A PROCESS FOR THE PREPARATION OF
(f?)-9-r2-(PHOSPHONO ETH-OXY)PROPYUADENINE (PMPA)
BACKGROUND OF THE INVENTION
THIS invention relates to a process for the preparation of (R)-9-[2- (phosphonometh-oxy)propyl]adenine (PMPA). This invention also relates to the preparation of Tenofovir disoproxil fumarate (TDF), a compound which can be produced from PMPA.
PMPA and TDF are highly potent antiviral agents having particular uses in treating retroviral infections, including HIV, HSV1 , HSV2 and other viral infections. The HIV/AIDS pandemic poses a great challenge to the world at large. Treatment of the HIV/AIDS involves the use of RT inhibitors.
Reverse transcriptase inhibitors target the HIV reverse transcriptase (RT) enzyme which is required for the replication of the virus. Examples of RT inhibitors include nucleoside RT inhibitors (NRTIs), nucleotide RT inhibitors (NtRTIs) and non-nucleoside RT inhibitors (NNRTIs). HIV-infected patients are routinely treated with triple therapy, which is a three-drug regimen containing one, two or three RT inhibitors in combination with other inhibitors such as protease or integrase inhibitors.
Clinical studies have shown that three-drug combinations of these anti-HIV drugs are much more effective than one drug used alone or two-drug combinations in preventing disease progression and death. Numerous studies of drug combinations with various combinations of such drugs have established that such combinations greatly reduce disease progression and deaths in people with HIV infections. The name now commonly given to
combinations of anti-HIV drugs is HAART (Highly Active Anti-Retroviral Therapy).
Tenofovir disoproxil fumarate (TDF) (({[(2R)-1-(6-amino-9H-purin-9- yl)propan-2-yl]oxy}methyl)phosphonic acid) is a nucleotide reverse transcriptase inhibitor approved for the treatment of HIV-1 infection in combination with other antiretroviral agents.
1
Chemical Formula: C^HsoNgO-ioP-C^C^
Critical in the production of TDF is the use of PMPA, a key intermediate in the process.
There are several prior art documents which deal with the synthesis of PMPA and TDF.
U.S. Pat. No. 5,733,788 discloses the process for the preparation of PMPA 3 which involves condensation of (RJ-9-[2-(hydroxyl)propyl]adenine (HPA 6) and diethyl p-toluenesulfonyloxymethylphosphonate (DESMP) in presence of lithium hydride in dimethylformamide (DMF) followed by dealkylation with bromotrimethylsilane (TMSBr) in acetonitrile.
U.S. Pat. No. 5,922,695 discloses the synthetic route for the preparation of PMPA 3 by condensation of HPA 6 with DESMP in presence of lithium tert- butoxide in tetrahydrofuran followed dealkylation with bromotrimethylsilane in acetonitrile. Further, the TD base is obtained as an oil which is further converted to TDF 1.
US 2004/0018150 discloses a process for the preparation PMPA 3 where DES P is condensed with HPA 6 in the presence of magnesium iso- propoxide or MTB in DMF medium followed by deallylation with TMSBr in acetonitrile with an overall yield 48%.
U.S. Pat. No. 6,465,649 discloses a process for the preparation of PMPA 3 by dealkylation of PPA 2 with TMSCI in chloroform under pressure.
U.S. 2009/0286981 describes the preparation of a novel TDF 1 salt, prepared by dealkylation of phosphonate esters by using mineral acids followed by esterification and crystallization to afford crystalline TDF 1. The conversion to the fumarate salts is also described.
WO 9905150A1 describes the compound TDF 1. The invention also provides methods to make PMPA 3 and intermediates in the PMPA 3 synthesis. Including lithium tert-butoxide, HPA 6 and DESMP. Method details improvements in the by-product profile of diethyl PMPA 3.
WO 2006/133632A1 describes TDF 1 , the derivation and use thereof. The patent also includes the synthesis of TD and TD compositions route involving the use of LiH, DMF to prepare PPA 2 and TMSBr, CH3CN to afford PMPA 3.
WO 201 1/111074A2 describes the synthesis of TD and TDF 1. Synthesis of TD is achieved through the use of MTB or potassium iert-butoxide in either cyclohexane, toluene, benzene or 1 ,4-dioxane. PMPA 3 is prepared using HBr (aq).
FR 2908133A1 describes novel routes to phosphonates and thiophosphonates, PPA 3 is employed as a precursor and is prepared using potassium t-butoxide.
WO 2008/157657A1 describes methods for preparing deuterium enriched TDF 1 , process involves the preparation of PPA 2 with NaH and PMPA 3 with TMSBr.
WO 2008/134578A2 describes the synthesis of isotopically labelled reverse transcriptase inhibitors, including acyclic nucleotide analogue. The process makes use of NaH/DMF to prepare compounds structurally related to PPA 2 these are then hydrolyzed using TMSBr in DMF:
PCT Int. Appl., 2008005555 relates generally to compounds and pharmaceutical compositions which selectively activate toll-like receptor 7 (TLR7), and methods of making and using them. The general process used for the preparation of PPA 2 and related derivatives involves the use of MTB in DMF.
WO 2008/007392A2 describes the use of MTB in DMF for the preparation of PPA 2. Patent claims the use of HBr, HCI (aq), HBr in acetic acid or HCI gas in IPA for the preparation of PMPA 3.
US 2006/122391 A1 describes the use of nucleotide analogues and their use as antiviral agents and inhibitors of ribonucleic acid (RNA) viral polymerases. Nucleotides are prepared from compounds structurally related to PPA 2 using NaH/DMF.
WO 9403467A2 describes the use of NaH for the preparation of compounds structurally related to PPA 2.
CN 101531679A describes a process for the preparation of PMPA 3 by the de-ethylation of PPA 2 using TMSCI/Acetonitrile in the presence of catalytic amounts of sodium halides.
CN 101531680A describes a method for preparing PPA 2 using dialkylmagnesium.
CN101574356A describes the use of sodium fert-butoxide for the preparation of PPA 2 and TMSCI/KI for the preparation of PMPA 3.
CN 101870713A describes a method for preparing TDF, the described route makes use of MTB to prepare PPA 2 and TMSBr to prepare PMPA 3.
CN 102295660A describes the synthesis of TDF using sodium te/i-butoxide to access PPA 2 and aqueous HBr to access PMPA 3.
CN 102093417A describes compounds and the preparation thereof that are capable of being used for the preparation of nucleoside phosphoric acids. The process makes use of lithium te/t-butoxide followed by TMSBr/CH3CN.
IN 2010CH03791A describes a process for the preparation of TD or TD-salt and its pharmaceutical composition. The use of NaNH2/DMF with catalytic MgCI2 in toluene or BuMgCI is described for the preparation of PPA 2. The subsequent de-ethylation to afford PMPA 3 is achieved using HBr (aq).
CAN 155:241012 AN 2011 :507772 describes a route to TDF.
CAN 156:54913 AN 2011 :504437 describes a route to TDF from adenine using a ring-opening condensation with f ^-propylene oxide, etherification and hydrolysis to give PMPA 3 which is subjected to reaction with CMIC followed by salt formation.
CN 2008-10015780 describes a method for the preparation of medical salts and/or derivatives of TD, and their application for preventing and/or treating HBV and HIV/AIDS infections.
IN 2008MU292 20080212 describes the deprotection of TD-phosphate esters by heating PPA 2 in the presence of an inorganic acid preferably HBr.
Tetrahedron 66 (2010) 8317-8144 describes a rapid, low temperature hydrolysis of PPA 2 mediated by TMSCI and NaBr which was demonstrated to be superior to the TMSBr mediated hydrolysis. The mild hydrolysis could then be coupled to alkylation of the phosphonic acid, providing a one-pot procedure for the formation of PMPA 3.
Tetrahedron Letters 39 (1998) 1853-1856 shows that the anti-HIV nucleotide analogue PMPA 3 can be prepared on a kilogram scale by a three step sequence: i) condensation of adenine with RJ-propylene carbonate, ii) alkylation of the resulting PPA 2 with DESMP using lithium ferf-butoxide and iii) cleavage of the phosphonate ester functionalities with TMSBr.
Nucleosides, Nucleotides and Nucleic Acids, 20(4-7), 1299-1302 (2001) shows that TMSCI completely dealkylates phosphonate esters at elevated temperatures in a sealed reaction vessel. These conditions are tolerated by a variety of functional groups and lead to high conversions of dimethyl, diethyl and diisopropyl phosphonates to their corresponding phosphonic acids.
Journal of the American Chemical Society 1996, 118, 7420-7421 describes the conversion of HPA 6 to PMPA 3 using NaH followed by TMSBr.
Journal of Medicinal Chemistry 2006, 49 (26), 7799-7806 describes the synthesis of 9-[2-(boranophosphonomethoxy)ethyl]adenine and (R)-9-[2- (boranophosphonomethoxy-)propyl]adenine. HPA 6 and PPA 2 are used as intermediates and PPA 2 is prepared using sodium fe/f-butoxide.
Organic Process Research and Development 2010, 14, 1 194-1201 describes the improved synthesis of TDF 1. The process involves the condensation of adenine with (Rj-propylene carbonate to afford HPA 6. HPA 6 is coupled with DESMP in the presence of MTB, followed by hydrolysis to PMPA 3 in the presence of TMSCI/NaBr. PMPA 3 is converted to TDF 1 by treatment with CMIC in the presence of triethylamine
and tetra-butylammonium bromide followed by salt formation with fumaric acid.
Journal of Medicinal Chemistry 2010, 53 (19), 6825-6837 describes the synthesis of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyI]-2,6- diaminopurine (HPMPDAP) and its cyclic form. The process involves the coupling of a bromophosphonate to an alcohol in the presence of NaH/DMF.
Journal of Labelled Compounds and Radiopharmaceuticals, 51 (4), 187- 194; 2008 describes the synthesis of compounds structurally related to PPA 2 employing the use of NaH/DMF.
Nucleosides, Nucleotides & Nucleic Acids, 24 (10-12), 1569-1585; 2005 describes the synthesis 9-[-1-(substituted)-2-
(phosphonomethoxy)ethyl]adenine derivatives as possible antiviral agents, the synthesis makes use of NaH/DMF to form structurally related compounds to PPA 2.
Journal of Medicinal Chemistry, 2006, 49, 3955-3962 describes the activation murine RNase L by isopolar 2, phosphate analogues of 2,5 oligoadenylates. The route involves the use of NaH/DMF to prepare compounds structurally related to PPA 2 and TMSBr, 2,6-lutidine to prepare analogues of PMPA 3.
