WO1997014703A1

WO1997014703A1 - Synthesis of polymerisable phosphodiesters

Info

Publication number: WO1997014703A1
Application number: PCT/GB1996/002540
Authority: WO
Inventors: Michael John Driver; Jeremy Colin Russel; Judith Elizabeth Browne; Peter G. Sammes
Original assignee: Biocompatibles Limited
Priority date: 1995-10-16
Filing date: 1996-10-16
Publication date: 1997-04-24
Also published as: EP0874857A1; AU7312196A; JPH11513681A; CN1202899A; CA2233161A1; GB9521234D0; KR19990064281A

Abstract

A mono- or di-functional phosphoramidite phosphitylating agent is used to phosphitylate an ethylenically unsaturated alcohol. The product may be oxidised to form the corresponding phosphate ester which may be reacted in further steps to form phosphoryl choline derivatives. The process is of value in the synthesis of 2-(Methacryloyloxyethyl)-2'-(trimethylammoniumethyl)phosphate, inner salt. It has the advantage over prior art processes in that the starting materials and intermediates are more stable and consequently easier to handle.

Description

Biocompatibles Limited

Synthesis of Polymerisable Phosphodiesters

The present invention relates to a process for synthesising polymerisable ethylenically unsaturated phosphodiester compounds using phosphite reactants.

Phospholipids are phosphate diester compounds, in which one of the alcohol residues is generally a glycerol derivative, and the other is a derivative of a different alcohol which may include a nonionic, cationic or, rarely, anionic functionality. Phospholipids and phospholipid analogues are of increasing interest, for instance to allow mimicking of cell membrane surfaces to provide useful properties in respect of biocompatibility, he ocompatibility, and to influence the reactions of various biomolecules with surfaces. Our previous publications, such as EP-A-0032622, EP-A-0157469, EP-A-

-0555295, EP-A-0601041, EP-A-0593561, EP-A-0639989, WO-A-

9416748 and O-A-9416749 describe various synthetic phospholipid analogues and their use to produce devices having biocompatible and hemocompatible surfaces. These specifications in particular describe zwitterionic phospholipid analogues, especially phosphoryl choline derivatives. It is desirable to produce these phospholipid analogues using wholly synthetic methods.

Thuong and Chabrier, Bull. Soc. Chim. de France 1974 (3 to 4) 667 to 670 and in FR-A-2270887 describe the synthesises of phosphoryl choline derivatives in which 2- chloro-2-oxo-l, 3,2-dioxaphospholane (CCP) is firstly reacted with an alcohol and is subsequently ring opened using trimethylamine in organic solvent to form the zwitterionic diester compound. The phospholane compound was produced by oxidising the corresponding 2-chloro-l,3,2- dioxaphospholane compound as described by Edmundson in Chem. and Ind. (1962) 1828 to 1829. The starting phospholane material is in turn formed by reaction of ethylene glycol with phosphorus trichloride. Analogous processes have been used to form polymerisable phospholipids analogues as described first by

Nakaya et al in JP-A-58-154591 (1983). Subsequently the process described by Nakaya has been improved as described in our copending applications numbers WO-A-9514701 and

WO-A-9514702. All these publications describe the synthesis of 2 - (methacryloyloxyethyl ) -2 ' - (trimethylammonium) ethyl phosphate inner salt (HEMA-PC) . This compound can subsequently be polymerised or copolymerised with other copolymerisable ethylenically unsaturated monomers to form polymers.

HEMA-PC itself was first described by Nakabayashi et al in JP-A-54-063025 (1979). In that publication the synthesis involved the reaction of 2-hydroxy ethylmethacrylate with 2-bromoethylphosphoryl dichloride, followed by amination of the bromine substituent of the phosphodiester. Although the process described by Nakaya

* was an improvement in the original synthesis of Nakabayashi et al, there are still difficulties with the synthetic process. In particular the halo phospholane intermediate, CCP, is very unstable. Furthermore as a multi-step process, optimum yields are obtained with different solvents in sequential steps, thereby requiring removal of solvent between the steps. Furthermore the reaction can be slow and produce a relatively low yield of product.

Phosphoramidite chemistry has been developed in the recent past for the synthesis of oligonucleutides. These synthesis are generally carried out on an insoluble support to which a nucieoside is covalently bound. Nucleotides are sequentially added using nucieoside phosphoramidites as relatively stable monofunctional phosphitylating agent synthons, as described first by Beaucage and Caruthers, Tet. Letts. 22, 1859 (1981) and McBride and Caruthers Tet. Letts. 24, 245 (1983). Originally the intermediate was made by reacting chloro-N,N-dimethylaminomethoxyphosphine with an appropriately protected nucieoside. Subsequently different alkyl groups were used in place of the methyl substituents on the amine nitrogen atom. The N,N- dimethylamino group was replaced by N,N-diisopropylamino, morpholino, pyrrolidino and 2,2,6,6-tetramethylpiperidino groups and a variety of other groups received by Beaucage et al in Tetrahedron (1992) 48(12), 2223-2311. Also the methoxy protecting group for the phosphorus atom was replaced with improved protecting groups such as beta- cyanoethoxy groups (Sinha et al, Tet. Letts 24, 5843 (1983) ) . Other phosphate protecting groups have been used, as reviewed by Beaucage et al (1992) op cit.

In Chemical Abstracts 72(14), 107309 (1970) and in the full reference Khim.Khim.Telchol. (21WWAB) , 68, 18-25 (1968) Podashova et al describe phosphitylation of hydroxy alkyl methacrylate by chlorophosphites. The product phosphite is polymerised and the polymers are alleged to have very low flammability.

It would be desirable to use, for the synthesis of polymerisable phosphodiester compounds, such as HEMA-PC, starting materials or intermediates which are relatively stable and do not, therefore, have to be synthesised and used immediately. The present inventors have achieved this objective by using phosphine reactants as phosphitylating agents for ethylenically unsaturated alcohols. In a new process which involves an alcoholysis step according to the invention, a compound of the formula I

Z - P - NR¹- I Y in which the groups R¹ are the same or different and each represents branched or straight alkyl groups

(including cycloalkyl) or, together with the nitrogen atom to which they are attached, form a heterocycle optionally containing other heteroatoms and being saturated or unsatuurraatteedd,, aanndd NNRR ₂₂ ooppttiioonnaallllyy bbeeiinngg iinn qquuaalternised form, Y is a phosphate protecting group, and Z is either NR 2₂ i .n which the groups R 2 are selected from the same groups as R and is the same or different to NR ₂ or is group OR in which R is selected from C ₄₀ straight and branched-alkyl groups, -alkenyl groups and - alkynyl groups, or R and Y together with the oxygen atom and phosphorus atom to which they are (respectively) attached, form a 5 to 15 membered heterocyclic ring, optionally containing additional heteroatoms and/or substituents at ring carbon and/or nitrogen atoms (if any) , any of which groups R may be unsubstituted or substituted by aryl groups, hydroxyl groups, halogen atoms, amine (including mono-, di- or tri-alkyl substituted amine) groups, sulphonium or phosphonium groups, is reacted in solution with an alcohol of the formula BR OH, in which R is a straight or branched alkylene, oxoalkylene or oligo-oxaalkylene chain and B is an ethylenically unsaturated polymerisable group selected from

wherein: R is hydrogen or a C,-C₄ alkyl group;

A is -O- or -NR 8- where R8 is hydrogen or a C₁-C₄ alkyl group; and

K is a group -(CH₂)_p0C(0)-, -(CH₂)_pC(0)0-, -(CH₂)_pOC(0)0-, -(CH₂)_pNR⁹-, - (CH₂) _p0C (O) NR⁹- , -(CH₂)_NR⁹C(0)NR⁹-, (in which the groups R⁹ are the same or different), -(CH₂)_pO-, -(CH₂)_pS0₃ -, or a valence bond and p is from 1 to 12 and R 9 is hydrogen or a C_|-C₄ alkyl group to form a compound of the formula II

Z - P - OR⁷B II

in which Z, ϊ, R and B represent the same groups as in the compound of the formula I and the alcohol. In one embodiment of the process Z is OR . In this embodiment, although R³ may be an alkyl, alkenyl or alkynyl group, for instance a C ₆ - alkyl group, preferably R and Y are joined together, thereby forming a heterocyclic ring with the phosphorus and oxygen atoms. Preferably they represent a C₂_₁₂, preferably C₂.₄ - alkylene chain, most preferably (CH₂)₂.

In another embodiment, where Z is a group NR₂, after the first phosphitylating step i) , the compound of the formula II is reacted in a second step, ii) , in solution in the presence of a weak acid with a second alcohol R OH, in which R' is selected from BR (as defined above) and C.,.₄₀ straight or branched -alkyl groups, -alkenyl groups and - alkynyl groups, any of which may be unsubstituted or substituted by aryl groups, halogen atoms, hydroxyl groups, amine (including mono-, di- or tri-alkyl substituted amine) groups, sulphonium or phosphonium groups, to form a compound of the formula III

R⁴0 - P - OR⁷B I III

Y in which R 4, Y, R7 and B are as in the respecti.ve starti.ng materials.

