ZWITTERIONIC COMPOUNDS AND THEIR USE TO CROSS-LINK COLLAGENOUS MATERIALS
The present invention relates to the synthesis of phosphate compounds and to their use to form biocompatible materials, especially to react with proteinaceous materials to cross link and biocompatibilise them.
It is known that compounds having phosphoryl choline groups and other zwitterionic groups, have useful biocompatibilising properties. Coatings of polymers with pendant phosphoryl choline groups have been shown to be useful as coatings to render blood contacting devices non-thrombogenic. Contact lenses formed from hydrogel polymers with pendant PC groups are less subject to protein deposition, lipid deposition and bacterial adhesion than contact lenses having similar water contents.
We have also described a variety of reagents which are useful for derivatising preformed surfaces, for instance of polymeric substrates, to introduce PC groups. Such reagents are generally mono functional, that is each reagent molecule includes a single PC group and a single reactive group. Examples of some such reagents are described in EP-A-0157469, EP-A-0515895 andEP-A-0556216. In EP-A-0515895 reagents which are capable of reacting with amino groups at surfaces to give amine linkages are described. InEP-A-0556216 compounds which react with surface amino groups include activated amine groups.
In WO-A-9301221 we described copolymers of ethylenically unsaturated PC group containing monomers and copolymerisable comonomers selected so as to give suitable surface binding characteristics. One class of comonomers includes a reactive group by which covalent bonding to an underlying surface may be carried out. Examples of covalent reactive groups include an aldehyde group.
It has been reported that PC may have significant benefits in extending the life and reducing calcification of tissue valves, although the means by which PC had been incorporated into such heart valve was not disclosed.
Monomers for use in forming condensation polymers, such as polyesters and polyurethanes have been described. For instance in EP-A-0199790 and EP-A-0275293 , monomers comprising two hydroxyl groups are used to react with di-isocyanates and dicarboxylic acids respectively to form polyurethanes and polyesters.
Processes for synthesising dialdehyde compounds have been described. For instance ribose derivatives when reacted with sodium periodate are oxidised at the 3 and 4 hydroxyl groups with the C3-C4 bond being cleared to generate two aldehyde groups.
In US-A-4203893 (& EP-A-000T 197) cytidine diphosphocholine, which comprises a ribose moiety, is periodated and the dialdehyde reaction product reacted with compounds having free primary amine groups, such as polypeptides or proteins comprising lysine moieties or amino polysaccharides. The diphosphocholine moiety is zwitterionic but has an overall anionic charge (it is not neutral). In EP-A-0140640 inositol phosphate dialdehyde derivative is reacted with an amine containing hemoglobin derivative.
In J.C.S. Perkin II (1976) 1162-1165 Astin, K. et al describe phosphate derivatives of various terpene derivatives, including some cyclic alkenes and acyclic alkadienes, for solvolysis. Biological tissues can be "fixed" by allowing them to soak for some minutes in a dilute solution of glutaraldehyde. The glutaraldehyde is used to cross link proteins by their amine groups. Lysine residues in the proteins are the most common source of these amine groups. The aldehyde and amine undergo a condensation reaction to form an imine with the subsequent loss of water. This process is described further in Cheung, D. T. and Nimni, M. E., Connective Tissue Research, 10, 187-216 (1982).
The major constituent of bioprosthetic implants is the protein collagen. To increase the stability of such implants against biodegradation and to reduce the antigenicity, collagen is cross-linked with glutaraldehyde. The problem is that, after implantation, calcification onto the implant gradually occurs, rendering it necessary to replace the implant. The present inventors have established that a PC derivative having two aldehyde groups, or aldehyde group precursors, is usefiil to replace glutaraldehyde in tissue fixing.
The present inventors have synthesised novel functional phosphate diester compounds, which are suitably phosphoryl choline derivatives, which can be used to replace glutaraldehyde to fix tissue and have devised a synthetic method for producing such compounds.