Tetrahedron, 56(29), 5077-5083; 2000 shows the synthesis of acyclic nucleoside and nucleotide analogues from amino acids, the paper describes the use of lithium ferf-butoxide in DMF to prepare compounds that are structurally related to PPA 2.
Nucleosides and Nucleotides (1995), 14(3-5), 695-702 describes the synthesis of acyclic nucleotide analogues bearing amino- and N-substituted amino groups; structurally similar compounds to PPA 2 are prepared using NaH.
Collection of Czechoslovak Chemical Communications (1993), 58(3), 649- 674 describes the synthesis of enantiomeric N-(3-hydroxy-2- phosphonomethoxypropyl) derivatives of purine and pyrimidine bases. Route uses NaH/DMF to prepare analogous compounds to PPA 2.
Nucleoside and Nucleotides (1989), 8(4), 619-624 shows a bis-trityl route to (S)-HPMPA, which makes use of Na THF to prepare compounds similar to PPA 2.
Tetrahedron Letters (1987), 28(42), 4963-4964 shows a convenient synthesis of S-HPMPA using Na/DMF to prepare structures similar to PPA 2.
Nucleic Acids Symposium Series 18 (Symp. Chem. Nucleic Acid Compon., 7th 1987), 33-6; 1987 describes the synthesis of HPMPA.
Dier Junyi Daxue Xuebao, 26(10), 1186-1189; 2005 shows the synthesis of acyclic nucleoside phosphonates based on HIV/HBV, route prepares similar compounds to PPA 2 using sodium t-butoxide in DMF.
Zhongguo Yiyao Gongye Zazhi, 38(1), 4-6; 2007 describes the synthesis of adefovir dipivoxil, the route uses NaH/DMF to prepare compounds similar in structure to PPA 2.
Yaoxue Shijian Zazhi, 27(1), 31-32, 45; 2009 shows an improved synthesis of TDF using sodium ferf-butoxide in DMF to prepare PPA 2.
Zhongguo Yiyao Gongye Zazhi, 17(22), 1937-1939; 2008 shows the synthesis of adefovir dipivoxil, the route makes use of NaH/DMF to prepare compounds structurally related to PPA 2.
The above prior art processes describe procedures to prepare either TDF 1 , structurally related analogues of TDF 1 or structurally related analogues of either PPA 2 or PMPA 3 making use of expensive reagents like DESMP
or related phosphonates. The coupling of HPA 6 and DESMP (and structurally related analogues) is typically characterised by the use of bases like MTB, potassium f-butoxide and sodium hydride in low yielding reactions which result in a high overall cost of goods.
On the basis of the findings in the literature available to date, the synthesis of the compound PMPA and the subsequent synthesis of related drugs, including TDF, GS-7340 and CMX-157, still presents numerous problems. There is consequently a need to develop a novel synthesis process which is more economical.
SUMMARY OF THE INVENTION
According to a first aspect of the invention there is provided a process for the synthesis of (R -9-[2-(phosphonometh-oxy)propyl]adenine (PMPA) (3),
3 the process including the steps of:
a) reaction between compound (6) (HPA)
and a phosphosulfonate compound (7)
wherein; is selected from alkyl, cycloalkyl, substituted or unsubstituted phenyl, substituted or unsubstituted benzyl or wherein the R-i groups together with the oxygen atoms to which they are attached form a heterocyclic group. R-i is preferably ethyl, methyl or benzyl;
R2 is selected from alkyl or substituted aryl. Preferably R2 is methyl or 4- methyphenyl; wherein the reaction in step a) takes place in an organic solvent in the presence of an organomagnesium species to yield intermediate (PPA) (2)
2
Ri having the meanings as defined above; and b) substitution of the R-i groups with H in compound (2) to yield the final product (3) (PMPA).
ln a preferred embodiment, R-i in compounds (7) and (2), is ethyl or methyl. R2 in compunds (7) and (2), is a sulfonyl group, preferably p-toluenesulfonyl or methanesulfonyl.
In a preferred embodiment, the organomagnesium species is used at a concentration of between 0.5 M and 2M. Step a) is preferably carried out at a temperature in the range of 55-75 °C.
Compound (6)
may be prepared by the rea nine (4)
and ( )-4-methyl-1 ,3-dioxolan-2-one (5)
to yield the intermediate (6) (HPA).
The reaction to yield intermediate 6 (HPA) is preferably carried out in the presence of a base such as a metal hydroxide, more specifically an alkali metal or alkali-earth metal hydroxide, tetraalkylammonium hydroxide, a metal carbonate or metal hydrogen carbonate. The metal hydroxide may be selected from lithium, sodium, potassium, calcium or magnesium
hydroxide. These metal hydroxides may be present as hydrates or in anhydrous forms. The tetraalkylammonium hydroxide may be selected from tetramethyl, ethyl or butyl ammonium hydroxide. The metal carbonates may be selected from lithium, sodium or potassium carbonate. The metal hydrogen carbonate may be selected from lithium, sodium or potassium hydrogen carbonate. Most preferably the base employed is sodium or potassium hydroxide.
The base is used in a catalytic amount in the range of about 0.001 mole % to 100 mole % and most preferably in the range 8 mole % to 10 mole %. The reaction is also carried out in polar aproticorganic solvent such as NMP, DMF, CH3CN or DMSO but most preferably in DMF at a temperature range of about 70-85 °C for 24 hours.
When potassium hydroxide is employed as the base 1 mole % is used in DMF as the solvent with a temperature for the reaction of about 75 °C for 16 hours.
In step a) the organic solvent is preferably selected from NMP, dimethoxyethane, toluene, acetone, ethyl acetate, chloroform, acetonitrile, THF, dichloromethane and cyciohexane. The organic solvent can further include an alcohol that may be selected from methanol, ethanol, iso- propanol, ferf-butanol, /so-butanol, cyclopentanol, octanol, cyclohexanol, phenol and benzyl alcohol. The reaction as set out in step a) is carried out at a temperature of about 120 °C for around 24 h.
The organomagnesium species may be selected from phenyl magnesium chloride, phenyl magnesium bromide, methyl magnesium chloride, methyl magnesium bromide, so-propyl magnesium chloride, 2,2,6,6- tetramethylpiperidinylmagnesium. When the organomagnesium species is used in the presence of an alcohol, then the alcohol may be selected from methanol, ethanol, /so-propanol, terf-butanol, /so-butanol, cyclopentanol, octanol, cyclohexanol, but most preferably tert-butanol.
The solvent used in step a) may be selected from NMP, dimethoxyethane, toluene, acetone, ethyl acetate, chloroform, acetonitrile, THF, dichloromethane and cyclohexane. Most preferably the solvent is cyclohexane. When cyclohexane is used as the solvent, then the reaction in step a) preferably takes at a temperature range of about 50 - 85°C for 3- 16 hours.
When methyl magnesium chloride is employed as the organomagnesium species te/i-butyl alcohol is the preferred alcohol with both reagents employed in about a 1 :1 molar ratio relative to compound 6. In this embodiment the preferred solvent is cyclohexane with a preferred temperature for the reaction in the range of about 75 °C.
The substitution reaction in step b) may be carried out as follows:
In one embodiment the (PPA) (2) is treated with 33% HBr in acetic acid in a molar ratio of about 6.7:1 to 20:1 and at a temperature of about 75 °C for 3 hours.
In another embodiment the (PPA) (2) is treated with aqueous 48% HBr in a molar ratio of about 20:1 and at a temperature of about 75 °C for 1 hour.
In another embodiment the (PPA) (2) is treated with aqueous 4.0 M HCI in dioxane in a molar ratio of about 6:1 to 20:1 and at a temperature of about 75 °C to 16 hours.
In another embodiment the (PPA) (2) is treated with a trialkylsilyl chloride in a molar ratio of about 5:1 to 20:1 in an organic solvent such as acetonitrile and at a temperature of about 75 °C for 16 hours. Preferably the trialkylsilyl chloride is trimethylsilyl chloride (TMSCI).
In another embodiment the (PPA) (2) is treated with a trialkylsilyl chloride in a molar ratio of about 5.3:1 to 20:1 and sodium or potassium bromide in a molar ratio of about 3.5:1 in an organic solvent such as NMP, DMSO or
acetonitrile and at a temperature of about 75 °C for 16 hours. Preferably the trialkylsilyl chloride is TMSCI.
In another embodiment the (PPA) (2) is treated with a trialkylsilyl chloride in a molar ratio of about 5:1 in an organic solvent such as acetonitrile in a sealed tube vessel and at a temperature of about 100-120 °C for 16 hours. Preferably the trialkylsilyl chloride is TMSCI.
In another embodiment the (PPA) (2) is treated with a trialkylsilyl chloride in a molar ratio of about 5:1 and sodium or potassium bromide in a molar ratio of about 3.5:1 in an organic solvent such as acetonitrile in a sealed tube vessel and at a temperature of about 120 °C for 16 hours. Preferably the trialkylsilyl chloride is TMSCI.
In another embodiment the (PPA) (2) is treated with a trialkylsilyl chloride in a molar ratio of about 5:1 in a sealed tube vessel and at a temperature of about 120 °C for 16 hours. Preferably the trialkylsilyl chloride is TMSCI.
According to a preferred embodiment (PPA) (2) is treated with 33% HBr in acetic acid in a molar ratio of about 6.7:1 to 20:1 and at a temperature of about 75 °C for 3 hours.
In an alternative embodiment, the product from step a) can be used directly in step b) without isolation of compound 2. The crude material obtained from step a) is treated with any of the embodiments described for step b), either before or after work-up of step a).
According to a second aspect of the invention there is provided a process for the synthesis of TDF (1)
The process comprises the steps, of treating compound (3) (PMPA) as synthesized above with chloromethyl isopropyl carbonate (CMIC) in the presence of a base to yield compound 1 (TDF).
The base may be selected from metal hydroxides, more specifically an alkali metal or alkali-earth metal hydroxide, metal carbonates, metal hydrogen carbonates, trialkylamine bases, guanidine bases, pyridine derived bases or tetraalkylammonium hydroxides.
The metal hydroxides may be selected from lithium, sodium, potassium, calcium or magnesium hyrdoxides either as their hydrates or in anhydrous forms. The metal carbonates include lithium, sodium or potassium carbonate. The metal hydrogen carbonates may be selected from lithium, sodium or potassium hydrogen carbonate. The trialkylamine bases include triethylamine, /V./V-diisopropylamine tripropylamine, dispropylethylamine and tributylamine. The guanidine bases include tetramethylguanidine. The pyridine derived bases include 2,6-lutidine. The tetraalkylammonium hydroxides may be selected from tetramethyl, ethyl or butyl.