The process may involve the further iii) step of oxidising the compound of the formula III or of the formula II in which Z is OR to form the corresponding phosphate of the general formula IV

in which Y, R and B represent the same groups as in the starting phosphite triester material and R is either R or R⁴.

An oxidation step of the process of the invention is generally followed by the following step: iv) the compound of the formula IV is deblocked so that the substituent Y is replaced by O^* or OH. In the alcohol used in the first phosphitylating step,

BR OH which is an acrylic type ethylenically unsaturated group, preferably R is hydrogen, methyl, or ethyl, more preferably methyl, so that the alcohol BR OH is an acrylic acid, methacrylic acid or ethacrylic acid derivative.

In used alcohol BR OH which is a styrene derivative, where K is a group (i.e. other than a valence bond) then preferably p is from 1 to 6, more preferably 1,2 or 3 and most preferably p is 1. When K is a group -(CH₂)_pNR⁹-, 0(CH₂)_pNRC(0)-, -(CH₂)_pC(0)NR⁹, -(CH₂)_pNR⁹C(0)0-,

(CH₂)_pOC(0)NR⁹- or 0(CH₂)_pNRC(0)NR⁹- then R⁹ is preferably hydrogen, methyl or ethyl, more preferably hydrogen. In such compounds preferably the vinyl group is para to the group -K-R OH. Preferably R is: an alkylene group of formula -(CR¹ ₂)_a-, wherein the groups -(CR ₂)- are the same or different, and in each

-group -(CR -)- the groups R are the same or different and each group R is hydrogen, fluorine or C^ alkyl or fluoroalkyl, preferably hydrogen, and a is from 1 to 12, preferably 1 to 6; an oxaalkylene group such as alkoxyalkyl having 1 to 6 carbon atoms in each alkyl moiety, more preferably - CH₂0(CH₂)₄-; or an oligo-oxaalkylene group of formula

-[(CR ₂)_b0]_c(CR ₂)_b- where the groups -(CR ₂) - are the same or different and in each group -(CR¹¹ ₂)- the groups R are the same or different and each group R is hydrogen or C,_₄ alkyl, preferably hydrogen, and b is from 1 to 6, preferably 2 or 3 and c is from 2 to 11, preferably 2 to 5. Preferred groups R include alkylene, oxaalkylene and oligo-oxaalkylene groups of up to 12 carbon atoms preferably (CH₂)₂.₆.

Most preferably the first alcohol is a hydroxy alkyl methacrylate compound, preferably hydroxyethyl methacrylate. Where the group Z of the compound of the formula I is a group NR₂, that is where the compound of the formula I is a difunctional phosphitylating agent and where the product compound is intended to contain one ethylenically unsaturated moiety, then in step 1 the process is such that only one of the groups NR₂ and NR² ₂ is replaced by the alcohol moiety. Where the groups NR¹ ₂ and NR² ₂ are different, this is achieved by using reaction conditions such that only one of the groups will react. Any alcoholysis step is conveniently carried out in the presence of a weak acid.

Where the groups NR¹ ₂ and NR² ₂ are the same, selective activation, for instance of bisphosphoramidite compounds of the formula I is achieved by appropriate selection of weak acid activating agents for the group NR¹ ₂ and NR² ₂ in the presence of a suitable solvent for the reaction step. For instance an acid that is capable of activating the first - group NR ₂ or NR ₂ should be used for the first step, whilst a different acid can be used for the activation of the second group NR ₂ or NR ₂ in the second step.

The use of a difunctional phosphitylating agent to produce zwitterionic phosphate, diesters is believed to be novel. According to a second aspect of the invention there is provided a process comprising the steps of v) a compound of the general formula V

R¹,N - P - NR²,

I V

Y in which the groups R and R² are the same or different and each represents branched or straight alkyl groups

(including cycloalkyl) or the R¹ groups and/or the R² groups and the nitrogen to which the respective groups are attached form a heterocycle optionally containing other heteroatoms and being either saturated or unsaturated, and in which the groups NR ₂ and NR²- may be in the quaternised form and

Y is a phosphate protecting group, is reacted in solution with a first alcohol R³ OH in which R³ is selected from C,_₄₀ straight or branched -alkyl groups, -alkenyl groups and -alkynyl groups, any of which may be unsubstituted or substituted by aryl groups, hydroxyl groups, halogen atoms, amine (including mono-, di- or tri¬ alkyl substituted amine) groups, sulphonium or phosphonium groups, in the substantial absence of water to form a compound of the general formula VI

R³θ - P - N

VI Y in which R , R and Y have the same meanings as in the compound of the formula V and the first alcohol vi) the compound of the formula VI is then reacted with a second alcohol R 4 OH, in which R4 is selected from Cι-₄o straight or branched -alkyl groups, -alkenyl groups and alkynyl groups, any of which may be unsubstituted or substituted by aryl groups, halogen atoms, hydroxyl groups amine (including mono-, di- or tri-alkyl substituted amine) groups, sulphonium or phosphonium groups, provided that at least one of R 3 and R4 represents a C,_₁₀-alkyl group having a halogen, sulphonium, amino or phosphonium substituent, in the presence of a weak acid and in the substantial absence of water to form a compound of the formula VII

R³0 - P - OR⁴ | VII

Y in which R3, R4 and Y have the same meani.ngs as i.n the compound of the formula VI and the second alcohol. vii) a compound of the formula VII is oxidised to form a compound of the formula VIII

O

R ₃O - P II - OR

| VIII Y in which R 3, R4 and Y have the same meanings as in the compound of the formula VII viii) the compound of the formula VIII is deblocked so that the substituent Y is replaced by O^* or OH, and if necessary, a cationic substituent is introduced into one of the groups R or R to form the product compound which is a zwitterionic phosphate diester.

In this aspect the introduction of a cationic group is necessary where neither of groups R 4 or R3 contains a sulphonium, amino (including an ammonio group) or phosophonium group, but does contain a halogen substituent. The reaction is generally the amination of such a halo¬ alkyl compound using a tertiary amine, so as to provide a quaternary ammonium group. Where it is necessary to react the product of step vii) so as to introduce a cationic group, it is often possible to combine the deblocking step (step viii) and the step of introducing a cationic group. For instance where one of R 3 and R4 i.s a halogen substituted alkyl group, this can be aminated to replace the halogen atom by a quaternary ammonium group using a tri lower alkylamine, for instance the same tri lower alkylamine used as the base in the

-deblocking step. The same solvents are suitable.

This aspect of the invention is of particular value for producing phosphonium derivatives and in one particularly preferred embodiment of the second aspect of this invention the cationic group is a phosphonium group.

It is often convenient for this group to be a substituent on one of the alcohols R 3OH or R4OH. In such an embodiment, there is no requirement for the introduction of a cationic substituent in step viii) .

This aspect is also of value for synthesising phospholipid analogues, that is glyceryl derivatives. In this aspect at least one of R OH and R⁴OH is a disubstituted glycerol preferably an α,_/.-disubstituted glycerol, such as an α,jS-diacyl, α,/J-dialkyl, α,/3-dialkenyl or α,j8-dialkynyl glycerol. The acyl, alkyl, alkenyl or alkynyl groups are, usually, 12-24 carbon atoms in length, preferably 12 to 18 carbon atoms in length and may be different or, preferably, the same as one another.

In the processes of the present invention the groups R and R represent any of those groups which have been used in phosphoramidite based nucleic acid synthesis processes. Although the groups may all be methyl or other lower alkyl, it is preferred for the groups each to represent alkyl having at least 3 carbon atoms, usually branched alkyl or for the groups R and the nitrogen to which it is attached (and/or R and the nitrogen to which they are attached) to represent saturated or unsaturated heterocyclic groups. The groups R or R may thus represent a straight or branched C₂.₁₂ -alkylene group for instance a (CH₂)₂.₁₂ group, or an alkylene group interrupted by a heteroatom, such as an oxygen, sulphur or N- lower alkyl nitrogen atom. Preferred examples of the groups R^N and R² ₂N are N,N- diisopropylamino, morpholino, pyrrolidino, triazolyl, tetrazolyl, imidazole, and 2,2,6,6-tetramethylpiperidino. In preferred quaternised derivatives of the phosphitylating agent the quaternising group may be the same as or different from the group R or R . Examples of

^•quaternised derivatives of groups NR ₂ and NR ₂ are N,N,N- trimethylammonium and N,N,N-triisopropyl ammonium group. Preferred unsaturated heterocyclic groups NR ₂ or NR ₂ are pyridinium and 2,6-dimethylpyridinium.

Any of the weak acids used in phosphate diester synthesis can be used in the or each phosphitylating step, for instance lH-tetrazole, 1,2,4-triazole, 3-chlorotriazole or diisopropylammonium tetrazolide or 4,5- dichloroimidazole.

Surprisingly we have found that other weak acids which are cheaper and more readily available than those conventionally used in phosphoramidite phosphitylation reactions can be used successfully. Such weak acids are, for instance, organic acids, preferably carboxylic acids and may be mono- or di-basic.