According to the invention there is provided a new process in which a zwitterionic crosslinker of the formula I
X-. (R1) (CHO)n I in which X is a zwitterionic pendant group having an overall neutral charge, R1 is an organic group having n+m functionality, m is at least 1 and n is at least 2, or a gem-diol, hemiacetal or acetal derivative thereof, is contacted in aqueous solution with a substrate having pendant primary amine groups, under conditions allowing reaction of the CHO groups of the compound of the formula I with the primary amine groups of the substrate.
It is generally preferred for the reaction conditions to be such that all of the aldehyde groups (or acetal derivative thereof) react with primary amine groups. Thus the conditions under which the compound of the formula I is contacted with the substrate having pendant amine groups will include lower than stoichiometric amounts of the compound of the formula I. Alternatively, where, following contact of the compound of the formula I with substrate having pendant amine groups results in a product having residual aldehyde groups, the treated substrate may be subjected to a post-treatment step in which residual aldehyde groups are reacted with amine group containing compounds in solution.
Preferably the compound of the formula I is used to replace glutaraldehyde in a tissue fixing process. Thus the proteinaceous substrate preferably comprises primarily coUagenous material, of which the lysine residues are reacted with the aldehyde groups.
The substrate is preferably proteinaceous, and is most preferably a coUagenous substrate. The process of the invention provides crosslinking between pendant amine groups of lysine moieties of the substrate.
The coUagenous material may be skin, connective tissue, bone or the organic matter of teeth. Tissues which may usefully be treated in the invention include heart valves tissue and vessels, veins and arteries, ligaments, tendons and Farcia lata, dura matter, pericardium, nerves and cornea implants.
According to the present invention there is also provided crosslinked proteinaceous materials produced by the process of the present invention. The use of
crosslinking compounds incorporating a zwitterionic pendant group has been found to produce crosslinked coUagenous products which retain good mechanical properties and are expected to have reduced tendency to calcification.
The group X may be a betaine group, for instance a sulpho-, carboxy- or phospho-betaine. A betaine group must have no overall charge and is preferably therefore a carboxy- or sulpho-betaine. If it is a phosphobetaine the phosphate terminal group must be a diester, ie be esterified with an alcohol. Such groups may be represented by the general formula II
-X3-R24-N+(R25)2-R26-V II
in which X3 is a valence bond, -O-, -S- or -NH-, preferably -O-;
V is a carboxylate, sulphonate or phosphate (diester-monovalently charged) anion; R24 is a valence bond (together with X3) or alkylene -C(O)alkylene- or -
C(O)NHalkylene preferably alkylene and preferably containing from 1 to 6 carbon atoms in the alkylene chain; the groups R25 are the same or different and each is hydrogen or alkyl of 1 to 4 carbon atoms or the groups R25 together with the nitrogen to which they are attached form a heterocyclic ring of 5 to 7 atoms; and
R26 is alkylene of 1 to 20, preferably 1 to 10, more preferably 1 to 6 carbon atoms provided that when V is a sulphonate anion, R26 is alkylene of 6 or more carbon atoms. One preferred sulphobetaine monomer has the formula III
R16
I
-*N-(CH2)d-SO3 e III
R I I6
where the groups R
16 are the same or different and each is hydrogen or C
M alkyl and d
Preferably the groups R16 are the same. It is also preferable that at least one of the groups R16 is methyl, and more preferable that the groups R16 are both methyl.
Preferably d is 2 or 3, more preferably 3.
