCl, acetic acid or sulphuric acid or an acid resin such as Amberlyst 15. The thus formed aldehyde is then reduced to the corresponding alcohol by the addition of an aldehyde reducing agent such as sodium borohydride, RaNi/H 2  or borane t-butylamine complex). However these reducing agents are well known to require a non-acidic operating environment, as specifically brought out at page 30 line 8 and Examples 14 &amp; 30 of WO98/34917 and Example 2 of WO00/08025. Accordingly the prior art processes require the addition of a base such as sodium bicarbonate or potassium carbonate or triethylamine or pyridine or KOH and the like to neutralise the acid which has been used for acetal hydrolysis, prior to addition of the aldehyde reducing agent.  
         BRIEF DESCRIPTION  
         [0007]    We have now discovered that, even without purifying the aldehyde, dramatically improved purity of the resultant alcohol can be obtained if a borane or borohydride aldehyde reducing agent is introduced under acid conditions.  
           [0008]    Accordingly, the invention provides a method comprising the acid hydrolysis of an intermediate of the formula II:  
                         
 
           [0009]    where X is a leaving group or an optionally protected guanine moiety,  
           [0010]    R1 is a hydroxy protecting group or a —C(═O)C 1 -C 22  alkyl ester group;  
           [0011]    R2 and R3 are independently lower alkyl or benzyl, or R2 and R3 taken together are —CH 2 CH 2 — or —CH2CH2CH2— or —CH2CH2CH2CH2—;  
           [0012]    to the corresponding aldehyde of the formula III:  
                         
 
           [0013]    and the reduction of the aldehyde to the corresponding alcohol of the formula IV:  
                         
 
           [0014]    by the addition of a borohydride or borane aldehyde reducing agent, characterised in that the aldehyde reducing agent is introduced under acid conditions.  
           [0015]    The resultant alcohol IV is then esterified with an activated N-protected alpha amino acid derivative as described in the above prior art patent applications to form valomaciclovir stearate XX. If necessary the alcohol IV or its N-protected amino acyl derivative can be reacted with the guanine base as also described in the prior art and/or the alcohol protecting group R1 can be deprotected and replaced with a fatty acid ester by conventional acylation.  
           [0016]    By the use of the present invention, the alcohol IV can be obtained at a purity greater than 85%, preferably greater than 90%. The purity of this intermediate has proven to contribute significantly to the overall yield of valomaciclovir stearate.  
           [0017]    Preferably the process of the invention is carried out without isolating the intermediate aldehyde of formula III. An optional preliminary step may comprise the esterification of the hydroxy group of the corresponding alcohol I  
                         
 
           [0018]    with an activated C 1 -C 22 COOH derivative, such as an activated stearic acid, by conventional acylation techniques, preferably without isolation of the resultant acetal II. Convenient activated derivatives for the esterification includes acid halides such as acid chlorides, and activated esters including, but not limited to, mixed anhydrides of steraric and pivalic and/or acetic acid derived anhydrides, anhydrides derived from alkoxycarbonyl halides such as isobutyloxycarbonylchloride and the like, N-hydroxysuccinimide derived esters, N-hydroxyphthalimide derived esters, N-hydroxybenzotriazole derived esters, N-hydroxy-5-norbornene-2,3-dicarboxamide derived esters, 2,4,5-trichlorophenyl derived esters, sulfonic acid derived anhydrides (for example, p-toluenesulonic acid derived anhydrides and the like) and the like. Alternatively the activated ester may comprise the acid in conjunction with a coupling agent such as DCC/DMAP or EDAC/DMAP.  
           [0019]    Preferably, when R1 in the start material of formula IV is an alkanoic acid ester such as a stearic acid, the material is purified for excess stearic acid in order to minimize the production of undesired impurities, such as (R)-9-[2-stearoyloxymethyl-4-(stearoyloxy)butyl]guanine. Purification may comprise one or more refluxing steps, for example between 10 minutes and 6 hours, such as 1 hour, in an organic solvent able to dissolve the alkanoic acid, such as acetone, typically followed by cooling to room temperature, filtration, washing with the same or similar solvent and drying. Conveniently the alkanoic acid content is reduced to &lt;about 2%, preferably less than about 1%, such as &lt;0.5%.  
           [0020]    Following reaction of the borohydride or borane aldehyde reducing agent under acid conditions, it is convenient to neutralise the reaction mixture with bases such as 0.1 to 10.0 molar equivalents of sodium hydrogen carbonate, potassium carbonate, triethylamine, pyridine, NaOH KOH and the like, especially sodium bicarbonate.  
           [0021]    Convenient strong acids for the hydrolysis of the acetal II include from about 0.1 to 10.0 molar equivalents of triflic, hydrochloric, formic or acetic/formic, sulphuric, sulphonic, p-toluene sulfonic or an ion exchanger sulphonic, especially hydrochloric. Solvents for the hydrolysis step include inert solvents such as THF/H 2 O or methylene chloride/H 2 O or ethylacetate/H 2 O or ethanol/H 2 O or methanol/H 2 O or water, especially THF. Suitable reaction temperatures include from about −25° C. to 100° C., such as room temperature  
           [0022]    Convenient borohydrides for the reduction step include potassium borohydride and especially sodium borohydride. Convenient boranes include BH 3 .py,  
                         