It is believed that the use of such acids in a phosphoramidite phosphitylation reactions is novel. According to a further aspect of the present invention there is provided a process in which a compound of the formula Z - P - NR¹ ₂ I

in which the groups R are the same or different and each represents branched or straight alkyl groups (including cycloalkyl) or, together with the nitrogen atom to which they are attached, form a heterocycle optionally containing other heteroatoms and being saturated or unsaturated, and NR ₂ optionally being in quaternised form,

Y is a phosphate protecting group, and

Z is either NR ₂ in which the groups R are selected from the same groups as R and is the same or different to

NR ₂ or is group OR in which R is selected from C,_₄₀ straight and branched-alkyl groups, -alkenyl groups and - alkynyl groups, or R and Y together with the oxygen atom and phosphorus atom to which they are (respectively) attached, form a 5 to 15 membered heterocyclic ring, optionally containing additional heteroatoms and/or substituents at ring carbon and/or nitrogen atoms (if any) , any of which groups R may be unsubstituted or substituted by aryl groups, hydroxyl groups, halogen atoms, amine (including mono-, di- or tri-alkyl substituted amine) groups, sulphonium or phosphonium groups, is reacted in solution with an alcohol of the formula RH in which R is selected from C,.₄₀ straight or branched - alkyl groups, -alkenyl groups and -alkynyl groups, any of which may be unsubstituted or substituted by aryl groups, hydroxyl groups, halogen atoms, amine (including mono-, di- or tri-alkyl substituted amine) groups, sulphonium or phosphonium groups, in a substantially water-free organic solvent and in the presence of a weak acid and amine HNR ₂ is removed from the reaction mixture, characterised in that the weak acid is a carboxylic acid which is soluble in the organic solvent and which forms a salt with the amine HNR ₂ which is insoluble in the organic solvent and the precipitated salt is removed from the liquid product mixture. The weak carboxylic acid may be, for instance, a mono basic acid such as benzoic acid, or, preferably, dibasic alkanedioic acid or aromatic diacid such as glutaric acid, phthalic acid, adipic acid or cyanuric acid. In an oxidation step, step iii) and step vii) , conventional oxidising systems and agents may be used. Provided that the substituent groups do not include water sensitive groups, it may be suitable to carry out the oxidation using aqueous iodine with 2,6-lutidine or pyridine catalyst. Alternatively organic oxidising agents such as peroxides, such as tert-butylhydroperoxide, di-te_τt butylhydroperoxide, bis-trimethylsilylhydroperoxide, perbenzoic acid derivatives such as meta-chloroperbenzoic acid or iodobenzene diacetate may be used. Alternatively ozone or molecular oxygen in benzene or other dry organic solvent optionally with free radical generators, or dinitrogen tetroxide or other nitrogen oxide oxidising - agents (such as tertiary amine oxides) may be used.

Preferably an amine oxide oxidising agent is used especially where the alcohol which is phosphitylated is BR OH since such oxidising agents give relatively good yields of desired product without by-product formation, especially without formation of by-products of side reactions where the ethylenic bond in a group B is attacked.

Suitable solvents for steps i) , v) and vi) are tetrahydrofuran, l-methyl-2-pyrrolidinone, chloroform, or, preferably dichloromethane or acetonitrile. Since the phosphite compounds are often water sensitive, it is desirable for the reaction mixtures for steps i) and ii) to be carried out in the substantial absence of water. It may be necessary to dry the reagents and solvents and/or to carry out the reaction under dry inert gas.

Y is a protecting group, preferably one which, where the process involves subsequent steps, can subsequently be removed from the product of an oxidation step iii) and step vii) , by the use of base. It is usually R⁵0- in which R⁵ is a C,_₆ alkyl group or an aryl group, preferably substituted with an electron withdrawing group, such as halogen atoms, nitro groups, sulphonyl groups, and aryl (including heteroaryl) groups. Substituents in R may additionally provide steric hindrance (for instance 1,1- dialkyl substituents on an alkyl group) . Although R⁵ may represent a methyl group, it preferably represents a lower alkyl group with an electron withdrawing substituent such as an aryl group, a cyano group, a vinyl group. Preferred groups R are allyl, benzyl, phenyl, phenylsulphoethyl, methyl-sulphoethyl, para-nitrophenylethyl, 2,2,2- trihaloethyl (especially trichloro-and tribromo-ethyl) , 2,2, 2-trihalo-l, 1,-dialkylethyl (especially 2,2,2,- trichloro-1, 1-dimethylethyl, 2'-and 4'-pyridylethyl, m- methylbenzyl, p- or m-halobenzyl, m,p-dichlorobenzyl, pentahalophenyl (especially pentafluoro- and pentachloro¬ phenyl) 2, 6-dimethylphenyl and 2,4-dinitrophenyl and, most ^* preferably beta-cyano ethyl.

The deblocking step, step iv) and step viii) , is carried out using conventional means. Although thiolates may be used, for instance using the processes described by Daub et al in JACS, 99, 3526 (1977) and by Andreus et al in Tet. Letts, 29, 5479 (1988), it is preferred to avoid the difficult subsequent recovery from, for instance thiophenol byproduct, by using organic base, especially tri lower alkylamine, usually trimethylamine, in organic solvent. Suitable solvents are acetonitrile, as well as mixtures of acetonitrile and dichloromethane, methanol/chloroform mixtures or even aqueous lower alcohol mixtures. Where Z is OR and the group Y in the compound of the formula I is joined to the group R³, a deblocking step effectively opens the ring to leave an -o^" and a group derived from Y-R -0-. Such a ring opening step may simultaneously aminate the distal end of the moiety Y, so as to form an amine group, usually a quaternary group. The simultaneous reaction thus involves reaction with a trialkylamine in an organic solvent, and is thus an analogous to the ring opening step described by Thuong and Chabrier (op cit) , by Nakaya et al and in our co-pending applications Nos WO-A-9514701 and WO-A-9514702.

The inventors have found that the use of amidite phosphitylating agents allow the process of the invention to proceed with yields of more than 90% or higher whereas the phosphitylation using chlorophosphite phosphitylating agents as described by Podashova et al (op. cit.) can give lower yields e.g. 60%. The reaction is, in addition, easier to control.

The following examples illustrate the inventions:

Example 1

2 - ( Mβthacryloyloxyethv l ) - 2 ' -tr imethy lammon iumethy l ) phosphate, inner salt

Hydroxyethyl methacrylate (0.43 g, 3.32 mmol) and 4,5- dichloroimidazole (0.45 g, 3.32 mmol) were stirred in dry acetonitrile (10 ml) in the presence of 4A molecular sieves (1 g) . Bis(diisopropylamino)-cyanoethylphosphine (1 g, 3.32 mmol) was added to the mixture which was stirred under nitrogen at room temperature for 4 hours to give the crude monophosphoramidite.

H-nmr, 200 MHz, (CDC1-) δ = 1.18 (d) , 1.94 (s) , 2.62 (t) , 3.60 (m) , 3.84 (m) , 4.30 (t) , 5.60 (s) , 6.14 (s)

Bromoethanol (redistilled, 0.41 g, 3.32 mmol), 4,5- dichloroimidazole (0.45 g, 3.32 mmol) and 4A molecular sieves (1 g) were added to the reaction mixture and stirred at room temperature for 16 hours. A further quantity of bromoethanol (redistilled, 0.05 g, 0.40 mmol) was added and the mixture was stirred at 40°C for four hours. After filtering through celite and a 0.2 ,_m glass fibre filter, the solvent was removed to give crude triphosphite.

¹H-nmr, 200 MHz, (CDC1₃) δ - 1.43 (d) , 1.94 (s) , 2.68 (t) , 3.50 (t) , 4.11 (m) , 4.34 (t) , 5.62 (s) , 6.17 (s)

The crude triphosphite was dissolved in dichloromethane (20 ml) and treated with meta-chloroperbenzoic acid (0.57 g,

3.32 mmol) and stirred at room temperature for ten minutes.

^* The solution was extracted with saturated aqueous sodium bicarbonate ( 4 x ca 20 ml) . The combined aqueous layers were back extracted with dichloromethane ( ca 40 ml) which was pooled with the earlier organic fraction and re- extracted with saturated aqueous sodium bicarbonate ( 2 x ca 20 ml) . The mixture was dried over magnesium sulphate and evaporated to give the crude triphosphate, 1.18 g, 3.19 mmol, 96% yield from hydroxyethyl methacrylate.

¹H-nmr, 200 MHz, (CDC1₃) δ = 2.00 (s) , 2.80 (t) , 3.54 (t) , 4.37 (m) , 5.65 (s) , 6.19 (s)

The crude triphosphate (1.18 g, 3.19 mmol) was dissolved in dry acetonitrile (ca 30 ml) and treated with trimethylamine (1.57 g, 26.5 mmol) and heated at 75°C for 48 hours. 4A molecular sieves (1.5 g) were added to the mixture and heating was maintained for a further two hours before filtering through celite and a 0.2 _/xm glass fibre filter . The solvent was evaporated and the residue dissolved in methanol and purified by column chromatography on silica gel (ca 20 g) eluting with methanol. Fractions containing product were combined and evaporated to dryness to give pure 2-(methacryloyloxyethyl)-2'-trimethylammoniumethyl) phosphate, inner salt, 0.29 g, 0.97 mmol, 30% yield.