The addition of a phase transfer catalyst such as tetraalkylammonium halides such as te/ifabutylammonium bromide, terfabutylammonium chloride, tertabutylammonium iodide, benzyldibutylammonium bromide, tetrabutylammonium tosylate, sodium tosylate may also be employed. The reaction is conducted in a polar aprotic organic solvent such as NMP, DMF, CH3CN or DMSO and at a temperature of about 45-70 °C for 5-6 hours.
The preferred embodiment treats PMPA (4) with chloromethyl isopropyl carbonate in a molar ratio of about 5:1 in the presence of triethylamine in a molar ratio of about 4:1 and tetrabutylammonium bromide in a molar ratio of about 1 :1 in NMP as the organic solvent at a temperature of about 50 °C for 5.5 h.
In an alternative embodiment the product from step a) can be used directly for the production of compound 1 (TDF), without the isolation of compound (3) (PMPA). The crude material obtained from step b) is treated with any of the embodiments described for the production of compound 1 , before or after work-up of step b).
PMPA (3) may also be used to prepare several other related pro-drugs including GS-7340 and CMX-157.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic diagram showing the reaction for the synthesis of PMPA and TDF.
DESCRIPTION OF PREFERRED EMBODIMENTS
The present invention relates to a process for the production of (R)-9-[2- (diethylphosphonomethoxy)propyl]adenine (PMPA) (3). The route utilises the combination of an organo-magnesium reagent and an alcohol in the key coupling protocol.
The invention will now be described with reference to figure .
(R -9-(2-Hydroxypropyl)adenine (HPA) (6) is prepared by the reaction between adenine 4 (available commercially from Shenzhen Penking Biochemical Technology Co., Ltd.) and 4-methyl-1 ,3-dioxolan-2-one (5) (available commercially from Shenzhen Penking Biochemical Technology Co., Ltd.). The reaction is preferably carried out in the presence of a base such as a metal hydroxide such as lithium, sodium, potassium, calcium or magnesium hydroxides either as their hydrates or in anhydrous forms alternatively, tetraalkylammonium hydroxides such as tetramethyl, ethyl or butyl maybe used or metal carbonates such as lithium, sodium or potassium carbonate or alternatively metal hydrogen carbonates such as lithium, sodium or potassium hydrogen carbonate. Most preferably the base employed in this transformation is sodium or potassium hydroxide. The base is used in a catalytic amount in the range of about 0.001 mole % to 100 mole % and most preferably in the range 8 mole % to 10 mole %. The reaction is also carried out in polar aproticorganic solvent such as NMP, DMF, CH3CN or DMSO but most preferably in DMF at a temperature range of about 70-85 °C for 24 hours.
When potassium hydroxide is employed as the base 1 mole % is used in DMF as the solvent with a temperature for the reaction of about 75 °C for 16 hours.
6 2
PPA 2 is prepared by the reaction between HPA (6) and a phosphosulfonate (7) in the presence of a base such as a metallic alkoxide for example, an organomagnesium species or an organomagnesium species in the presence of an alcohol in an organic solvent such as NMP, dimethoxyethane, toluene, acetone, ethyl acetate, chloroform, acetonitrile, THF, dichloromethane and cyclohexane and an alcohol such as methanol, ethanol, /so-propanol, te/f-butanol, /so-butanol, cyclopentanol, octanol, cyclohexanol, phenol and benzyl alcohol can be used. The reaction is carried out at a temperature of about 75 °C for around 2-3 h.
Ri can be alkyl, cycloalkyl, phenyl, substituted phenyl, benzyl, substituted benzyl or the groups together with the oxygen atoms to which they are attached form a heterocyclic group. Preferably Ri is ethyl, methyl or benzyl. R2 can be alkyl or substituted aryl. R2 is preferably methyl or 4- methylphenyl.
The reaction is preferably carried out in the presence of an organomagnesium species in the presence of an alcohol in an organic solvent. The organomagnesium species can be phenyl magnesium chloride, phenyl magnesium bromide, methyl magnesium chloride, methyl magnesium bromide, /so-propyl magnesium chloride, 2,2,6,6- tetramethylpiperidinylmagnesium most preferably methyl magnesium chloride or bromide. The alcohol can be methanol, ethanol, /so-propanol, ferf-butanol, /so-butanol, cyclopentanol, octanol, cyclohexanol, but most preferably te i-butanol. The organic solvent includes NMP,
dimethoxyethane, toluene, acetone, ethyl acetate, chloroform, acetonitrile, THF, dichloromethane and cyclohexane or solvent free but most preferably in cyclohexane at a temperature range of about 50 - 85°C for 3-16 hours.
When methyl magnesium chloride is employed as the organomagnesium species fe/f-butyl alcohol is the preferred alcohol with both reagents employed in about a 1 :1 molar ratio relative to HPA (6). In this embodiment the preferred solvent is cyclohexane with a preferred temperature for the reaction in the range of about 75 °C.
In the preferred embodiment DESMP or dimethyl-p toluenesulfonyloxymethylphos-phonate are used.
Stage 2b
3
PMPA (3) is prepared from PPA (2) by substitution of the dialkyl phosphonate.
In one embodiment the PPA (2) is treated with 33% HBr in acetic acid in a molar ratio of about 6.7:1 to 20:1 and at a temperature of about 75 °C for 3 hours.
In another embodiment the PPA (2) is treated with aqueous 48% HBr in a molar ratio of about 20:1 and at a temperature of about 75 °C for 1 hour. In another embodiment the PPA (2) is treated with aqueous 4.0 M HCI in dioxane in a molar ratio of about 6:1 to 20:1 and at a temperature of about 75 °C to 16 hours.
ln another embodiment the PPA (2) is treated with a trialkylsilyl chloride in a molar ratio of about 5:1 to 20:1 in an organic solvent such as acetonitrile and at a temperature of about 75 °C for 16 hours. Preferably the trialkylsilyl chloride is trimethylsilyl chloride.
In another embodiment the PPA (2) is treated with a trialkylsilyl chloride in a molar ratio of about 5.3:1 to 20:1 and sodium or potassium bromide in a molar ratio of about 3:5:1 in an organic solvent such as NMP, DMSO or acetonitrile and at a temperature of about 75 °C for 16 hours. Preferably the trialkylsilyl chloride is TMSCI.
In another embodiment the PPA (2) is treated with a trialkylsilyl chloride in a molar ratio of about 5:1 in an organic solvent such as acetonitrile in a sealed tube vessel and at a temperature of about 100-120 °C for 16 hours. Preferably the trialkylsilyl chloride is TMSCI.
In another embodiment the PPA (2) is treated with a trialkylsilyl chloride in a molar ratio of about 5:1 and sodium or potassium bromide in a molar ratio of about 3.5:1 in an organic solvent such as acetonitrile in a sealed tube vessel and at a temperature of about 120 °C for 16 hours. Preferably the trialkylsilyl chloride is TMSCI.
In another embodiment the PPA (2) is treated with a trialkylsilyl chloride in a molar ratio of about 5:1 in a sealed tube vessel and at a temperature of about 120 °C for 6 hours. Preferably the trialkylsilyl chloride is TMSCI.
The preferred embodiment is as follows:
PPA (2) is treated with 33% HBr in acetic acid in a molar ratio of about 6.7:1 to 20:1 and at a temperature of about 75 °C for 3 hours. Preferably the trialkylsilyl chloride is TMSCI.
Stage 3
TDF 1 is prepared from PMPA 3 by treatment with CMIC in the presence of a base such as a metal hydroxide such as lithium, sodium, potassium, calcium or magnesium hyrdoxides either as their hydrates or in anhydrous forms. Alternatively, metal carbonates such as lithium, sodium or potassium carbonate or metal hydrogen carbonates such as lithium, sodium or potassium hydrogen carbonate may be used. . Additionally trialkylamine bases such as triethylamine, A/,A/-diisopropylamine tripropylamine, dispropylethylamine and tributylamine may be used. Also guanidine bases such as tetramethylguanidine may be used, or pyridine derived bases such as 2,6-lutidine, Alternatively, tetraalkylammonium hydroxides such as tetramethyl, ethyl or butyl maybe used. The addition of a phase transfer catalyst such as tetraalkylammonium halides such as te/ abutylammonium bromide, ferfabutylammonium chloride, ferfabutylammonium iodide, benzyldibutylammonium bromide, tetrabutylammonium tosylate, sodium tosylate may also be employed. The reaction is conducted in a polar aprotic organic solvent The reaction is also carried out in polar aprotic organic solvent such as NMP, DMF, CH3CN or DMSO and at a temperature of about 45-70 °C for 5-6 hours.
The preferred embodiment treats PMPA 3 with CMIC in a molar ratio of about 5:1 in the presence of triethylamine in a molar ratio of about 4:1 and tetrabutylammonium bromide in a molar ratio of about 1 :1 in NMP as the organic solvent at a temperature of about 50 °C for 5.5 h. The crude material is converted to the fumarate salt TDF (1) by treatment with fumaric acid at a temperature of about 50°C for 2 hours.
Stage 2a/2b telescoped process
The product from Stage 2a can be used directly in Stage 2b without isolation of PPA 2. The reaction is typically carried out using the embodiment described for Stage 2a. The crude material obtained from Stage 2a is typically treated with any of the embodiments described for Stage 2b either before or after work-up of Stage 2a.
Stage 2b/3 telescoped process
The product from Stage 2b can be used directly in Stage 3 without isolation of PMPA 3 after stage 2b.
The reaction is typically carried out using the embodiment described for Stage 2b. The crude material obtained from Stage 2b is typically treated with any of the embodiments described for Stage 3 either before or after work-up of Stage 2b.
The invention will now be described with reference to the following non- limiting examples.
Experimental
General Methods
All reagents were purchased from Sigma Aldrich with the exception of adenine 4, RPA 5, DESMP and MTB which were purchased from Shenzhen Penking Biochemical Technology Co., Ltd.
Analytical analysis was performed on an Agilent 1200 HPLC with a flow- rate of 1ml/min on a Waters Xbridge C18 (50 mm x 4.6 mm x 5 microns) coupled to a UV detector (260 nm) and an Agilent 6120 Quadrupole mass spectrophotometer in the positive mode. All ΊΗ NMR and 31 P NMR data were recorded on a Bruker AVANCE III 400 MHz spectrometer.