Alternatively the group X may be an amino acid moiety in which the alpha carbon atom (to which an amine group and the carboxylic acid group are attached) is joined through a linker group to the group R1. Such groups may be represented by the general formula IV
NR28 3
-X4. rv
•R27 H "C02
in which X4 is a valence bond, -O-, -S- or -NH-, preferably -O-, R27 is a valence bond (optionally together with X4) or alkylene, -C(O)alkylene- or -C(O) Halkylene, preferably alkylene and preferably containing from 1 to 6 carbon atoms; and the groups R28 are the same or different and each is hydrogen or alkyl of 1 to 4 carbon atoms, preferably methyl, or two of the groups R28, together with the nitrogen to which they are attached, form a heterocyclic ring of from 5 to 7 atoms, or the three group R28 together with the nitrogen atom to which they are attached form a fused ring structure containing from 5 to 7 atoms in each ring. X is preferably of formula V
oΘ
in which the moieties X1 and X2, which are the same or different, are -O-, -S-, - NH- or a valence bond, preferably
-O-, and W+ is a group comprising an ammonium, phosphonium or sulphonium cationic group and a group linking the anionic and cationic moieties which is preferably a C^- alkylene group.
Preferably W contains as cationic group an ammonium group, more preferably a quaternary ammonium group.
The group W+ may for example be a group of formula -W!-N+R23 3, -W'-P+R23^ -W'-S+R23^ or -W'-Het* in which:
W1 is alkylene of 1 or more, preferably 2-6 carbon atoms optionally containing one or more ethylenically unsaturated double or triple bonds, disubstituted-aryl, alkylene aryl, aryl alkylene, or alkylene aryl alkylene, disubstituted cycloalkyl, alkylene cycloalkyl, cycloalkyl alkylene or alkylene cycloalkyl alkylene, which group W1 optionally contains one or more fluorine substituents and/or one or more functional groups; and either the groups R23 are the same or different and each is hydrogen or alkyl of 1 to 4 carbon atoms, preferably methyl, or aryl, such as phenyl or two of the groups R23 together with the nitrogen atom to which they are attached form a heterocyclic ring containing from 5 to 7 atoms or the three groups R23 together with the nitrogen atom to which they are attached form a fused ring structure containing from 5 to 7 atoms in each ring, and optionally one or more of the groups R23 is substituted by a hydrophilic functional group, and the groups R23a are the same or different and each is R23 or a group OR23, where
R23 is as defined above; or
Het is an aromatic nitrogen-, phosphorus- or sulphur-, preferably nitrogen-, containing ring, for example pyridine.
Preferably W1 is a straight-chain alkylene group, most preferably 1,2-ethylene. Preferred groups X of the formula V are groups of formula VI:
where the groups R12 are the same or different and each is hydrogen or C alkyl, and e is from 1 to 4.
Preferably the groups R12 are the same. It is also preferable that at least one of the groups R12 is methyl, and more preferable that the groups R12 are all methyl.
Preferably e is 2 or 3, more preferably 2.
Alternatively the ammonium phosphate estergroup VI may be replaced by a glycerol derivative of the formula VB, VC or VD defined in our earlier publication no WO-A-93/01221.
The compound of the formula I may be a small, non-polymeric compound. Such compounds may have more than one pendant group X, that is m may be more than 1, although more usually m is 1. In such compounds, although n may be more than 2, it is generally found that adequate crosslinking takes place, as for glutaraldehyde, where there are two aldehyde groups per molecule, that is n is 2. In such non-polymeric compounds, the group R1 is generally a C2.12-n+m-functional optionally substituted alkane group. The group R1 may be an alkyl or an aralkyl group and may be interrupted by heteroatoms such as oxygen atoms, amido and/or sulphonamido, groups, or by carbonyl groups and may be substituted by alkyl, alkoxy, aryl, aralkyl, aralkoxy, hydroxyl or alkylamido groups, or halogen atoms. Generically any substituents should have no ionic charge, under aqueous conditions at around pH 7, eg in the range 6-8 preferably 4-10. Most preferably R1 is a C3.24-alkyl group, especially a C3.12-alkyl group.