¹H-nmr, 200 MHz, (D-O) δ = 1.96 (s) , 3.23 (s) , 3.67 (m) , 4.17 (m) , 4.31 ( ) , 4.40 (m) , 5.76 (s) , 6.20 (s)

The overall yield of 2-(methacryloyloxyethyl)-2'- - trimethylammoniumethyl) phosphate, inner salt from hydroxyethyl methacrylate was 29%.

Example 2

2- (Methacryloyloxyethyl ) -6 ' -triphenylphosphoniumhexyl) phosphate , inner salt

To diisopropylammonium tetrazolide (0.43 g, 2.49 mmol) in dichloromethane (70 ml) was added bis(diisopropylamino)- cyanoethylphosphine (0.75 g, 2.49 mmol) in dry acetonitrile (5 ml) followed by 6-hydroxyhexyltriphenylphosphonium bromide (1.11 g, 2.49 mmol) in acetonitrile (3.3 ml). The mixture was stirred at ambient temperature for 40 minutes under an atmosphere of argon. The solvents were evaporated aanndd tthhee ttrriipphheennyyllpphhoosspphhoonniiuumm ssalt was analysed by H-nmr before using in the next stage.

H-nmr, 200 MHz, (CDC1₃) δ = 1.16 (m) , 1.29 (m) , 1.43 (d) , 1.66 (m) , 2.63 (t) , 2.39 - 3.87 (m) 4.11 (m) , 4.30 ( ) , 7.74 (m)

- The triphenylphosphonium salt in acetonitrile (75 ml) was treated with tetrazole (0.18 g, 2.49 mmol) and 2- hydroxyethyl methacrylate (0.43 g, 3.32 mmol) and stirred at room temperature for 18 hours. The solvents were evaporated and the residue partitioned between dichloromethane/chloroform (9 : 1, ca 60 ml) and water ( ca 60 ml) . The organic layer was re-extracted with water (2 x ca 60 ml) and evaporated to give crude triphenylphosphonium phosphite methacrylate, 1.8 g.

H-nmr, 200 MHz, (CDC1-) δ = 1.29 (m) , 1.63 (m) , 1.89 (s) , 2.66 (t) , 3.80 (m) 4.03 (m) , 4.29 (m) , 5.60 (s) , 6.11 (s) , 7.73 (m)

Crude triphenylphosphonium methacrylate (1.8 g, ca 2.5 mmol) was dissolved in dichloromethane : chloroform (1 : 1, 80 ml) and treated with meta-chloroperbenzoic acid (0.69 g, 2.8 mmol) . The mixture was stirred at room temperature for one hour and then extracted with saturated sodium bicarbonate solution ( 3 x 80 ml) . The organic layer was evaporated to give crude triphenylphosphonium phosphate methacrylate, which was used without further purification.

¹H-nmr, 200 MHz, (CDC1-) δ = 1.29 (m) , 1.59 (m) , 1.91 (s) , 2.76 (t) , 3.71 (m) 4.01 (m) , 4.26 (m) , 5.60 (s) , 6.14 (s) , 7.70 ( )

Triphenylphosphonium phosphate methacrylate was dissolved in methanol (15 ml) and treated with aqueous ammonia (5%, 35 ml) and stirred at room temperature for thirty minutes. The solvents were evaporated and the residue was azeotroped with benzene (2 x 100 ml) and dried over phosphorus pentoxide under vacuum for 16 hours. The crude residue was purified by column chromatography on silica gel (31 g) eluting with chloroform : methanol (4 : 1) . Fractions containing product were combined, evaporated and dried under vacuum to give 2-(methacryloyloxyethyl)-6'- triphenylphosphoniumhexyl) phosphate, inner salt, 0.34, 0.61 mmol, 25% overall yield from 6-hydroxyhexyl-l- triphenylphosphonium bromide.

¹H-nmr, 200 MHz, (CDC1-) δ = 1.34 (m) , 1.53 (m) , 1.83 (s) , 3.31 (m) , 3.86 (m) 4.09 (m) , 4.20 (m) , 5.46 (s) , 6.03 (s) , 7.71 (m) Example 3

Large scale preparation of 2-(methacryloyloxyethylϊ-2'- trimetbyi_*wmnnjumethyl) hosphate, inner salt

3.1 N,N-Diisopropylaminoethylene phosphite

Diisopropylamine (463 g, 4518 mmol) was added dropwise over one hour to a stirred solution of 2-chloro-l,3,2- dioxaphospholane (distilled, 115.8 g, 915 mmol) in anhydrous ether (4000 ml) at -10°C under nitrogen. The mixture was allowed to return to room temperature and was

- stirred for 16 hours, filtered to remove diisopropylammonium chloride and the filter bed washed with ether (400 ml) . The filtrate and washings were evaporated to ca 500 ml and the mixture was refiltered and the filter bed was washed with ether (1400 ml) . The solvent was removed to give a mobile liquid which was filtered through a sinter funnel and fractionally distilled (40°C, 0.1 mbar) to give pure N,N-diisopropylaminoethylene phosphite , 104.2 g, 545 mmol, 60% yield.

H-nmr , 200 MHz , (CDC1-) δ = 1. 17 (d) , 3 . 46 (m) , 3 . 89 (m) 4 . 14 (m) 3.2 2- (Methacryloyl) ethyloxy 1 -1.3 .2-dioxapho3Pholane

A mixture of 4,5-dichloroimidazole (70 g, 511 mmol) and 2- hydroxyethyl methacrylate (68.0 g, 523 mmol) was dissolved in dry acetonitrile (500 ml) . N,N-Diisopropylaminoethylene phosphite (100 g, 523 mmol ) in dry acetonitrile (100 ml) was added dropwise over one hour under a nitrogen atmosphere at room temperature and was then stirred for ten minutes. The mixture was filtered through a tightly packed bed of Celite and the pad was washed with acetonitrile (200 ml) to give a solution of 2-[ (methacryloyl)ethyloxy] - 1,3,2-dioxaphospholane which was used immediately.

3.3 2-f (Methacryloyl) ethyloxy1 -2-oxo-l .3.2- dioxaphospholane

The solution of 2-[ (methacryloyl)ethyloxy] -1,3,2- dioxaphospholane in acetonitrile was cooled to -10°C and a solution of trimethylamine-N-oxide (35.4 g, 471 mmol) in acetonitrile (200 ml) was added dropwise under nitrogen over 90 minutes. The mixture was allowed to warm to room temperature and the solvent was evaporated to ca 800 ml to give a solution of 2-[ (methacryloyl)ethyloxy]-2-oxo-l,3,2- dioxaphospholane which was used immediately. A sample was analysed by H-nmr.

H-nmr, 200 MHz, (CDC1₃) δ - 1.97 (s) , 4.43 (m) , 5.66 (s) 6.21 (s) 3.4 2-(Methacryloγloxyethyl)-2 '-trimethv.»«""«»-"m-ethyl) phosphate, inner salt

The solution of 2-[ (methacryloyl)ethyloxy]-2-oxo-l,3,2- dioxaphospholane was treated with trimethylamine (49.9 g, 845 mmol) in dry acetonitrile (200 ml) and stirred at 50°C for 16 hours. The mixture was cooled to ambient temperature and some of the excess trimethylamine was removed under reduced pressure. The solution was warmed to 80°C, filtered through a bed of Celite under nitrogen . After cooling a total of 300 ml of acetonitrile was removed under reduced pressure, and the solid was isolated after crystallisation to give pure 2-(Methacryloyloxyethyl)-2 '- trimethyl-ammoniumethyl) phosphate, inner salt, 35.0 g, 119 mmol, 23 % yield from 2-hydroxyethyl methacrylate.

H-nmr, 200 MHz, (D₂0) δ = 1.94 (s) , 3.21 (s) , 3.66 (m) , 4.17 ( ) , 4.31 (m) , 4.39 (m) , 5.76 (s) , 6.19 (s)

13 C-nmr, 50.1 MHz, (D₂0) δ = 20.2, 56.8, 62.3, 66.7, 67.3, 68.8, 129.9, 138.7, 172.4

Example 4 2- r (Methacryloyl) ethyloxVl -2-oxo-i .3.2-dioxaphospholane using dinitrogen tetroxide oxidising agent

Dinitrogen tetroxide was purified using the method of MS Anson and C McGuigan (J. Chem. Soc. Perkin Trans. 1, 1989, 715). A solution of dinitrogen tetroxide (0.175 g, 1.92 mmol) in dry dichloromethane (12 ml) was added dropwise to a solution of 2-[ (methacryloyl)ethyloxy] -1,3,2- dioxaphospholane (produced as in example 7.2) (1.69 g, 7.68 mmol) in dry dichloromethane at -60°C. After leaving for five minutes the mixture was allowed to warm to room temperature and the solvent was evaporated to give crude 2- [ (methacryloyl)ethyloxy]-2-oxo-l,3,2-dioxaphospholane (1.86 g, 7.8 mmol) which was generally used immediately (for example in a step corresponding to example 7.4).