 
           [0023]    and especially borane-tert-butylamine complex.  
           [0024]    To minimize the formation of undesired regioisomers, such as (R)-9-[2-hydroxymethyl-4-stearoyloxybutyl]guanine by transesterification during the acid hydrolysis of the start material of formula II, when R1 is an alkanoic acid ester, it is convenient if the acid hydrolysis is made under mild conditions, such as at a low temperature, for a short time and/or with a low concentration of acid. It is furthermore convenient if the reaction mixture is neutralised as soon as all of the acetal of formula II has been hydrolysed and/or extra borohydride or borane aldehyde reducing agent is added to react with the last of the aldehyde present.  
           [0025]    Convenient leaving groups for X in formulae I-IV include sulphonates such as methane sulphonate, triflate, p-toluenesulphonate, benzene sulphonate, especially TsO. Preferably, however, X in formulae I-IV is a guanine, a guanine derivative such as 6-deoxyguanine or 6-chloroguanine or iodoguanine, an N-protected and/or hydroxy-proteceted guanine such as 2-N-acetyl-guanine, 6-benzyloxyguanine or 2-N-acetyl-6-diphenyl-carbamoylguanine.  
           [0026]    The term “lower alkyl” as used in conjunction with R2 and R3 refers to straight or branched chain alkyl radicals containing from 1 to 7 carbon atoms including, but not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, t-butyl, n-pentyl, 1-methylbutyl, 2,2-dimethylbutyl, 2-methylpentyl, 2,2-dimethylpropyl, n-hexyl and the like. Preferably R2 and R3 are the same species of lower alkyl, especially methyl and most preferably ethyl.  
           [0027]    Preferred —C(═O)C 1 -C 22  alkyl ester groups for R1 include acetyl, palmityl, cetyl, eicosanyl and especially stearyl. Alternative Hydroxy protecting groups for R1 are extensively discussed in Greene, “Protective Groups In Organic Synthesis,” (John Wiley &amp; Sons, New York (1981)). O-protecting groups comprise substituted methyl ethers, for example, methoxymethyl, benzyloxymethyl, 2-methoxyethoxymethyl, 2-(trimethylsilyl)ethoxymethyl, t-butyl, benzyl and triphenylmethyl; tetrahydropyranyl ethers; substituted ethyl ethers, for example, 2,2,2-trichloroethyl; silyl ethers, for example, trimethylsilyl, t-butyldimethylsilyl and t-butyldiphenylsilyl; and esters prepared by reacting the hydroxyl group with a carboxylic acid, for example, acetate, propionate, benzoate and the like. 
       