Preparation of (7 )-9-(2-Hydroxypropyl)adenine
Example 1 (Stage 1 using NaOH)
Adenine 4 (10 g, 74.0 mmol, 1.0 eq) and sodium hydroxide (0.237 g, 5.92 mmol, 0.08 eq) were mixed with DMF (47.5 ml) at 25-30 °C and the mixture was stirred for 10 min at 25-30 °C. (R)-propylene carbonate 5 (8.4 ml, 9.97 g, 98 mmol, 1.32 eq) was added drop-wise to the reaction mixture over 10- 15 min at 25-30 °C. The mixture was heated to 120 °C and held at that temperature for 24 h. A clear solution resulted, and the reaction mixture was cooled to 70 °C. A mixture of methanol (30 ml) and /'so-propanol (30 ml) was added drop-wise to the reaction mixture over 10 min, during which time the reaction mixture was allowed to cool to 55 °C, and precipitation of product was observed. The reaction mixture was cooled to ~ 5 °C and held at this temperature for 1 h. The product was isolated by filtration and the cake was washed with a chilled mixture of methanol (10 ml) and /so- propanol (10 ml). The resulting solid was dried under vacuum at 70-75 °C, affording HPA as an off-white solid (9.03 g, 63.2 %). Proton NMR (98% purity) indicated the formation of the desired product 6, with no visible trace of the unwanted regioisomer. H1 NMR (d6-DMSO) 400 MHz 8.14 (1 H, s, H- 4), 8.06 (1H, s, H-9), 7.20 (2H, s broad, -NH2), 5.05 (1 H, d, H-1'a), 4.12- 3.98 (3H, m, H-1'b, H-2', OH), 1.07 (3H, d, H-3'). LCMS retention time 1.650 min. m/z = 194.0 [M+H]+ Purity 98.5%
Example 2 (Stage 1 using KOH)
Adenine 4 (40 g, 296 mmol, 1.0 eq) and potassium hydroxide (1.66 g, 29.6 mmol, 0.1 eq) were mixed with DMF (190 ml) at 25-30 °C and the mixture
was stirred for 10 min at 25-30 °C. (R)-propylene carbonate 5 (33.6 ml, 39.9 g, 391 mmol, 1.32 eq) was added drop-wise to the reaction mass over 10-15 min at 25-30 °C. The mixture was heated to 120 °C and held at that temperature for 48 h. A clear solution resulted, and the reaction mixture was cooled to 70 °C. A mixture of methanol (120 ml) and /so-propanol (120 ml) was added drop-wise to the reaction mixture over 10 min, during which time the reaction mixture was allowed to cool to 55 °C, and precipitation of product was observed, the reaction mixture was cooled to ~ 5 °C and held at this temperature for 1 h. The product was isolated by filtration and the cake was washed with a chilled mixture of methanol (10 ml) and /so- propanol (10 ml). The resulting solid was dried under vacuum at 70-75 °C, affording HPA 6 as an off-white solid (37 g, 65%).
Example 3 (Stage 1 Base Screen)
Adenine 4 (0.5 g, 3.70 mmol, 1.0 eq) and base/acid (0.08 eq) were mixed with solvent (DMF, DMSO or NMP) (5 ml) at 25-30 °C and the mixture was stirred for 10 min at 25-30 °C. (R)-propylene carbonate 6 (0.42 ml, 0.499 g, 4.88 mmol, 1.32 eq) was added drop-wise to the reaction mass over 10-15 min at 25-30 °C. The mixture was heated to 120 °C and held at that temperature for 24 h. The reaction was monitored by LCMS thoughout this period to determine the percentage conversion of Adenine 4 to HPA.
Table of solvents and bases tested (the time taken until the solution homogenised is given in brackets after the mass of the catalyst used).
Base/Solvent DMSO (5 ml) NMP (5 ml) DMF (5 ml)
NaOH 24.0 mg (55 min) 24.0 mg (-300 min) 24.0 mg (340 min)
LiOH.H20 12 mg (70 min) 12 mg (> 480 min) 12 mg (> 480 min)
KOH 16.6 mg (50 min) 16.6 mg (60-90 min) 16.6 mg (150 min) nBu4NOH.30H2O 236.8mg (60-90 min) 236.8 mg (150 min) 236.8 mg
TsOH 50.9 mg 50.9 mg 50.9 mg
K2C03 41.0 mg (30 min) 41.0 mg (60 min) 41.0 mg (75 min)
Results for UOH.H20 and NaOH screens
Example 4 (Stage 1 Temperature Screen)
Adenine 4 (0.5 g, 3.70 mmol, 1.0 eq) and base (0.08 eq) were mixed with solvent (DMF, DMSO or N P) (5 ml) at 25-30 °C and the mixture was stirred for 10 min at 25-30 °C. (R)-propylene carbonate 5 (0.42 ml, 0.499 g, 4.88 mmol, 1.32 eq) was added drop-wise to the reaction mass over 10-15 min at 25-30 °C. The mixture was heated to the indicated temperature and held at that temperature for 24 h. The reactions were monitored by LCMS to determine the percentage conversion of Adenine 4 to HPA.
Matrix of base/solvent/temperature tested
Results @ 160 °C
Results @ 140 °C
Time NaOH/ NaOH/ NaOH/ KOH/ KOH/ KOH/
DMSO NMP DMF DMSO NMP DMF
0 0 0 0 0 0 0
15 32.02 27.53 18.28 45.29 39.86 28.31
30 52.39 47.72 28.95 77.38 62.63 44.25
45 61.83 56.48 36.37 87.32 67.88 51.35
60 76.26 67.24 47.36 92.54 82.33 60.87
90 94.24 85.3 64.37 94.62 94.24 73.06
120 94.81 94.06 74.45 94.75 94.55 81.48
150 94.95 94.16 82.54 94.9 94.75 94.53
Regioisomer 5.1 5.90 4.70 5.46
Results @ 120 °C
Results @ 100 °C
Example 5 (Stage 1 base stoichiometry screen)
Adenine 4 (0.5 g, 3.70 mmol, 1 .0 eq) and potassium hydroxide (various equivalents) were mixed with DMSO (5 ml) at 25-30 °C, the reaction mixture was heated to 160 °C and left to equilibrate for 30 min. (R)- propylene carbonate 5 (0.42 ml, 0.499 g, 4.88 mmol, 1.32 eq) was added in a single portion to the reaction mass and an aliquot was withdrawn every 60 s for 12 min for catalyst loading of 1 .0 eq and every 60 s for 20 min for catalyst loadings of 0.1 eq.
Catalyst loadings analysed
0.10 eq 0.021 g
1.00 eq 0.210 g
Stage 1 using 10 mol% KOH
Stage 1 using 100 mol% KOH
Preparation of 9-[2-(phosphonomethoxy)propyl]Adenine 4
2
Example 1 (Stage 2a using MTB in DMSO)
A 0.5M solution of HPA 6 (1 g, 5.18 mmol, 1.0 eq) in DMSO (10.3ml) was prepared. MTB (2.65 g, 15.53 mmol, 3.0 eq) was added to the solution at ambient temperature. The resulting slurry was heated to 70 °C and DESMP (2.5 g, 7.76 mmol, 1.5 eq) was added dropwise over 10-15 min. The resulting mixture was stirred at 70 °C for 3 h after which time LCMS showed ~ 90% conversion to product. LCMS after 16 h indicated ~ 96% conversion (both regioisomers). LCMS retention time 3.403 min, m/z 344.2 [M +1]+.
Example 2 (Stage 2a using MTB in NMP)
A 0.5M solution of HPA 6 (10 g, 25.9 mmol, 1.0 eq) in NMP (50 ml) was prepared. MTB (26.5 g, 155 mmol, 3.0 eq) was added to the solution at ambient temperature. The resulting slurry was heated to 70 °C and DESMP (25.0 g, 78 mmol, 1.5 eq) was added dropwise over 10-15 min. The resulting mixture was stirred at 70 °C for 16 h after which time the solution was treated with acetic acid to adjust the pH to ~6-7. Ethyl acetate (600 ml) was added drop-wise to the rapidly stirring solution and the magnesium salts were allowed to precipitate out, the salts were removed by filtration through a sintered glass funnel under vacuum. The salt cake was re- extracted into ethyl acetate (300 ml) at 50 °C for 30 min. The mixture was again filtered and the filtrate was combined with that obtained from the initial extractions. The combined organic fractions were concentrated in vacuo to afford a solution of (R -diethyl (((1-(6-amino-9H-purin-9-yl)propan- 2-yl)oxy)methyl)phosphonate in NMP as shown by LCMS. The solution was taken up in ethyl acetate (600 ml) and washed with 3x300 ml 25% brine
solution, the organic phase was then evaporated in vacuo to afford an orange oil. Mass balance indicated that ~60-70% of the product had remained in the aqueous layer. The aqueous layer was heated in a beaker to 80 °C and compressed air was blown over it to remove the water/NMP mixture, the sodium chloride that precipitated was removed by filtration every hour until most of the volume of water/NMP had been removed. Once no more water/NMP appeared to be coming off, the solution was filtered and taken up in ethyl acetate (300 ml), the precipitate was taken up in ethyl acetate (300 ml) and again filtered. The filtrates were combined, dried (Na2S04), filtered and evaporated in vacuo to afford a yellow oil. The yellow oil was triturated with diethyl ether (100 ml) at 0 °C, after ~ 1 h the product precipitated as an off white solid which was then collected by filtration and dried under vacuum affording PPA 2 (2.55 g, 14.4%). LCMS retention time 3.403 min, m/z 344.2 [M +1f.
Example 3 (Stage 2a using PhMgBr in DMSO)
A mixture of HPA 6 (0.5 g, 2.59 mmol, 1.0 eq) in DMSO (2.5 ml) was prepared. Phenyl magnesium bromide (2M, 3.88 ml, 7.76 mmol, 3.0 eq) was added to the solution at ambient temperature. A violent reaction occurred and an off-white precipitate was formed. THF (5 ml) was added to form a slurry. The resulting slurry was heated to 70 °C and DESMP (12.51 g, 38.8 mmol, 1.5 eq) was added dropwise over 10-15 min. The resulting mixture was stirred at 70 °C for 3 h after which time LCMS showed some product formation, the reaction was stirred overnight at 70 °C, the LCMS still showed no further product formation. The reaction was not worked up or purified. LCMS retention time 3.399 min, m/z 344.2 [M +1]+.
Example 4 (Stage 2a Grignard and solvent screen)
A mixture of HPA 6 (0.5 g, 2.59 mmol, 1.0 eq) and terf-butanol (0.384 g, 5.18 mmol, 2.0 eq) in solvent (2.5 ml) was diluted with THF (5 ml). Phenyl magnesium chloride was added to the solution at 0 °C. The slurry was stirred at 0 °C for 30 min, thereafter it was heated to 75 °C and DESMP
(1.251 g, 3.88 mmol, 1.5 eq) was added dropwise. The resulting mixture was stirred at 75 °C for 18.5 h after which time a sample was withdrawn for LCMS analysis.