H-nmr, 200 MHz, (CDCl₃) δ = 1.97 (s) , 4.44 (m) , 5.63 (s) , 6.21 (s)

Example 5

2- (Methacryloyl)ethyloxyl-2-oxo-l.3.2-dioxaphospholane using t-butγl hydroperoxide oxidising agent

A solution of tert-butylhydroperoxide in decane (5.5 M, 1.40 ml, 7.68 mmol) in dry dichloromethane (15 ml) was added dropwise to a solution of 2-[ (methacryloyl)ethyloxy] -1,3,2-dioxaphospholane (1.69 g, 7.68 mmol) (produced in example 7.2) in dry dichloromethane (15 ml) at 0°C. After leaving for five minutes the mixture was allowed to warm to room temperature and the solvent was evaporated and the residue dried under vacuum to give crude 2- [ (methacryloyl)ethyloxy]-2-oxo-l,3,2-dioxaphospholane which was generally used immediately (for instance for a step corresponding to example 7.4). ¹H-nmr , 200 MHz , (CDC1₃) δ = 1. 97 (s) , 4 . 46 (m) , 5. 63 (s) , 6. 21 (s)

Example 6 l - ( Hydroxymethy l - / r ( hvdroxγphosph inyl ) oxyl -N . N . N- trimethyletha inium hydroxide , inner salt! ) 2 -dimethyl propane

2,2-Dimethylpropan-l-ol (0.44 g, 5.0 mmol) and - diisopropylammonium tetrazolide (0.43 g, 2.5 mmol) were dissolved in dry dichloromethane (25 ml) and stirred under an atmosphere of nitrogen. Bis(diisopropylamino)- cyanoethylphosphine (1.66 g, 5.5 mmol) was added and stirring was maintained for one hour. The mixture was diluted with dichloromethane (100 ml) and extracted with aqueous sodium carbonate (2%, 2 x 125 ml) and washed with saturated sodium chloride solution (125 ml) . The combined aqueous phases were back extracted with dichloromethane

(125 ml) and the pooled organic phases were dried over sodium sulphate at 4°C for one hour. The mixture was filtered to remove the solid which was washed with dry dichloromethane (50 ml) . The solvent was removed by evaporation to give the 2,2-dimethylpropan-l-yl phosphite adduct (1.46 g, 5.0 mmol, quantitative yield).

¹H-nmr, 200 MHz, (CDC1₃) δ = 0.91 (s), 1.21 (d) , 2.68 (t) , 3.26 (m) , 3.60 (m) , 3.85 (m)

2,2-Diιαethylρropan-l-yl phosphite adduct (1.46 g, 5.0 mmol) was added to a mixture of bromoethanol (0.95 g, 7.6 mmol) and lH-tetrazole (sublimed, 0.18 g, 2.5 mmol) in dry acetonitrile (40 ml) under nitrogen and stirred at ambient temperature for sixteen hours. Further bromoethanol (0.16 g, 1.3 mmol) and lH-tetrazole (0.09g, 1.3 mmol) were added and the mixture was stirred for 30 minutes. The solvent was evaporated and the residue was dissolved in dichloromethane (ca 75 ml) and extracted with aqueous sodium carbonate (2%, 2 x ca 100 ml) and washed with saturated sodium chloride solution (ca 100 ml) . The combined aqueous phases were back extracted with dichloromethane ( ca 100 ml) and the pooled organic phases were dried over sodium sulphate at 4°C for one hour. The mixture was filtered to remove the solid which was washed with dry dichloromethane ( ca 50 ml) . the solvent was removed by evaporation to give the crude 2,2- dimethylpropan-1-yl bromoethyl phosphite adduct which was purified by column chromatography eluting with hexane : acetone : triethylamine (63:32:5) to give the pure material (1.09 g, 3.5 mmol, 69% yield).

H-nmr, 200 MHz, (CDC1₃) δ = 0.94 (s) , 2.71 (t) , 3.53 (d) , 3.56 (t) , 4.12 (m)

2,2-Dimethylpropan-l-yl bromoethyl phosphite adduct (1.23 g, 3.9 mmol) was dissolved in dry dichloromethane (ca 15 ml) and treated with meta-chloroperbenzoic acid (0.68 g, 3.9 mmol) . the mixture was stirred at room temperature for ten minutes when further meta-chloroperbenzoic acid (0.07 g, 0.4 mmol) was added and stirring maintained .for fifteen minutes. The solution was extracted with aqueous sodium bicarbonate (IM, 2 x ca 30 ml) and washed with saturated sodium chloride solution (ca 30 ml) . The combined aqueous phases were back extracted with dichloromethane (ca 30 ml) and the pooled organic phases were dried over sodium sulphate, filtered to remove the solid and washed with dry dichloromethane ( ca 15 ml) . The solvent was removed by evaporation to give the crude 2,2-dimethylpropan-l-yl bromoethyl phosphate adduct (1.12 g, 3.4 mmol, 87% yield).

H-nmr, 200 MHz, (CDC1-) δ = 0.96 (s) , 2.79 (t) , 3.56 (t) 3.76 (d) , 4.34 (m)

mass spec. CI. , m/2 = 328 (3%) E.I. , m/2 = 328 (14%)

2,2-Dimethylpropan-l-yl bromoethyl phosphate adduct (0.2 g, 0.61 mmol) and trimethylamine (0.87 g, 14.6 mmol) in anhydrous acetonitrile (ca 30 ml) were heated at 46°C for 16 hours. The solvent and excess trimethylamine was evaporated and the residue was purified by column -chromatography on silica gel (ca lOg) eluting with methanol. Fractions containing product were combined and evaporated to give 1 - ( h y d r o xy m e t h y 1 -

{ [ (hydroxyphosphinyl) oxy] -N, N , N-trimethylethaminium hydroxide, inner salt}) 2 -dimethyl propane (O.lOg, 0.40 mmol, 65% yield) .

H-nmr, 200 MHz, (CDC1- /CD₃OD) <S = 0.94 (s) , 3.22 (s) ,

3.54 (d) , 3.63 (m) , 4.24 (m)

mass spec: C.I., m/2 = 254 (32%).

The overall yield of 1 - ( hydroxymethy 1- { [ (hydroxyphosphinyl) oxy] -N, N , N-trimethylethaminium hydroxide, inner salt}) 2-dimethyl propane from starting phosphoramidite was 39%. Example 7

1.3 -Dihexadecyloxy-2 - ( hvdroxymethyl- f r (hydroxyphosphinyl) oxyl -N. .N-trimethylethaminium hydroxide, inner salt}) 2 -methyl propane

1,3-Dihexadecycloxy-2-(hydroxymethyl)-2-methyl propane (0.38 g, 0.66 mmol) and diisopropylammonium tetrazolide (0.06 g, 0.33 mmol) were dissolved in dry dichloromethane (25 ml) and stirred under an atmosphere of nitrogen. Bis(diisopropylamino)-cyanoethylphosphine (0.20 g, 0.66 mmol) was added and stirring was maintained for three hours. The solvent was removed by evaporation to give the crude dialkyl phosphite adduct.

The crude dialkyl phosphite adduct was added to a mixture of bromoethanol (0.15g, 1.16 mmol) and lH-tetrazole (sublimed, 0.035g, 0.50 mmol) in dry acetonitrile (10 ml). The mixture was stirred at ambient temperature for 64 hours. The solvent was evaporated and the residue was dissolved in dichloromethane (ca 30 ml) and extracted with aqueous sodium carbonate (2%, 2 x ca 30 ml) and saturated sodium chloride solution (ca 30 ml) . The combined aqueous extracts were back extracted with dichloromethane (ca 30 ml) and the pooled organic phases were dried over sodium sulphate and evaporated to give the crude dialkyl phosphite, 0.30g, 0.37 mmol, 56% yield.

¹H-nmr, 200 MHz, (CDC1₃) δ = 0.88 (t) , 0.94 (s) , 1.20 (m) , 1.53 (m) , 2.68 (t) , 3.22 (s) , 3.38 (t) , 3.53 ( ) , 3.74 (t) , 4.12 (m)

To the crude dialkyl phosphite (0.30 g, 0.37 mmol) in dry dichloromethane (5 ml) was added meta-chloroperbenzoic acid

(0.064 g, 0.37 mmol) in dry dichloromethane (ca 5 ml) . The mixture was stirred at room temperature for 15 minutes and then stored at 4°C for 16 hours before it was extracted with saturated sodium bicarbonate solution (2 x ca 15 ml) and saturated sodium chloride (ca 15 ml) . The combined aqueous phases were back extracted with dichloromethane

(ca . 15 ml) and the pooled organic phases were dried over sodium sulphate, filtered and evaporated to give the crude dialkyl phosphate, 0.25 g, 0.31 mmol, 83 % yield.

¹H-nmr, 200 MHz, (CDC1₃) δ = 0.88 (t) , 0.97 (s) , 1.24 (m) , 1.53 (m) , 2.76 (t) , 3.24 (s) , 3.38 (t) , 3.56 (t) , 3.74 (t) , 4.00 (m) , 4.29 (m)

Dialkyl phosphate adduct (0.25 g, 0.31 mmol) and trimethylamine (0.66 g, 11.2 mmol) in anhydrous acetonitrile (5 ml) were heated at 46°C for 48 hours. A solid precipitated which was removed by filtration and washed with acetonitrile. The solid was dissolved in chloroform and was purified by column chromatography on silica gel (ca lOg) eluting with chloroform to chloroform : methanol : ammonia (25%) (690 : 270 : 64) . Fractions containing product were combined and evaporated to give 1 , 3 - D ihex ade cy l oxy - 2 - ( hydr o xymethy l - { [ (hydroxyphosphinyl) oxy] -N, N, N-trimethylethaminium hydroxide, inner salt}) 2-methyl propane (0.126g, 0.17 mmol, 56% yield) . H-nmr, 200 MHz, (CDC1₃ /CD₃OD) δ = 0.91 (t) , 0.97 (s) , 1.26 (m) , 1.54 (m) , 3.29(t,s), 3.35 (s) , 3.65 (m) , 3.75 (m) , 4.24 (m)

mass spec: +ve FAB, m/2 = 735 (m+1) .