    
    
     DETAILED DESCRIPTION  
       [0028]    The invention will now be illustrated by way of example only with reference to FIG. 1 which tabulates the results from Example 1  
       EXAMPLE 1  
       [0029]    A flat bottomed reactor equipped with magnetic stirring, thermometer and a pH electrode was charged with:  
         [0030]    1.0 g (1.7 mmol) (R)-9-[4,4-diethoxy-2-(stearyloxymethyl)butyl]guanine  
         [0031]    [0031] 95 % tetrahydrofuran (25 ml)  
         [0032]    The suspension was mixed for about 20 minutes to give a slightly turbid solution. To the solution was added portionwise over 4 hours:  
         [0033]    37% hydrochloric acid (1.0 ml, 10 mmol)  
         [0034]    Borane-tert-butyl-amine complex suspended in 3 ml 95% tetrahydrofuran  
         [0035]    Borane-tert-butyl-amine complex (0.2 g, 2.3 mmol).  
         [0036]    The reaction mixture was adjusted to pH=1.6 by addition of:  
         [0037]    0.4 g sodium hydrogen carbonate (4.8 mmol)  
         [0038]    20 ml water  
         [0039]    The next day the suspension was adjusted to pH=5 by addition of:  
         [0040]    10 ml water  
         [0041]    3N sodium hydroxide (about 1 ml)  
         [0042]    The stirred suspension was cooled and kept at 0° C. for 1 hour. The suspension was filtered and the filter cake washed with acetone (2×2.5 ml) and sucked dry. The wet filter cake was dried in a forced ventilation cupboard (50° C., 4.5 h) to give 0.72 g (82% yield) of (R)-9-[4-hydroxy-2-(stearyloxymethyl)butyl]guanine with 95.9% purity. The above example and the corresponding trial with 79% THF is summarised in Table 1 in the accompanying FIG. 1. The roman numeral II is the start material, III is the aldehyde and IV is the alcohol.  
       EXAMPLE 2  
     Reduction of Distearate  
       [0043]    A 50 ml reaction flask with magnetic stirring was charged with:  
         [0044]    Stearate alcohol (0.4 g, 0.77 mmol)  
         [0045]    Tetrahydrofuran (27 ml)  
         [0046]    3N HCl (3 ml, 9 mmol)  
         [0047]    The stirred homogenous solution was kept at 41° C., while samples (25 μl) were taken, diluted to 1.0 ml with 90% THF contaning 2.5% Et 3 N and analysed by HPLC.  
                                                                                         RT   RT           T   Reaction   Reaction   16 min   21 min   Total area       Time   Temp ° C.   time (h)   IV %   VII %   AU * 10 8                                  12.58   20   0.00                   13.05   27   0.07   95.4   1.2   16.2       13.45   41   0.47       14.10   41   1.12   89.1   8.6   13.9       15.15   41   2.17   79.6   16.85   10.7       17.00   41   4.02   69.4   25.88   7.7       12.25   41   23.27   31.9   28.4   0.48       9.10   41   68   0   0   0.18                  
 
         [0048]    It will be apparent that after 4 hours 25% of the regioisomer was formed by transesterification of the stearate alcohol, while 50% of the stearate alcohol and corresponding regioisomer were hydrolysed. After about 23.5 h, about 3% of the stearate alcohol and regioisomer were present. The rest was apparently hydrolysed to stearic alcohol and guanine dialchohol. The conclusion is that the formation of the regioisomer is minimised if the hydrolysis of the acetal alkanoate is done under mild conditions, ie low temperature and/or short time and/or low acid concentration. Additionallly, it is helpful if the reaction mixture is neutralised without undue delay. Similarly, it assists if extra borane complex is added to react with any remaining aldehyde present.  
       EXAMPLE 3  
     Preparation of (R)-9-(2-stearoyloxymethyl-4-hydroxybutyl)guanine  
       [0049]    This example shows the preparation of the title compound from guanine alcohol without isolation of the intermediate guanine stearate and stearate aldehyde:  
                         