Quantities of Phenyl magnesium chloride/Solvent added:
DMSO
Example A 1.29 ml, 2.59 mmol, 1.0 eq
Example B 2.59 ml, 5.18 mmol, 2.0 eq
Example C 3.88 ml, 7.76 mmol, 3.0 eq
NMP
Example D 1.29 ml, 2.59 mmol, 1.0 eq
Example E 2.59 ml, 5.18 mmol, 2.0 eq
Example F 3.88 ml, 7.76 mmol, 3.0 eq
Results
The presence of both the product PPA 6 (m/z 344) as well as the half- hydrolyzed product ethyl hydrogen ((( /? 1-(6-amino-9H-purin-9-yl)propan- 2-yl)oxy)methyl)phos-phonate (m/z 316) were observed. The total conversion given is the area % by LCMS corresponding to both products.
Example A
Total conversion: 73.6%. LCMS retention times 2.434 min (m/z 316.1 [M +1]*.) and 3.382 min (m/z 344.1 [M +1]+.).
Example B
Total conversion: 76.2%. LCMS retention time 2.205 min (m/z 316.1 [M +1]+.) and 3.385min (m/z 344.1 [M +1]+.).
Example C
Total conversion: 54.9%. LCMS retention time 2.431 min (m/z 316.1 [ +1f.) and 3.386 (m/z 344.1 [M +1f.).
Example D
Total conversion: 61.8%. LCMS retention time 3.386 min, m/z 344.1 [M +1]+ and 3.386 (m/z 344.1 [M +1f .).
Example E
Total conversion: 57.8%. LCMS retention time 2.422 min, m/z 316.1 [M +1]+and 3.382 (m/z 344.1 [M +1]+.).
Example F
Total conversion: 36.8%. LCMS retention time 2.452 min, m/z 316.1 [M +1]+ .
Example 5 (Stage 2a using optimised Grignard stoichiometry in DMSO)
A mixture of HPA 6 (1 g, 5.18 mmol, 1.0 eq) and ferf-butanol (0.767 g, 0.983 ml, 10.35 mmol, 2.0 eq) in DMSO (10 ml). Phenyl magnesium chloride (2.6 ml, 5.18 mmol, 1.0 eq) was added to the solution at 0 °C. The slurry was stirred at 0 °C for 30 min, thereafter the reaction mixture was warmed to ambient temperature and DESMP (2.50 g, 7.76 mmol, 1.5 eq) was added in one portion and the reaction mixture was heated to 75 °C. The resulting mixture was stirred at 75 °C, and a sample was withdrawn every 30 min for LCMS analysis. The stirring was continued for 210 min, after which time the reaction mixture was cooled to 0 °C. LCMS showed the formation of the desired product (52%) and the half-hydrolysis product (15%). LCMS retention times 3.407 min and 2.560 min respectively
Example 6 (Stage 2a Grignard screen in NMP)
A mixture of HPA 6 (0.5 g, 2.59 mmol, 1.0 eq) and te/t-butanol (0.384 g, 0.491 ml, 5.18 mmol, 2.0 eq) was suspended in NMP (5 ml). Grignard was added to the solution at 0 °C. The slurry was stirred at 0 °C for 30 min, thereafter the reaction mixture was warmed to ambient temperature and DESMP (1.251 g, 3.88 mmol, 1.5 eq) was added in one portion, and the reaction mixture was heated to 75 °C. The resulting mixture was stirred at 75 °C for 16 h.
Quantities of Grignard's used
PhMgBr (3M, 0.863 ml, 2.59 mmol)
Me gBr (1M, 2.59 ml, 2.59 mmol)
MeMgCI (3M, 0.863 ml, 2.59 mmol)
iPrMgCI (2M, 1.294 ml, 2.59 mmol)
2,2,6,6-Tetramethyl-piperidinylmagnesium (1 , 2.59 ml, 2.59 mmol)
LC S traces were obtained after 90 min and 16 h. Average LC S retention times 2.15-2.5 min (m/z 316.1 [M +1 .) and 3.396-3.421 min (m/z 344.1 [M +1f.).
Solvent: NMP
Conditions: HPA (1.0eq), DESMP (1.5 eq), -BuOH (2.0 eq), PhMgCI (1.0 eq), NMP (2 M), 75 °C, T=90 min
Conditions: HPA (1.0eq), DESMP (1.5 eq), -BuOH (2.0 eq), PhMgCI (1.0 eq), NMP (1 M), 75 °C, T= 16 h
*Very messy LCMS trace
# Reaction is clean but there are 2 additional peaks with m/z ions of 463 [M +1]+ and (176/372/491)
Example 7 (Stage 2a time screen)
A mixture of HPA 6 (2 g, 10.35 mmol, 1.0 eq) and tert-butanol (1.535 g, 1.965 ml, 20.70 mmol, 2.0 eq) was suspended in NMP (5.2 ml). Methyl magnesium chloride (3M in THF) (1.97 ml, 20.70 mmol, 1.0 eq) was added to the solution at 0 °C. The slurry was stirred at 0 °C for 30 min, thereafter the reaction mixture was warmed to ambient temperature and DESMP (5.00 g, 15.53 mmol, 1.5 eq) was added in one portion and the reaction mixture was heated to 75 °C. The resulting mixture was stirred at 75 °C, a sample was withdrawn every 15 min for LCMS analysis as part of a time screen. After 150 min a further portion of methyl magnesium chloride (3M in THF) (0.79 ml, 0.4 eq) was added and monitoring was continued. Average LCMS retention times 2.150-2.538 min (m/z 316.1 [M +1]+) and 3.406-3.456 min (m/z 344.1 [M +1f)
The results are given below
Stage 2a screen using 1.5 eq DESMP at 120 °C
Example 8 (Stage2a time screen)
A mixture of HPA 6 (2 g, 10.35 mmol, 1.0 eq) and te/i-butanol (1.535 g, 1.965 ml, 20.70 mmol, 2.0 eq) was suspended in NMP (5.2 ml). Methyl magnesium chloride (3M in THF) (1.97 ml, 20.70 mmol, 1.0 eq) was added to the solution at 0 °C. The slurry was stirred at 0 °C for 30 min, thereafter the reaction mixture was warmed to ambient temperature and DESMP (5.00 g, 15.53 mmol, 1.5 eq) was added in one portion and the reaction mixture was heated 120 °C. The resulting mixture was stirred at 120 °C. The reaction was monitored by LCMS. Average LCMS retention times 2.438-2.463 min (m/z 316.1 [M +1]+) and 3.316-3.513 min (m/z 344.1 [M +1f).
The results are given below
Stage 2a screen using 1.5 eq DESMP at 120 °C
Time Half Unhydrolyzed Total
Hydrolyzed product Conversion product
30 0 4.3 4.3
60 9 46.2 55.2
90 29.5 42.4 71.9
120 35.7 36.5 72.2
150 31.1 36.7 67.8
Example 9 (Stage 2a using 3 eq DESMP @ 75°C)
A mixture of HPA 6(2 g, 10.35 mmol, 1.0 eq) and te/f-butanol (1.535 g, 1.965 ml, 20.70 mmol, 2.0 eq) was suspended in NMP (5.2 ml). Methyl magnesium chloride (3M in THF) (1.97 ml, 20.70 mmol, 1.0 eq) was added to the solution at 0 °C. The slurry was stirred at 0 °C for 30 min, thereafter the reaction mixture was warmed to ambient temperature and DESMP (5.00 g, 15.53 mmol, 1.5 eq) was added in one portion and the reaction mixture was heated to 75 °C. The resulting mixture was stirred at 75 °C for 16 h, a sample was withdrawn every 15 min for LCMS analysis. Average LCMS retention times 2.180-2.594 min (m/z 316.1 [M +1]+) and 3.433-3.476 min (mz 344.1 [M +1]+).