The overall yield of 1, 3-Dihexadecyloxy-2-(hydroxymethyl- { [ (hydroxyphosphinyl) oxy] -N, N, N-trimethylethaminium hydroxide, inner salt}) 2-methyl propane from starting phosphoramidite was 39%.

Example 8

1 , 3 -Dihexadecyloxy-2 - ( hvdroxymethyl- ■f r (hydroxyphosphinyl) oxyi-N.N.N-guinuclidinylethaminium hvdroxide, inner salt}) 2 -methyl propane

To bromoethyl dialkyl phosphate (0.558 g, 0.69 mmol) (produced in the third step of Example 2) was treated with quinuclidine (0.76 g, 6.9 mmol) in a mixture of acetonitrile and dichloromethane (1 : 1, 10 ml) and heated at 45°C for 16 hours. A solid precipitated which was removed by filtration. The mother liquors were evaporated and the residue was dissolved in acetonitrile (ca 10 ml) . The resulting solid was filtered and added to the previously isolated material, to give crude product (0.59 g) , which was purified by column chromatography on silica gel (15 g) eluting with chloroform to chloroform : methanol : water (65 : 25 : 4) . Fractions containing product were combined and evaporated to give the title compound , 0.28 g, 0.48 mmol, 69 % yield.

¹H-nmr, 200 MHz, (CDC1₃) 5 = 0.88 (t) , 0.96 (s) , 1.26 (m) , 1.51 (m) , 2.01 (m) , 2.16 (m) , 3.29(s), 3.35 (t) , 3.78 (m) , 4.27 (m)

¹³C-nmr, 75 MHz (CDC1-) , δ = 14.1, 17.1, 19.7, 22.7, 24.1, 26.2, 29.7, 31.9, 40.8, 56.0, 58.3, 64.6, 68.5, 71.6, 73.0.

³¹P-nmr, 84.5 MHz, (CDC1-) , δ = 0.695 (S) .

Mass spec: CI. m/2 = 786 (1.3%), +ve FAB m/2 = 788 (m+1, 50%)

Overall yield of this compound from 1,3-dihexadecyloxy-2- (hydroxymethyl)-2-methyl propane is 59%.

Example 9

1 . 3 -D i hexa d e cv l oxy- 2 - ( hvdroxymethv l - { r (hydroxyphosphinyl) oxy 1-N.N.N-triphenγlhexaphosphonium hydroxide, inner salt}) 2-methyl propane

OH * ^{v v v} OH

Br^*

A mixture of 6-bromohexan-l-ol (18.5 g, 102 mmol) and triphenylphosphine (26.9 g, 102 mmol) in dry acetonitrile (360 ml) was heated at 70°C for 184 hours. The solvent was evaporated and the residue dissolved in chloroform (270 ml) and added dropwise to diethyl ether (910 ml) with stirring. After stirring for one hour, the solid was removed by filtration, washed with ether (100 ml) and dried under vacuum for 16 hours to give 6- hydroxyhexyltriphenylphosphonium bromide, 35.6 g, 80.3 mmol, 79 % yield.

¹H-nmr, 200 MHz, (CDC1₃) δ = 1.51 (m) , 1.69 (m) , 3.68 (m) , 3.77 (m) , 7.80 (m)

Br-

6-Hydroxyhexyl-l-triphenylphosphonium bromide (35.6 g, 80.3 mmol) was dissolved in dichloromethane (ca 300 ml) and subject to Dean and Stark drying at 70°C for sixteen hours. A sample was removed (0.29 g, 0.65 mmol) in dichloromethane (4 ml) and mixed with lH-tetrazole (sublimed, 0.046 g, 0.65 mmol) and diisopropylphosphoramidite (produced as in the first step of example 7) (0.5 g, 0.65 mmol) in acetonitrile (10 ml) and stirred at room temperature for sixteen hours. The product was analysed by H-nmr and used immediately in the next stage.

H-nmr, 200 MHz, (CDC1-) δ = 0.88 (t) , 0.90 (s) , 1.29 (m) , 1.46 (d) , 1.63 (m) , 2.63 (t) , 3.23(s), 3.36 (t) , 3.40 - 3.90 (m) , 3.97 (m) , 7.78 (m)

The solution of triphenylphosphonium triphosphite, containing diisopropylaminium salts was treated with meta- chloroperbenzoic acid (0.11 g, 0.65 mmol) in dichloromethane (10 ml) and stirred for fifteen minutes. The solvents were evaporated and the residue was dissolved in dichloromethane (ca 20 ml) and extracted with a solution of saturated sodium bicarbonate (3 x ca 20 ml) . The combined aqueous layers were back extracted with dichloromethane ( ca 50 ml) and the pooled organic phases were dried over magnesium sulphate, filtered and evaporated to give the triphenylphosphonium triphosphate compound, 0.53 g, 0.47 mmol, 72% yield from 6-hydroxyhexyl-l- triphenylphosphonium bromide.

^-nmr, 200 MHz, (CDC1-) <S « 0.84 (t) , 0.91 (s) , 1.23 (m) , 1.56 ( ) , 2.77 (t) , 3.20(s), 3.31 (t) , 3.57 - 4.29 (m) , 7.71 (m)

Triphenylphosphonium triphosphate compound (0.53 g, 0.47 mmol) was dissolved in methanol (40 ml) and chloroform (7 ml) and treated with aqueous ammonia (5%, 9 ml, 25 mmol) at room temperature for four hours. The solvent was evaporated and the residue was azeotroped with benzene (x

5) and dried under high vacuum to give the crude product.

The material was purified by column chromatography on silica gel (40 g) and eluted with chloroform to chloroform

: methanol (3 : 1) . Fractions containing product were combined and evaporated to give pure 1,3-dihexadecyloxy-2-

(hydroxymethyl-{ [ (hydroxyphosphinyl) oxy]-N,N,N- triphenylhexaphosphoniu hydroxide, inner salt}) 2-methyl propane, 0.26 g, 0.26 mmol, 56% yield.

¹H-nmr, 200 MHz, (CDC1-) 5 = 0.87 (t,s), 1.23 (m) , 1.46 (m) . 3.25(s,t), 3.53 - 3.97 (m) , 7.74 ( )

Mass spec: FAB (+ve) 994 (m+1, 100%)

Example 10

2-Hydroxyethyl methacrylate (0.15g, 1.03 mmol) and 1H- tetrazole (0.07g, 1.03 mmol) in dry acetonitrile (ca 10 ml) were added to the reaction mixture and stirred at room temperature for 16 hours. After filtering through celite, the solvent was removed to give crude triphosphite.

H-nmr, 200 MHZ, (CDC1₃) 8 = 1.45(d), 1.94(s), 2.68(t), 3.52(t) 4.14 (m) , 4.35 (t) , 5.65(s), 6.17(s)

The crude triphospite was dissolved in dichloromethane (ca 20 ml) and treated with meta-chloroperbenzoic acid (0.18 g, 1.03 mmol) and stirred at room temperature for ten minutes. The solution was extracted with saturated aqueous sodium bicarbonate (2 x ca 20 ml) . The organic phase was dried over magnesium sulphate and evaporated to give the crude triphosphate, 0.35 g, 91% yield for hydroxyethyl methacrylate.

H-nmr, 200 MHz (CDC1₃) 8=1.98(s) , 2.80(t) 3.52(t), 4.35(m), 5.64(s), 6.17(s).

The crude triphosphate (0.35g, 0.95 mmol) was dissolved in dry acetonitrile (ca 10 ml) and treated with trimethylamine (0.31g, 5.2 mmol) and heated at 75°C for 16 hours. The solvent was evaporated and the residue dissolved in methanol and purified by column chromatography on silica gel (ca 30 g) eluting with methanol. Fractions containing product were combined and evaporated to dryness to give pure 2- (methacryloyloxyethy1) -2 ' - trimethylammoniumethyl) phosphate, inner salt, 0.09 g, 0.31 mmol, 30% yield.

¹H-nmr, 200 MHz, (D₂0) 8=1.97(s) , 3.22(s) , 3.68(m) , 4.17 (m) , 4.32(m) , 4.41(m) , 5.82(s), 6.23(s)

The overall yield of 2-(methacryloyloxyethyl)-2'- trimethylammoniumethyl) phosphate, inner salt from hydroxyethyl methacrylate was 30%. Example 11 11. 1 2- (N.2. 6-dimethylpyrid i n-i ιιm ) -1. 3 .2 -dioxaphospholane chloride

Anhydrous lutidine (0.313g, 2.92 mmol) was added to a chilled (-10°C) stirred solution of 2-chloro-l,3,2- dioxaphospholane (0.369g, 2.92 mmol) in dry d₃ acetonitrile (4ml) . The resulting solution was stirred at

-10°C for 1 hour and analysed by NMR spectroscopy.