 
         [0050]    The following were charged in a reaction flask:  
         [0051]    Stearic acid (26.0 g, 0.0913 mol), 99% pure  
         [0052]    Tetrahydrofuran (500 g)  
         [0053]    Triethyl amine (10.5 g, 0.104 mol)  
         [0054]    Pivaloyl chloride (11.0 g, 0.913 mol), 99% pure  
         [0055]    Guanine alcohol (27 g, 0.083 mol)  
         [0056]    4-dimethylaminopyridine (2 g, 0.0163 mol) 99% pure  
         [0057]    The stirred suspension was analysed after 2 days and found to contain 99.0% guanine stearate. Thereafter was added:  
         [0058]    6N Hydrochloric acid (50 ml, 0.300 mol)  
         [0059]    Borane-t-butyl amine complex (7.25 g, 0.083 mol) in portions of about 0.3 g over two hours, while the temperature was maintained at about 24°.  
         [0060]    To this reaction mixture was added:  
         [0061]    Water 800 ml  
         [0062]    1.2N Ammonium hydroxide (195 ml, 0.234 mol) over 10 minutes. The pH was 5.2. The white suspension was colled and kept at 5° C. for 2 hours and filtered on a G3 glass filter over 1.5 hours. The filter cake was washed with acetone (2×50 ml) and the wet filter cake (101 g) dried at 50° C. overnight, yielding 43.5 g (97%) stearate alcohol at 75.7% purity with 23.3% guanine stearate and 0.5% stearate aldehyde.  
         [0063]    To reduce the guanine stearate content, it was charged into a reaction flask:  
         [0064]    43 g of the material from the preceding step  
         [0065]    Tetrohydrofuran (600 g)  
         [0066]    Water (50 g)  
         [0067]    6N sulphuric acid (50 ml)  
         [0068]    Borane t-butyl amine complex (4.5 g, 0.052 mol) in 0.3 g portions was added over 7 hours while maintaining the temperature at about 24° C. The reaction was monitored for completion by HPLC, the last point being 0.18% guanine stearate. The reaction mixture was worked up as previously described to yield 39.54 g stearate alcohol, purity 94.4%, 2.6% stearate aldehyde and 0.17% guanine stearate.  
         [0069]    The product was cleaned twice by reflux in acetone. In a 500 ml flask was placed:  
         [0070]    Acetone (400 ml)  
         [0071]    Product (31.77 g)  
         [0072]    The stirred suspension was heated and kept at reflux for 1 hour, cooled to 27° C., filtered and washed with acetone (3×25 ml). The procedure was repeated. The resulting wet filter cake (33.9 g) was dried at 50° C. for 16 hours to give 27.9 g (82% relative to guanine alcohol) at 95.0% stearate alcohol, 0.1% guanine stearate, 2.7% stearate alcohol regioisomer, 1.4% of a product with RT=50-58 minutes and 2% steraric acid.  
         [0073]    Additional runs were performed using dried gaunaine alcohol, HCl and borane t-butylamine complex. The wet filtercake of stearate alcohol was refluxed twice with acetone to remove stearic acid:  
                                           Material &amp; step   30.2 g   75.5 g       Stearic acid       Tetrahydrofuran   535 g   1338 g       Triethylamine   14.8 g   37 g       Pivaloyl chloride   12.4 g   31 g       Guanine alcohol   27 g   67.5 g       Dimethylaminopyridine   2 g   5 g       Reaction time   18 hours   24 hours       3N HCl   110 ml   275 ml       Borane t-butyl amine complex   9.8 g   27 g       Temp. range during hydrolysis &amp;   29-33° C.   25-17° C.       reduction       Sodium hydrogen carbonate   15 g   26 g       Water   1000 ml   2500 ml*       Borane-t-butyl amine complex   1 g   4.5 g       Reflux with acetone   1000 ml   2500 ml       Content of stearic acid in acetone wash   4.9 g   9.9 g       Reflux with acetone   1000 ml   2500 ml       Content of stearic acid in acetone wash   2.5 g   2.0 g       Yield of dry stearate alcohol   37.85, 88%   95.45 g, 89%       Purity   96.8%   97.5%       Content of stearic acid   0%   0%                          
 