Stage 2a screen using 3.0 eq DESMP at 75 °C
Example 10 (Back screen of t-BuOH when using 1 eq Grignard)
A mixture of HPA 6 (0.5 g, 2.59 mmol, 1.0 eq) and terf-butanol was suspended in NMP (2.5 ml). Phenyl magnesium chloride (1.3 ml, 2.59 mmol, 1.0 eq) was added to the solution at 0 °C. The slurry was stirred at 0 °C for 30 min and DESMP (1.25 g, 3.88 mmol, 1.5 eq) was added in one portion and the reaction mixture was heated to 75 °C. The resulting mixture was stirred at 75 °C. The reaction was monitored by LCMS. Average LCMS retention times 2.302-2.453 min (m/z 316.1) and 3.439-3.444 min (m/z 344.1)
Equivalents of fe/f-butanol
Example A 0.5 eq (0.123 ml)
Example B 1.0 eq (0.246 ml)
Example C 1.5 eq (0.368 ml)
Example D 2.0 eq (0.491 ml)
Example E 2.5 eq (0.614 ml)
Example F 3.0 eq (0.737 ml)
Results when using 1.0 eq ferf-butanol
Example 11 (Stage 2a using 3 eq of DESMP @ 120 °C)
A mixture of HPA 6 (1 g, 5.18 mmol, 1.0 eq) and ferf-butanol (0.767 g, 0.983 ml, 10.35 mmol, 2.0 eq) in NMP (2.6 ml). Methyl magnesium chloride (3M in THF) (1.72 ml, 20.70 mmol, 1.0 eq) was added to the solution at 0 °C. The slurry was stirred at 0 °C for 30 min, thereafter the reaction mixture was warmed to ambient temperature and DESMP (5.00 g, 15.53 mmol, 3.0 eq) was added in one portion and the reaction mixture was heated 120 °C. The resulting mixture was stirred at 120 °C, a sample was withdrawn every 15 min for LCMS analysis. Average LCMS retention times 2.462-2.485 min (m/z 316.1) and 3.420-3.498 min (m/z 344.1)
Time (min) Half Hydrolyzed product Unhydrolyzed product Total
Conversion
0 0 0 0
15 0 4.4 4.4
30 2.6 64.1 66.7
45 6.4 69.8 76.2
75 6.5 71.1 77.6
105 6.9 58.7 65.6
Example 12 (Alcohol Screen)
A mixture of HPA 6 (0.5 g, 2.59 mmol, 1.0 eq) and an alcohol (1eq) was suspended in NMP (2.5 ml). Phenyl magnesium chloride (1.3 ml, 2.59 mmol, 1.0 eq) was added to the solution at 0 °C. The slurry was stirred at 0 °C for 30 min and DES P (1.25 g, 3.88 mmol, 1.5 eq) was added in one portion and the reaction mixture was heated to 75 °C. The resulting mixture was stirred at 75 °C. The reaction was monitored by LCMS. Average LCMS retention times 2.375-2.533 min (m/z 316.1) and 3.426-3.449 min (m/z 344.1)
Alcohols screened
Example A MeOH(0.105 ml)
Example B EtOH (0.151 ml)
Example C /so-PrOH (0.198 ml)
Example D 2-BuOH (0.238 ml)
Example E Cyclopentanol (0.235 ml)
Example F Octanol (0.409 ml)
Example G Cyclohexanol (0.269 ml)
Example H ie -BuOH (0.246 ml)
Alcohol Screened Half Hydrolyzed Unhydrolyzed Total Conversion product product
MeOH 2.7 9.8 12.5
EtOH 26.2 16.7 42.9
/'so-PrOH 24.8 7.2 32
2-BuOH 28.3 13.2 41.5
Cyclopentanol 21.9 18.1 40
Octanol 23.3 9.5 32.8
Cyclohexanol 25.6 12.3 37.9 tert-BuOH 43.5 23.1 66.6
Example 13 (DESMP stoichiometry screen)
A mixture of HPA 6 (0.5 g, 2.59 mmol, 1.0 eq) and ferf-butanol (0.192 g, 0.246 ml, 2.59 mmol, 2.0 eq) was suspended in NMP (2.6 ml). Methyl magnesium chloride (3M in THF) (0.86 ml, 2.59 mmol, 1.0 eq) was added to the solution at 0 °C. The slurry was stirred at 0 °C for 30 min, thereafter the reaction mixture was warmed to ambient temperature and DESMP was added in one portion and the reaction mixture was heated 120 °C. The resulting mixture was stirred at 120 °C, a sample was withdrawn every 15 min for LCMS analysis. Average LCMS retention times 2.363-2.575 min (m/z 316.1) and 3.466-3.488 min (m/z 344.1)
DESMP stoichiometry screen
90 min
DESMP HPA Total Conversion
equivalents
1.5 35 59
1.7 25.2 68.6
2 18 75.4
2.3 11.7 82.3
2.6 7.4 85.9
150 min
DESMP HPA Total Conversion
equivalents
1.5 35 59
1.7 21 70.5
2 16.4 79
2.3 11.2 85.1
2.6 6.8 86.2
16 h
Example 14 (Stage 2a Time Screen)
A mixture of HPA 6 (4.68 g, 24.22 mmol, 1.0 eq) and terf-butanol (1.795 g, 2.30 ml, 24.22 mmol, 2.0 eq) was suspended in NMP (12.1 ml). Methyl magnesium chloride (3M in THF) (8.1 ml, 24.22 mmol, 1.0 eq) was added to the solution at 0 °C. The slurry was stirred at 0 °C for 30 min, thereafter the reaction mixture was warmed to ambient temperature and DESMP (17.96 g, 14.31 ml, 55.7 mmol, 2.3 eq) was added in one portion and the reaction mixture was heated to 75 °C, a sample was withdrawn every 30 min for LCMS analysis. Average LCMS retention times 2.305-2.607 min (m/z 316.1) and 3.449-3.490 min (m/z 344.1)
Example 15 (Stage 2a Grignard Stoichiometry Screen 0.75eq)
A mixture of HPA 6 (1 g, 5.18 mmol, 1.0 eq) and terf-butanol (0.288 g, 0.37 ml, 3.88 mmol, 0.75 eq) was suspended in NMP (2.6 ml). Methyl magnesium chloride (3M in THF) (1.29 ml, 3.88 mmol, 0.75 eq) was added to the solution at 0 °C. The slurry was stirred at 0 °C for 30 min, thereafter the reaction mixture was warmed to ambient temperature and DESMP (3.84 g, 3.06 ml, 11.09 mmol, 2.3 eq) was added in one portion and the reaction mixture was heated to 75 °C, a sample was withdrawn every 30 min for LCMS analysis. Average LCMS retention times 2.676-2.696 min (m/z 316.1) and 3.533-3.537 min (m/z 344.1)
Example 16 (Stage 2a Grignard Stoichiometry Screen 0.5eq)
A mixture of HPA 6 (1 g, 5.18 mmol, 1.0 eq) and te/f-butanol (0.192 g, 0.246 ml, 2.59 mmol, 0.75 eq) was suspended in NMP (2.6 ml). Methyl magnesium chloride (3M in THF) (0.86 ml, 2.59 mmol, 0.5 eq) was added to the solution at 0 °C. The slurry was stirred at 0 °C for 30 min, thereafter the reaction mixture was warmed to ambient temperature and DESMP (3.84 g, 3.06 ml, 11.09 mmol, 2.3 eq) was added in one portion and the reaction mixture was heated 75 °C, a sample was withdrawn every 30 min for LCMS analysis. Average LCMS retention times 2.656-2.7 1 min (m/z 316.1) and 3.528-3.537 min (m/z 344.1)
Time SM Half Hydrolyzed Product Total Conversion Impurities
(HPA) product
0 100 0 0 0 0.0
30 96.1 0 2.6 2.6 1.3
60 60.5 1.5 33 34.5 5.0
90 64.7 1.2 34.1 35.3 0.0
120 61.65 2.3 36.1 38.4 0.0
Example 17 (Stage 2a order of reagent addition screen)
A mixture of HPA 6 (1 g, 5.18 mmol) in NMP (2.6 ml) was charged with tert- butanol (0.384 g, 0.491 ml, 5.18 mmol, 1.0 eq) and DESMP (4.17 g, 12.94 mmol, 2.5 eq). Phenyl magnesium chloride (13 ml, 25.9 mmol, 1.0 eq) was added to the solution at 0 °C and the reaction mixture was heated to 75 °C. The resulting mixture was stirred at 75 °C, and monitored by LCMS every 30 min for the first hour and thereafter every hour for 3h. LCMS indicates ~80% total conversion to product. Average LCMS retention times 2.723- 2.782 min (m/z 316.1) and 3.547-3.554 min (m/z 344.1)
Example 18 (Stage 2a order of reagent addition screen)
A mixture of HPA 6 (1 g, 5.18 mmol) in NMP (2.6 ml) was charged with DESMP (4.17 g, 12.94 mmol, 2.5 eq). Phenyl magnesium chloride (13 ml, 25.9 mmol, 1.0 eq) was pre-mixed ferf-butanol (0.384 g, 0.491 ml, 5.18 mmol, 1.0 eq) and stirred for 30 min prior to being added to the solution of HPA/DESMP at 0 °C and the reaction mixture was heated to 75 °C. The resulting mixture was stirred at 75 °C, and monitored by LCMS. LCMS indicated PPA 71.6% and HPA 20.4%. Average LCMS retention times 2.687-2.794 min (m/z 316.1) and 3.552-3.555 min (m/z 344.1)
Example 19 (Stage 2a solvent free screen)
HPA 6 (1 g, 5.18 mmol) in ferf-BuOH (0.384 g, 0.491 ml, 5.18 mmol, 1.0 eq) was charged with DESMP (4.17 g, 12.94 mmol, 2.5 eq). Phenyl magnesium chloride (2.6 ml, 5.18 mmol, 1.0 eq) was added dropwise at 0
°C and the reaction mixture was heated 75 °C. The resulting mixture was stirred at 75 °C, and monitored by LCMS every 30 min. LCMS indicated a 77.4% total conversion to PPA 2 and half-hydrolyzed PPA 2. The reaction mixture was cooled to ambient temperature and allowed to stand overnight. The reaction mixture formed a solid "glass" on standing. The "glass" was taken up in water (100 ml) and extracted with ethyl acetate (2 x 100 ml). The aqueous extract showed both un-reacted HPA 6 and PPA 2 but no unreacted DESMP. The organic extracts showed un-reacted HPA 6, PPA 2 and un-reacted DESMP. The organic fractions were combined, dried (Na2S04), filtered and evaporated in vacuo to afford a yellow oil. The aqueous fraction was again extracted with dichloromethane (2 x 100 ml), the organic fraction were combined, dried (Na2S04), filtered and evaporated in vacuo to afford a white solid. The original organic fraction and aqueous fraction left over from the dichloromethane extraction were combined and extracted with /'so-propyl acetate (2 x 50 ml). The organic fractions were combined, dried (Na2S04), filtered and evaporated in vacuo to afford a yellow oil. LCMS indicates a mixture of both unreacted HPA 6 and PPA 2 in both the aqueous and organic layers, in addition the organic fraction contained un-reacted DESMP. The aqueous phase and organic residue obtained were combined and extracted with dichloromethane (2 x 100 ml), the organic phases were combined dried (Na2S04), filtered and evaporated in vacuo to afford a yellow oil. LCMS indicated only PPA 2 and un-reacted DESMP.
The organic extracts from both dichloromethane extractions were combined and taken up in water (100 ml), the water layer was acidified to pH 2 using cone HCI and extracted with ethyl acetate (3 x 100 ml), the aqueous layer was neutralised using 1 M NaOH (aq) and re-extracted with dichloromethane (3 x 100 ml). The ethyl acetate extractions were dried (Na2S04), filtered and evaporated in vacuo to afford a yellow oil LCMS indicated un-reacted DESMP and traces of PPA 2. The dichloromethane extraction were combined, dried (Na2S0 ), filtered and evaporated in vacuo to afford a yellow oil, LCMS indicated only PPA 2 with no unreacted
DESMP present. Average LCMS retention times 2.647-2.763 min (m/z 316.1) and 3.547-3.563 min (m/z 344.1)
Example 20 (Stage 2a Phenyl magnesium chloride backscreen against optimised conditions for t-BuOH and DESMP)
A mixture of HPA 6 (0.5 g, 2.59 mmol) in NMP (1.3 ml) and t-BuOH (0.192 g, 0.246 ml, 2:59 mmol, 1.0 eq) was charged with DESMP (2.09 g, 6.47 mmol, 2.5 eq). Phenyl magnesium chloride (1.3 ml, 2.59 mmol, 1.0 eq) was added drop-wise at 0 °C and the reaction mixture was heated to 75 °C. The resulting mixture was stirred at 75 °C. The reaction was monitored by LCMS every 60 min for 2 h, and thereafter after 16 h. LCMS indicated PPA 2 (63%) and HPA 6 (30%). Average LCMS retention times 2.620 min (m/z 316.1) and 3.558-3.559 min (m/z 344.1)
Example 21 (Stage 2a order of reagent addition screen)
A mixture of HPA 6 (1 g, 5.18 mmol) was charged with methyl magnesium chloride (3M in THF) (1.73 ml, 5.18 mmol, 1.0 eq) in terf-BuOH (0.384 g, 0.491 ml, 5.18 mmol, 1.0 eq) which was added dropwise to the solid HPA 6 at 0 °C, the reaction mixture was stirred at 0 °C for 30 min before being charged with DESMP (4.17 g, 12.94 mmol, 2.5 eq). The reaction mixture was heated to 75 °C. The reaction was monitored by LCMS (Purity PPA 2 73.3%, HPA 6 10%). LCMS retention times 2.627 min (m/z 316.1) and 3.523 min (m/z 344.1)
Example 22 (Stage 2a order of reagent addition screen)
A mixture of HPA 6 (1 g, 5.18 mmol) in THF (5.2 ml) and tert-BuOH (0.384 g, 0.491 ml, 5.18 mmol, 1.0 eq) was charged with methyl magnesium chloride (3M in THF) (1.73 ml, 5.18 mmol, 1.0 eq) which was added dropwise at 0 °C, the reaction mixture was stirred at 0 °C for 30 min before being charged with DESMP (4.17 g, 12.94 mmol, 2.5 eq). The reaction mixture was heated to 75 °C. The reaction was monitored by LCMS (Purity
PPA 2 83.6% HPA 6 8.2%). LCMS retention times 2.743 min (m/z 316.1) and 3.566 min (m/z 344.1)
Example 23 (Stage 2a solvent screen)
A mixture of HPA 6 (0.5 g, 2.59 mmol, 1.0 eq) in solvent (5.2 ml, 0.5 M) and te/f-BuOH (0.192 g, 0.246 ml, 2.59 mmol, 1.0 eq) was charged with methyl magnesium chloride" (3M in THF) (0.86 ml, 2.59 mmol, 1.0 eq) which was added drop-wise at 0 °C, the reaction mixture was stirred at 0 °C for 30 min before being charged with DESMP (2.09 g, 1.66 ml, 6.47 mmol, 2.5 eq). The reaction mixture was heated to 75 °C for 16 h. The reaction was monitored by LCMS.