HNMR(CD₃CN) 58.10(lH,t,J=6) ,7.48(2H,d,J=6) ,4.65-3.95(4H,m) ,

2.75(6H,S) .

11.2 Preparation of 2- (methacrγloyloxyethyl) - 2' (trimethγla_nft"i"m«»thyl)phosphate inner salt

A solution of 2-chloro-l,3,2-dioxaphospholane (4.9g, 0.04M) in acetonitrile was cooled to -10°C under a nitrogen atmosphere when 2,6 - dimethylpyridine (4.0g, 0.04M) was added over 20 minutes. The mixture was stirred at -10°C for a further 20 minutes. A solution of 2-hydroxyethyl methacrylate (4.4g, 0.033M) in acetonitrile (δml) was added over 15 minutes at -10°C The mixture was allowed to warm to 0°C and was filtered. The filtrate was cooled to -10°C and treated with a solution of trimethylamine N-oxide (2.4g, 0.32M) in acetonitrile (25ml). A solution of trimethylamine (3.3g) in acetonitrile (10ml) was added. The mixture was heated in a closed system at 45°C for 16 hours to give the desired product which was isolated by concentrating the reaction mixture and crystallisation at 0°C

Tic showed that the product was clean and contaminated by low quantities of by products. ¹H-nmr, 200 MHz, (D₂0) δ = 1.94 (s), 3.21 (s), 3.66 (m) , 4.17 (m) , 4.31 (m) , 4.39 (m) , 5.76 (s) , 6.19 (s)

³C-nmr, 50.1 MHz, (D₂0) δ = 20.2, 56.8, 62.3, 66.7, 67.3, 68.8, 129.9, 138.7, 172.4

Thus the product was used in a process analogous to that of example 3, step 2, to react with HEMA except it was found that addition of acid activator was unnecessary. This is believed to be due to the fact that the amidite group is cationically charged and consequently activated to reaction with the alcohol.

EXAMPLE 12

2-(N-Pyridinium)-1.3.2-dioxaphospholane chloride

Anhydrous pyridine (0.228g, 2.88 mmol) was added to a chilled (-10°C) , stirred solution of 2-chloro-l,3,2 - dioxaphospolane (.0364g, 2.88 mmol) in dry d₃-acetonitrile (4ml) . The resulting solution was stirred at -10°C for 1 hour and analysed by NMR spectroscopy.

¹HNMR(CD₃CN)<S8.70(2H,d,J=4) ,8.38(1H, t, J=6) , 7.88(2H,m),

4.65-3.95(4H,m) .

¹³CNMR(CD-CN)<S66,56(CH-) , 124.81(CH) ,137.34(CH) ,150.04(CH) . As for example 11.2, this product was used to react with HEMA. Again, due to the fact the amidite group is cationically charged and activated for reaction with the alcohol, no weak acid activation is required.

Example 13

2-(Methacryloyloxyethyl)-2¹-(trimethyl»mmg iumethv1) phosphate, inner salt.

2-Hydroxyethylmethacrylate (1.64g, 12.5 mmol) and benzoic acid (1.52g, 12.4 mmol) were stirred under a nitrogen atmosphere at ambient temperature for approximately 0.25 hour in dry acetonitrile (15 ml). A solution of 2-(N,N- diisopropylamino)-1,3,2-dioxaphospholane (2.50g, 12.4mmol) in dry acetonitrile (2.5 ml) was added to the mixture over a period of 0.5 hour. Stirring was continued for a further two hours at ambient temperature, then the mixture was cooled to around 0°C and filtered under nitrogen. The filtrand was washed with dry acetonitrile (2 ml) . A sample of the filtrate was evaporated to dryness and shown to be the desired 2- (methacryloyloxyethyloxy) -1, 3 ,2- dioxaphospholane by thin layer chromatography and Hnmr analysis. Thus benzoic acid is a successful weak acid activator for the phosphitylating agent.

The bulk of the material was cooled to -20°C and treated with a solution of trimethylamine N-oxide (0.89g, 11 mmol) in dry acetonitrile (12 ml) . After one hour the mixture was allowed to warm to around 21°C when a sample was removed, evaporated and analysed by Hnmr. The Hnmr spectrum showed the presence of unoxidised material so the bulk reaction mixture was recooled to -20°C when a further portion of trimethylamine N-oxide (0.23g, 3.1 mmol) was added in dry acetonitrile (4 ml) . After 5 minutes the mixture was allowed to warm to around 21°C when a solution of trimethylamine (1.28g, 21.4 mmol) in dry acetonitrile (4 ml) was added. The mixture was heated to 45°C for 16 hours. The solution was filtered through Celite and evaporated to give the crude product. ^nmr, 200 MHz, (D₂0) δ 6.2 (IH,S), 5.8 (IH,S) , 4.4 (2H,m) , 4.3 (2H,m), 4.2 (2H,m) , 3.7 (2H,m) , 3.2 (9H,s) and 1.9 (3H,s) ppm.

Byam le 14

Preparation of 2-(methacryloyloxyethyloxy)-1.3.2- dioxaphospholane using carboxylic acid activators

14.1 Using glutaric acid

2-Hydroxyethylmethacrylate (1 eq) and glutaric acid (0.5 eq) in acetonitrile were warmed to 40°C and treated with 2-(N,N-diisopropylamino)-1,3,2- dioxaphospholane (1 eq) . The mixture was allowed to cool to ambient temperature and was filtered to give a solution of the title compound.

14.2 Using adipic acid

2-Hydroxyethylmethacrylate and adipic acid were reacted with 2-(N,N-diisopropylamino) -1,3,2- dioxaphospholane in a method analogous to that described above to give the title compound.

These examples show that dibasic aliphatic carboxylic acids can be successful activators for the phosphitylating agent.

EXAMPLE 15 (Comparative)

2-r (Methacryloyl)ethyloxy1-l.3.2-dioxaphospholane

2-Chloro-l,3,2-dioxaphospholane (0.425g, 3.36 mmol.) was added dropwise to a chilled (-10°C) stirred solution of anhydrous pyridine (0.269g, 3.40 mmol) and 2-hydroxyethyl methacrylate (0.437g, 3.36 mmol) in anhydrous diethyl ether (15ml) . The mixture was allowed to warm to room temperature, stirred up for a further 0.5 hours and filtered under nitrogen to remove pyridinium chloride, the filter bed being washed with further portions of anhydrous diethyl ether (2 x 5ml). The 2-[ (methacryloyl)ethyloxy]- 1,3,2-dioxaphospholane product was identified by thin layer chromatography.

Tic, silica gel, 10% ethylacetate/dichloromethane, visualisation 1% aqueous KMn04: Rf = 0.75. In addition a number of other spots were visible on the chromatogram, showing that the product mixture included significant quantities of by products. This shows the process of the invention using an amidite starting material is greatly preferred to the prior art process using a chlorophosphite phosphitylating agent.

EXAMPLE 16 (Comparative)

Reaction of 2-chloro-1.3.2-dioxaphospholane with 2- hvdroxyethyl methacrylate

2-Chloro-l,3,2-dioxaphospholane (12.6g, 0.1M) in anhydrous acetonitrile (5ml) was added dropwise to a ^* stirred solution of 2-hydroxyethyl methacrylate (13.0g,

0.1M) and N,N,N, 'N'-tetramethylethylene diamine (5.81g, 0.05M) at -30°C under a nitrogen atmosphere. The mixture was allowed to warm to 0°C over a further period of 0.5 hours. The reaction mixture was filtered and a sample of the filtrate evaporated in vacuo and examined by H and P nmr spectroscopy. 'H-nmr, 500 MHz, (CDC1₃) showed the desired product together with a number of methacrylate related additional products.

P-nmr showed the presence of the desired product

(peak at 137.3ppm) together with a large number of additional peaks including 140.9, 136.7, 18.9, 10.6, 9.6 and -24.8ppm.

Thus the process according to the prior art using a chlorophosphite phosphitylating agent results in a product which is not as clean as the process of the invention.

Claims

1. A process comprising an alcoholysis step whereby a compound of the formula I

Z - P - NR¹2 I

in which the groups R are the same or different and each represents branched or straight alkyl groups (including cycloalkyl) or, together with the nitrogen atom to which they are attached, form a heterocycle optionally containing other heteroatoms and being saturated or unsaturated, and optionally being in quaternised form, Y is a phosphate protecting group, and

Z is either NR 2₂ in which the groups R2 are selected from the same groups as R and is the same or different to

- N ^ or is group OR in which R is selected from C,.₄₀ straight and branched-alkyl groups, -alkenyl groups and - alkynyl groups, or R and Y together with the oxygen atom and phosphorus atom to which they are (respectively) attached, form a 5 to 15 membered heterocyclic ring, optionally containing additional heteroatoms and/or substituents at ring carbon and/or nitrogen atoms (if any) , any of which groups R may be unsubstituted or substituted by aryl groups, hydroxyl groups, halogen atoms, amine (including mono-, di- or tri-alkyl substituted amine) groups, sulphonium or phosphonium groups, is reacted in solution with an alcohol of the formula BROH, in which R is a straight or branched alkylene, oxoalkylene or oligo-oxaalkylene chain and B is an ethylenically unsaturated polymerisable group selected from

wherein:

R is hydrogen or a C₁-C₄ alkyl group; A is -O- or -NR⁸- where R⁸ is hydrogen or a C₁-C₄ alkyl group; and

K is a group -(CH₂)_pOC(0)-, -(CH₂)_pC(0)0-, -(CH₂)_pOC(0)0-, -(CH₂)_pNR⁹-, - (CH-).OC (O) NR⁹-, -(CH₂) NR C(0)NR -, (in which the groups R are the same or different), -(CH₂)_p0-, -(CH₂) S0₃ -, or a valence bond and p is from 1 to 12 and R is hydrogen or a C_|-C₄ alkyl group to form a compound of the formula II

Z - P - OR⁷B II

in which Z, Y, R and B represent the same groups as in the compound of the formula I and the alcohol.