       EXAMPLE 4  
     Avoiding Regioisomerism During Hydrolysis  
       [0074]    In a 50 ml reaction flaks with magnetic stirring was charged:  
         [0075]    Stearate alcohol (0.4 g, 0.77 mmol)  
         [0076]    Tetrahydrofuran (27 ml)  
         [0077]    3N HCl (3 ml, 9 mmol)  
         [0078]    The stirred homogenous solution was kept at 41° C. 25 μl sampes were taken, diluted to 1.0 ml with 90% THF containing 2.5% ET 3 N and analysed by HPLC:  
                                                                     Reaction   Reaction time   RT 16 min               temp ° C.   hours   stearate   RT 21 min   Total area       20   0:00   alcohol %   Regioisomer %   AU*10 8                                  27   0:07   95.4   1.2   16.2       41   0:47       41   1.12   89.1   8.6   13.9       41   2:17   79.6   16.85   10.7       41   4:02   69.4   25.88   7.7       41   23.27   31.9   28.4   0.48       41   68   0   0   0.18                  
 
         [0079]    After 4 hours, 25% of regiosiomer (R)-9-(2-hydroxymethyl-4-stearoyloxy)butylguanine had formed by transesterification of the intended stearate alcohol, while about 50% of the stearate alcohol and regioisomer had hydrolysed. After 23.5 hours, about 3% of the intended stearate alcohol and regioisomer were present. The rest was apparently hydrolysed to stearic acid and guanine dialcohol. It is thus apparent that the formation of the regioisomer is minimized if the acid hydrolysis of the guanine stearate is performed under mild conditions, that is at low temperature and/or short time and/or low acid concentration. Furthermore it may be advantageous that the reaction mixture is neutralised as soon as the guanine stearate is all hydrolysed and/or extra borane reagent added with any remaining stearate alcohol present.  
         [0080]    Plots (not depicted) of analystic results as a function of reaction time were drawn from further dual experiments starting with guanine stearate (1 g, 1.7 mmol) dissolved in 90% THF (25 ml) and acid (10 mmol). BH 3 , t-BuNH 2  (200 mg, 2.3 mmol) was added at constant rate, while the temperature was kept at 30° C. Samples (25 μl) were taken for HPLC analysis of guanine stearate, stearate aldehyde, stearate alcohol and guanine alcohol during the 4-5 hour reaction time. Both plots were similar with an intitial fast hydrolysis of guanine stearate, a high concentration of stearate aldehyde, and a linear increase in stearate alcohol, governed by the addition rate of BH 3 , t-BuNH 2 .  
       EXAMPLE 5  
     Preparation of Pure Guanine Stearate  
       [0081]    To prepare stearic acid free guanine stearate, an experiment was run with only 90 mol % stearic acid and pivaloyl chloride. The starting materials were stearic acid (21.3 g, 75 mmol), pivaloyl chloride (9.0 g, 75 mmol) and guanine alcohol (27 g, 83 mmol). The yield was 33.5 g (75% relative to stearic acid) of guanine stearate, purity 99.6% (HPLC). A stearic acid determination gave &lt;1%, as expected, as the guanine stearate is precipitated by addition of acetone (1000 ml) which will dissolve stearic acid.  
         [0082]    The filtrate (1350 ml) contained stearic acid 4.8 g, 17 mmol) corresponding to 23% of the charged amount. This could be explained by water present in the reaction mixture. The solvent tetrahydrofuran was almost anhydrous (6 mg H 2 O/l) but the guanine alcohol contained 28 mg H 2 /l, which corresponds to ½ mol crystal water. This means that the 27 g of guanine alcohol contained 42 mmol H 2 , which could react with the mixed anhydride of astearic acid and pivalic acid. Accordingly, it is advantageous if the guanine alcohol start material is dried by to reaction, for instance at 50° C. for 2 hours to reduce the water content to less than 20 mg H 2 O/g, preferably less than 10 mg/g, most preferably &lt;5 mg/g