Solvents Screened (PA = Polar aprotic, PP = Polar Protic, NP = Non-Polar)
1. Dichloromethane (PA) 40 °C
2. Acetone (PA) 56 °C
3. THF (PA) 66 °C
4. Ethyl Acetate (PA) 77 °C
5. Acetonitrile (PA) 82 °C
6. Methanol (PP) 65 °C
7. Ethanol (PP) 79 °C
8. Chloroform (NP) 61 °C
9. Cyclohexane (NP) 81 °C
10. Dimethoxyethane (NP) 85 °C
11. Dioxane (NP) 101 °C
12. Toluene (NP) 111 °C
HPA PPA HPA PPA HPA PPA
Solvent (1 h) (1 h) (2 h) (2 h) (16 h) (16 h)
Dioxane 86.3 0 86.3 0 82.9 0
Methanol 89.4 0.3 87.4 0.9 82.9 0.9
Ethanol 81.9 0.2 70.9 2.3 85.4 1.4
Dimethoxyethane 80.8 0.6 91.7 0 87.8 2.8
Toluene 66.8 9.2 44.3 24.6 66.8 9.2
Acetone 81.9 3.5 69.1 14.3 66.4 19.6
Ethyl Acetate 64 5.3 40.9 4.5 12.1 35.1
Chloroform 84.4 2.5 74.1 5.9 32 46
Acetonitrile 79.5 9.4 70 16.4 21.9 53.2
THF 80.4 6.6 75.6 13 26 56.8
Dichloromethane 64.8 22.8 16.9 63.6 2.4 67.3
Cyclohexane 16.5 28.9 39 45.6 0 85.6
Example 24 (Stage 2a solvent concentration screen)
A mixture of HPA 6 (1 g, 5.18 mmol, 1.0 eq) in cyclohexane (5 ml, 1 M) and te/f-BuOH (0.384 g, 0.491 ml, 5.18 mmol, 1.0 eq) was charged with methyl magnesium chloride (3M in THF) (1.73 ml, 5.18 mmol, 1.0 eq) which was added dropwise at 0 °C. The reaction mixture was stirred at 0 °C for 30 min before being charged with DESMP (4.17 g, 3.32 ml, 12.94 mmol, 2.5 eq). The reaction mixture was heated to 75 °C for 16 h. The reaction was monitored by LCMS. High concentration showed faster reaction rates, however formed a solid pellet more quickly. A 0.5M solution seems to be the best compromise along rapid reaction which goes to near completion and still has sufficient solvent to form a stirrable slurry. The 1.0 M solution formed a sticky solid pellet in seconds whereas the 0.25M remains as a slurry but does not proceed to completion.
Solvent concentrations Screened
Exampe A 1.0 (5 ml Cyclohexane)
Example B 0.5 M (10 ml Cyclohexane)
Example C 0.25M (20 ml Cyclohexane)
Example 25 (Best mode with work-up 1 on 10 g scale)
A mixture of HPA 6 (10 g, 51.8 mmol, 1.0 eq) in cyclohexane (100 ml, 0.52 M) and terf-BuOH (3.84 g, 4.91 ml, 51.8 mmol, 1.0 eq) was charged with methyl magnesium chloride (3M in THF) (17.25 ml, 51.8 mmol, 1.0 eq) which was added dropwise at 0 °C. The reaction mixture was stirred at 0 °C for 30 min, the reaction mixture was then charged with DESMP (41.7 g, 33.2 ml, 129 mmol, 2.5 eq) drop-wise. The reaction mixture was heated to 75 °C for 3 h. LCMS indicated ~ 85% conversion.
The solid pellet formed was dissolved in water (250 ml) and placed in a continuous extraction apparatus with chloroform (250 ml) for 24 h. The organic fraction was collected, dried (Na2S04), filtered and evaporated in vacuo to afford a yellow oil. LCMS indicated a mixture of HPA 6 (9.7%), PPA 2 (83.6%) and DESMP.
The organic fraction from the continuous extraction was taken up in dichloromethane (300 ml) and extracted with 2M HCI (aq) (3 x 200 ml), the organic fraction was collected, dried (Na2S04), filtered and evaporated in vacuo to afford a mixture of DESMP and the de-tosylated analogue of DESMP diethyl (hydroxymethyl)phosphonate (~20 g).
The aqueous phase was basified to pH 11 using 25% ammonia solution; the basified solution was extracted with dichloromethane (3 x 600 ml). The combined organic phases were dried (Na2S04), filtered and evaporated in vacuo to afford a yellow oil which solidified to a white solid under vacuum. LCMS indicated the white solid to be PPA 2 (13.5 g, 76%, Purity 94.3%)
The aqueous phase was submitted to LCMS, results indicated the presence of both HPA and PPA.
The aqueous phase was placed in a continuous extractor with dichloromethane and allowed to extract for 24h. The organic phase was isolated and evaporated in vacuo to afford a clear oil. LCMS indicated the presence of both HPA 6 and PPA 2. The organic phase was extracted with 2M HCI (aq) (3 x 200 ml), the aqueous phase was basified to pH 11 using 25% ammonia solution; the basified solution was extracted with dichloromethane (3 x 200 ml). The combined organic phases were dried (Na2S04), filtered and evaporated in vacuo to afford a yellow oil which solidified to a white solid under vacuum. LCMS indicated the white solid to be PPA 2 (1.65 g, 9%, Purity 97%)
The batches were combined affording PPA 2 as a white solid (15.15 g, 85%). LCMS retention times 3.362-3.551 min (m/z 344.1 )
Example 26 (Best mode with work-up 2 on 10 g scale)
A mixture of HPA 6 (10 g, 51.8 mmol, 1.0 eq) in cyclohexane (100 ml, 0.52 M) and fe/t-BuOH (3.84 g, 4.91 ml, 51.8 mmol, 1.0 eq) was charged with methyl magnesium chloride (3M in THF) (17.25 ml, 51.8 mmol, 1.0 eq) which was added dropwise at 0 °C. The reaction mixture was stirred at 0 °C for 30 min, the reaction mixture was then charged with DESMP (41.7 g, 33.2 ml, 129 mmol, 2.5 eq) drop-wise. The reaction mixture was heated to 75 °C for 3 h. LCMS indicated ~ 90% conversion.
The solid pellet formed was dissolved in water (250 ml) and placed in a continuous extraction apparatus with chloroform (250 ml) for 24 h. The organic fraction was collected, dried (Na2S04), filtered and evaporated in vacuo to afford a yellow oil.
The organic fraction from the continuous extraction was taken up in dichloromethane (300 ml) and extracted with 2M HCI (aq) (3 x 200 ml), the
organic fraction was collected, dried (Na2S04), filtered and evaporated in vacuo to afford a mixture of DESMP and the de-tosylated analogue of DESMP diethyl (hydroxymethyl)phosphonate (-20 g).
The aqueous phase was basified to pH 11 using 25% ammonia solution; the basified solution was extracted with dichloromethane (3 x 200 ml) and chloroform (3 x 200 ml). The combined organic phases were dried (Na2S04), filtered and evaporated in vacuo to afford a yellow oil which solidified to a white solid under vacuum. LCMS indicated that the white solid to be PPA 2 (12.8 g, 72%, 95% purity by LCMS). LCMS retention times 3.351 min (m/z 344.1).
Example 27 (Best mode with work-up 58 g scale)
A mixture of HPA 6 (58 g, 300 mmol, 1.0 eq) and te/f-BuOH (22.24 g, 28.5 ml, 300 mmol, 1.0 eq) was suspended in cyclohexane (300 ml, 1 M) in a 2L 3-necked round bottom flask fitted with an overhead stirrer. The suspension was charged with methyl magnesium chloride (100 ml, 300 mmol, 1.0 eq) which was added drop-wise at 0 °C to the rapidly stirring solution. The reaction mixture was stirred at 0 °C for 30 min before being charged with DESMP (242 g, 193 ml, 750 mmol, 2.5 eq) drop-wise. The reaction mixture was heated to 75 °C for 2 h. The resulting residue was dissolved in water (170 ml) and subjected to continuous extraction in chloroform (~2 L) for 24 h. The organic fraction from the continuous extraction was collected, dried (Na2S0 ), filtered and evaporated in vacuo to afford a yellow oil. The residue was re-dissolved in dichloromethane (300 ml) and extracted with 2M HCI (aq) (2 x 150 ml). The aqueous fraction was basified to pH 1 1 with 25% ammonia solution (-50 ml), and extracted with dichloromethane (9 x 100 ml). The combined organic fractions were dried (Na2S0 ) filtered and evaporated in vacuo to afford a yellow oil that solidified to an off-white solid on standing. H1 NMR (400 MHz, CDCI3) 8.34 (3H, s), 7.97 (3H, s), 5.80 (2H,s broad), 4.36 (0.9 H, dd), 4.00-4.18 (5H, m), 3.90-3.94 (1 H, m), 3.55- 3.86 (2H, ddd), 3.67 (0.4 H, d), 1.30 (3H, t), 1.25 (3H, t), 1.24 (3H, d), 1.35- 1.20 (12H, m), P31 NMR (400 MHz, CDCI3), C13 NMR (400 MHz, CDCI3)
155.7, 152.6, 149.8, 141.3, 118.9, 76.13 (d), 63.39, 62.13 (t), 61.72, 47.90, 16.29, 16.16 (t), 16.14 (t), LCMS retention time (3.369 min, 99%).