2. A process according to claim 1 where Z is a group X', and comprising a further step of reacting II in solution with a second alcohol R 4OH, in which R4 is selected from BR7 and C.,_₄₀ straight or branched -alkyl groups, -alkenyl groups and -alkynyl groups, any of which may be unsubstituted or substituted by aryl groups, halogen atoms, hydroxyl groups, amine (including mono-, di- or tri-alkyl substituted amine) groups, sulphonium or phosphonium groups, to form a compound of the formula III

R⁴0 P - OR B

III

in which Y, R and B represent the same groups as in the compound of the formula II and in the second alcohol.

3. A process according to claim 1 or claim 2 in which the or each alcoholysis step is carried out in the presence of a weak acid.

4. A process according to claim 2 or claim 3 in which Z is a group NR 2₂ and NR2₂ i.s the same as NR1₂.

5. A process according to claim 4 in which in the first and second alcoholysis steps, different weak acids are used.

6. A process according to claim 2 or claim 3 in which Z is a group NR ₂ and NR ₂ is different to NR¹ ₂.

7. A process according to claim 6 in which in the first and second alcoholysis steps, the same weak acid is used.

8. A process according to any of claims 3, 5 and 7 in which the weak acid is selected from lH-tetrazole, 1,2,4- triazole, 3-chlorotriazole, diisopropylammoniumtetrazolide and 4,5-dichloroimidazole.

9. A process according to any preceding claim comprising the subsequent step of oxidising the compound of the formula III or of the formula II in which Z is OR to form the corresponding phosphate of the general formula IV

O

in which R is either R or R , and Y, R and B are as defined in claim 1 or claim 2.

10. A process according to claim 9 wherein in the oxidation step, the agent is an organic oxidising agent preferably selected from peroxides, (such as tert- butylhydroperoxide di-tert butylhydroperoxide, bis- trimethylsilylhydroperoxide andperbenzoic acid derivatives such as meta-chloroperbenzoic acid) and iodobenzene diacetate, or is ozone or molecular oxygen in benzene or other dry organic solvent optionally with free radical generators, or is a nitrogen oxide oxidising agent (such as dinitrogen tetroxide) or a tertiary amine oxide.

11. A process according to any preceding claim in which B is a group

in which R is hydrogen or methyl, preferably methyl and A is preferably -0-; and

R⁷ is (CH₂)₂.₆, preferably (CH₂)₂.

12. A process according to any preceding claim in which Y is R⁵0- in which R is a C,_₆ alkyl group or an aryl group, preferably substituted with an electron withdrawing group. such as halogen atoms, nitro groups, sulphonyl groups, and aryl (including heteroaryl) groups, selected from allyl, benzyl, phenyl, phenylsulphoethyl, methyl-sulphoethyl, para-nitrophenylethyl, 2,2,2-trihaloethyl (especially trichloro- and tribromo-ethyl) , 2,2,2-trihalo-l,1,- dialkylethyl (especially 2 ,2 , 2 , -trichloro-1, l- dimethylethyl, 2 ' - and 4'-pyridylethyl, m-methylbenzyl, p- or m-halobenzyl, m,p-dichlorobenzyl, pentahalophenyl (especially pentafluoro- and pentachloro-phenyl) 2,6- dimethylphenyl and 2,4-dinitrophenyl and, most preferably beta-cyano ethyl.

13. A process according to any of claims 9 to 12 including a further step whereby the compound of the formula IV is deblocked so that the substituent Y is replaced by o^".

14. A process according to claim 12 in which Z is OR and the group Y in the compound of the formula I is joined to the group R to form a heterocyclic ring, a deblocking step

^"effectively opens the ring to leave an -0^* and a group derived from Y-R -0- joined to the phosphorus atom.

15. A process according to claim 14 in which the ring opening deblocking step involves simultaneously amination of the end of group Y distal from the phosphoryl group to form an amine group, preferably a quaternary amine group.

16. A process according to any preceding claim in which Z iiss OORR aanndd tthhee ggrroouuip Y is joined to R³ to provide a compound having the formula

in which R¹² is a C₂.₄ -alkylene, -alkenylene or -alkynylene group, any of which may be unsubstituted or substituted by one or more halogen atoms, hydroxyl, amine, alkyl or alkylene groups, is part of a fused ring system, and R¹ is as defined in claim 1.

17. A process of synthesising a zwitterionic phosphate diester comprising the steps of v) reacting a compound of the general formula V R¹ ₂N - P - NR² ₂

in which the groups R and R² are the same or different and each represents branched or straight alkyl groups (including cycloalkyl) or the R¹ groups and/or the R² groups and the nitrogen to which the respective groups are attached form a heterocycle optionally containing other heteroatoms and being saturated or unsaturated and Y is a protecting group, in solution with a first alcohol R OH in which R³ is selected from C_|.₄₀ straight or branched -alkyl groups, - alkenyl groups and -alkynyl groups, any of which may be unsubstituted or substituted by aryl groups, hydroxyl groups, halogen atoms, amine (including mono-, di- or tri¬ alkyl substituted amine) groups, sulphonium or phosphonium groups, in the substantial absence of water to form a compound of the general formula VI

R³0 P - N

VI

Y in which R , R and Y have the same meanings as in the compound of the formula V and the first alcohol, vi) the compound of the formula VI is then reacted with a second alcohol R 4 OH, in which R4 is selected from ^cι-₄₀ straight or branched -alkyl groups, -alkenyl groups and alkynyl groups, any of which may be unsubstituted or substituted by aryl groups, halogen atoms, hydroxyl groups amine (including mono-, di- or tri-alkyl substituted amine) groups, sulphonium or phosphonium groups, provided that at least one of R 3 and R4 represents a c,.₁₀-alkyl group having a halogen, sulphonium, amino or phosophonium substituent, in the substantial absence of water to form a compound of the formula VII

R³0 - P - OR⁴

VII in which R 1, Y and R4 have the same meani .ngs as i .n the compound of the formula VI and the second alcohol, vii) a compound of the formula VII is oxidised to form a compound of the formula VIII O

in which R 3, R4 and Y have the same meanings as in the compound of the formula VII viii) the compound of the formula VIII is deblocked so that the substituent Y is replaced by θ^", and if necessary, a cationic substituent is introduced into one of the groups R 3 or R4 form the product compound.

18. A process according to claim 17 in which each of steps v and vi are carried out in the presence of a weak acid, which is preferably lH-tetrazole, 1,2,4-triazole, 3-

_ chlorotiazole or diisopropylammonium tetrazolide or, preferably, 4,5-dichloroimidazole.

19. A process according to claim 17 or claim 18 in which Y is R O- in which R is a C,_₆ alkyl group or an aryl group, preferably substituted with an electron withdrawing group, such as halogen atoms, nitro groups, sulphonyl groups, and aryl (including heteroaryl) groups, selected from allyl, benzyl, phenyl, phenylsulphoethyl, methyl-sulphoethyl, para-nitrophenylethyl, 2,2,2-trihaloethyl (especially trichloro- and tribromo-ethyl) , 2,2,2-trihalo-l,l,- dialkylethyl (especially 2, 2 , 2 , -trichloro-1, 1- dimethylethyl, 2'- and 4'-pyridylethyl, m-methylbenzyl, p- or m-halobenzyl, m,p-dichlorobenzyl, pentahalophenyl (especially pentafluoro- and pentachloro-phenyl) 2,6- dimethylphenyl and 2,4-dinitrophenyl and, most preferably beta-cyano ethyl.

20. A process according to any of claims 17 to 19 in which the oxidising agent in step vii) is an organic oxidising agent preferably selected from peroxides, (such as tert- butylhydroperoxide di-tert butylhydroperoxide, bis- trimethyIsilylhydroperoxide and perbenzoic acid derivatives such as meta-chloroperbenzoic acid) and iodobenzene diacetate, or is ozone or molecular oxygen in benzene or other dry organic solvent optionally with free radical generators, or is a nitrogen oxide oxidising agent (such as dinitrogen tetroxide) or a tertiary amine oxide.

21. A process according to claim 17 in which R 3 and/or R4 has a phosphonium substituent.

22. A process according to any of claims 17 to 20 in which R OH and/or R OH are α,jS-disubstituted glycerols, preferably α,)3-di C₁₂.₂₄-acyl, -alkyl, -alkenyl or -alkynyl glycerol.