Preparation of (,i?j-(((1-(6-amino-9H-purin-9-yl)propan-2-yI)oxy)methyl)ph- osphonic acid (PMPA 3)
3
Example 1 (OPRD route)
A solution of PPA 2 (~ 2.59 mmol) in NMP (2.5 ml) was treated with sodium bromide (0.933 g, 9.07 mmol, 3.5 eq). The solution was cooled to 0-5 °C and TMSCI (1.491 g, 1.74 ml, 13.73 mmol, 5.3 eq) was added over ten min, and the reaction was then heated to 75 °C and stirred for 16 h at this temperature. The reaction mixture was cooled to 20-25 °C and diluted with water (5 ml) and washed twice by extraction with ethyl acetate (2 x 5 ml). The aqueous layer was cooled to 5 °C, the pH adjusted to between 2.8 and 3.2 with 40% NaOH (aq) crystallizing the product. The resulting precipitate was stirred at 5 °C for 2 h and the product isolated by filtration. The solids were washed with chilled water (5 °C, 0.75 ml) and dried under vacuum below 65 °C to yield PMPA 3 as a white solid (178.9 mg, 24%).
Example 2 (TMSCI/NaBr Stoichiometry screen)
A solution of PPA 2 (~ 10.35 mmol) in NMP (5.2 ml) was treated with sodium bromide. The solution was cooled to 0-5 °C and TMSCI was added over ten min, and the reaction was then heated to 75 °C and stirred for 2-3 h at this temperature. The reaction was monitored by LCMS. Average LCMS retention times 1.088-1.187 min (m/z 288.0, PMPA 3) and 2.257- 2.699 min (m/z 316.1 , Half-hydrolyzed PPA 2).
Quantities of TMSCI/NaBr added
Example A TMSCI (3.19 ml, 2.73 g, 25.1 mmol, 8.3 eq) + NaBr (1.715 g, 16.67 mmol, 5.5 eq)
Example B TMSCI (3.77 ml, 3.23 g, 29.7 mmol, 9.8 eq) + NaBr (2.036 g, 19.79 mmol, 6.53 eq)
Example A
Example B
Time Hydrolyzed Product Half Hydrolyzed product Unhydrolyzed product
0 0 0 84
60 0 19.06 59.39
90 0 26.13 59.43
120 7.14 34.24 42.16
180 16.06 46.79 21.55
240 22.4 52.14 10.76
300 30.61 49.11 4.72
360 38.85 44.03 2.45
420 45.01 38.42 1.58
480 50.3 33.94 1.2
690 62.62 20.34 0.67
750 65.82 16.82 0.85
810 67.73 15.13 0.53
Example 3 (32% HCI)
Crude PPA 2 (0.694 g) was diluted with NMP (1 ml) and treated with 32% HCI (aq). The solution was heated to 75 °C and stirred at this temperature. The reaction was monitored by LCMS. After 45 min the LCMS showed ~ 60% product 3, 20% half-hydrolyzed product and 17% unreacted HPA 6 from Stage 2a. Further analysis indicated no improvement in conversion. Average LCMS retention times 1.107-i .115 min
Example 4 (TMSCI/NaBr in NMP)
A solution of PPA 2 (25.9 mmol) in NMP (25 ml) was treated with sodium bromide (9.33 g, 91 mmol, 3.5 eq). The mixture was cooled to 0-5 °C before adding trimethylsilyl chloride (14.91 g, 17.55 ml, 137 mmol, 5.3 eq) dropwise over 10 min. The reaction mixture was heated to 75 °C for 16 h at which time LCMS indicated complete consumption of PPA 2. The reaction mixture was cooled to ambient temperature, diluted with water (50 ml) and extracted with ethyl acetate (2 x 50 ml). The aqueous layer was cooled to between 3 and 6 °C and the pH was adjusted to ~ 3.4 using 40% sodium hydroxide (aq), crystallizing the product. The resulting suspension was stirred at 0-6 °C for 2 h, after which time the PMPA 3 (20%) was isolated by filtration through a sintered glass funnel.
Example 5 (33% HBr/Acetic)
PPA 2 (51.8 mmol) was treated with 33% HBr/Acetic Acid (73.0 g, 518 mmol, 52.2 ml, 10 eq). The mixture was heated to 75 °C and stirred for 20 min. The reaction mixture was cooled to ambient temperature and diluted with water (50 ml). The mixture was added dropwise to rapidly stirring ethyl acetate (1 L), resulting in the precipitation of a white solid which was collected by filtration (LCMS indicated PPA 2, HPA-Acetate and some half- hydrolyzed product). The filtrate was concentrated in vacuo to afford a yellow solution the solution was diluted with water and extracted with ethyl acetate (2 x 100 ml), the precipitate was also taken up in water and
extracted with ethyl acetate (2 x 50 ml). The aqueous layers were combined, pH adjusted to 3 using 40% NaOH (aq) resulting in the precipitation of PMPA 3 (25%).
Example 6 (33% HBr/Acetic)
A solution of PPA 2 (3 g, 8.74 mmol, 1.0 eq) was treated with 33% HBr/Acetic Acid (8.4 g, 59.6 mmol, 6 ml, 6.82 eq). The mixture was heated to 75 °C and stirred for 3 h. The reaction mixture was cooled to ambient temperature and diluted with water (20 ml). Ethyl acetate was added to the reaction mixture resulting in the precipitation of a cream solid which was removed by filtration, the pH of the solution was adjusted to ~ pH 3 using 50% NaOH (aq). The solution was cooled to 0 °C and stirred for 1 h during which time a white precipitate of PMPA 3 formed. The precipitate was collected by filtration through a sintered glass funnel affording PMPA 3 as a white solid (1.8 g, 73%). Average LCMS retention time 1.066-1.080 min.
Example 7 (33% HBr/Acetic)
A solution of PPA 2 (0.8 g, 2.33 mmol, 1.0 eq) was treated with 33% HBr/Acetic Acid (6.57 g, 46.6 mmol, 4.7 ml, 20 eq). The mixture was heated to 75 °C and stirred for 20 min. The reaction mixture was cooled to ambient temperature and diluted with water (10 ml).
The mixture was added drop-wise to rapidly stirring ethyl acetate (50 ml), resulting in the precipitation of a white solid which was collected by filtration (LCMS indicated PPA 2, HPA-Ac and some half-hydrolyzed product). The filtrate was concentrated in vacuo to afford a yellow solution. The solution was diluted with water and extracted with ethyl acetate (2 x 50 ml), the precipitate was also taken up in water and extracted with ethyl acetate (2 x 50 ml). The aqueous layers were combined, pH adjusted to 3 using 40% NaOH (aq) resulting in the precipitation of product. The solid was collected by filtration (~20% PMPA 3), the filtrate was concentrated under airflow overnight. An additional quantity of material precipitated (~20% PMPA 3),
this was colected by filtration. The filtrate was allowed to stand for 72 hours and again more material precipitated, this was again collected by filtration through a sintered glass funnel (-22% PMPA 3). Overall PMPA 3 was isolated as a white solid (0.4865 g, 73%).
Example 8 (Sealed tube examples)
A solution of PPA 2 (0.2 g, 1.46 mmol, 1.0 eq) Was hydrolyzed using the conditions tabulated below. The reaction mixtures were heated at 120 °C for 16h in a sealed vessel.
Example A PPA, TMSCI (0.316 g, 0.372 ml, 2.91 mmol, 5 eq), NaBr (0.210 g, 2.039 mmol, 3.5 eq), CH3CN (2 ml)
Example B PPA, TMSCI (0.316 g, 0.372 ml, 2.91 mmol, 5 eq), CH3CN (2 ml)
Example C PPA, TMSCI (0.316 g, 0.372 ml, 2.91 mmol, 5 eq)
Example D PPA, TMSCI (0.316 g, 0.372 ml, 2.91 mmol, 5 eq), NaBr (0.210 g, 2.039 mmol, 3.5 eq), CMIC (0.444 g, 2.91 mmol, 5 eq), CH3CN (2 ml)
Example E PPA, TMSCI (0.316 g, 0.372 ml, 2.91 mmol, 5 eq), CMIC (0.444 g, 2.91 mmol, 5 eq), CH3CN (2 ml)
Example F PPA, CMIC (0.444 g, 2.91 mmol, 5 eq), CH3CN (2 ml) Example G PPA, CMIC (0.444 g, 2.91 mmol, 5 eq)
LCMS of examples A and C showed complete conversion to the desired PPA. Example B showed incomplete conversion, however the vessel did not seal completely and the low conversion is no doubt due to the rapid loss of TMSCI. The remaining examples showed none of the desired alkylated product.
Abbreviations
HIV Human immunodeficiency virus
AIDS Acquired immune deficiency syndrome
RT Reverse transcriptase
NRTIs nucleoside reverse transcriptase inhibitors
NtRTIs nucleotide reverse transcriptase inhibitors
NNRTIs non-nucleoside reverse transcriptase inhibitors
HAART Highly Active Anti-Retroviral Therapy
TDF Tenofovir disoproxil fumarate
PPA (R)-9-[2-(Diethylphosphonomethoxy)propyl]adenine
P PA ('R -9-[2-(Phosphonometh-oxy)propyl]adenine
XRPD X-Ray Powder Diffraction
DSC Differential scanning calorimetry
IR Infra Red
TD Tenofovir
DMF Dimethyl Formamide
TMSBr Bromo trimethylsilane
TLR7 Toll-like receptor 7
MTB Magnesium fe/f-butoxide
I PA /so-Propyl acetate
RNA Ribonucleic acid
TMSCI Chloro trimethylsilane
CMIC Chloromethyl-/so-propyl carbonate
DESMP Diethyl P-Toluenesulfonyloxy Methylphosponate
HPMPDAP 9-(S)-[3-Hydroxy-2-(phosphonomethoxy)propyl]-2,6- diaminopurine
HPMPA 9-(S)-(3-Hydroxy-2-phosphonoylmethoxypropyl)adenine
HPA ( )-9-(2-Hydroxypropyl)adenine
HBV Hepatitis B virus
DMSO Dimethylsulfoxide
NMP /V-methylpyrollidone
NMR Nuclear Magnetic Resonance
Liquid Chromatography Mass Spectrum
Tetrahydrofuran
Mass Spectrum