Preferably the compound of the formula I is a compound of the formula VII
in which X is as defined above; R2 is hydrogen or a C -alkyl group, each of the groups R3 and R4 is independently selected from hydrogen, halogen, optionally substituted Cι.24-alkyl, and hydroxyl groups, or two groups R3 or two groups R4 or one group R3 and one group R4 together represent an optionally substituted C^- alkylene, an optionally substituted C2.g-alkenylene or an optionally substituted C2.8-alkynylene group, or two groups R3 or two groups R4 attached to adjacent carbon atoms may, together with the carbon atoms to which they are attached form a 1 ,2-arylene group,
p is 1-4; q is 1-4;
R5 and R6 are each independently selected from hydrogen and C1.4-alkyl, each R7 is the same and is hydroxyl or C^-alkoxy, each R8 is the same and is selected from hydroxyl and C^ alkoxy, or the groups R7 and R8 attached to the same carbon atom together are =O or Cj. -oxaalkyleneoxy, and
R9 is a bond or a C^ alkylene group.
Compounds of the formula VII are thus dialdehyde compounds or the corresponding gem-diol, hemiacetal or acetal derivatives. Gem-diols, hemiacetal and acetal compounds are precursors to aldehydes in that they can react easily to form the corresponding aldehydes. These compounds are believed to be novel and form a further aspect of this invention. p and q are preferably each 1. Thus the group R1 is preferably a linear C3 alkane derivative, in which the CHO groups are joined at the 1 and 3-positions whilst the zwitterionic group is joined through the 2 position.
Preferably each group R3 and R4 is independently selected from hydrogen, optionally substituted C^-alkyl, hydroxyl, protected hydroxyl and amine.
Preferred substituents in alkyl groups R3 and R4 are hydroxyl, amino and halogen. Preferably each of R3 and R4 is unsubstituted C,.4-alkyl or hydrogen, most preferably hydrogen.
Preferably R5 and R6 each represent hydrogen.
The novel compounds may be made by a process in which a compound of the formula VIII
in which a) either R
10 is CR
12 2 and
R11 is CR5=CR12 2 or b) R10 and Rπ together are CR5; and in which each R12 is selected from hydrogen and C alkyl; R5 and R6 are each hydrogen; and
R3, R4, R9, X, q and p have the same meanings as in the compound of the formula VII, is oxidised to form the dialdehyde compound upon oxidation at the double bond(s). The dialdehyde compound may be reacted with a water, an alcohol or a glycol to form the corresponding gem-diol, hemiacetal or acetal, that is in which each of R7 and R8 represents hydroxyl or alkoxy. Such gem-diols, hemiacetals and acetals are found to be relatively stable to long term storage.
Compounds of the formula VIII believed to be novel are further claimed in our copending application filed even date herewith claiming priority from GB 97303847.4.
The oxidation reaction of the compound of the formula VIII to form the compounds of the formula VII has been found to produce stable products, where there are 3 carbon atoms between the groups R6C(R7)R8 and R5C(R7)(R8) (ie q=p=l ), or where there are more than 3 carbon atoms and the groups R6C(R7)R8 and R5C(R7)R8 are kept apart by steric means to prevent an intramolecular alcohol condensation taking place.
Where q+p>2, it is preferred for one group R3 and one group R4 together to represent a Cj.2, preferably -alkylene, usually methylene. The dialdehyde product of the oxidation of such compounds is resistant to an internal aldol condensation and is relatively storage stable in the form of the gem-diol, hemiacetal or acetal derivative. The oxidation reaction may be carried out using an appropriate oxidising agent, such as ozone in conjunction with hydrogen and a palladium-carbon catalyst, with zinc and acetic acid, with iodide and acetic acid, with dimethyl sulphide, with thiourea, with triphenylphosphine, with trimethylphosphite, or with pyridine or periodate, for instance in the presence of a catalyst such as osmium tetroxide. Other oxidising agents known to generate aldehyde groups or precursors thereof from ethylenically unsaturated starting materials are potassium permanganate, sodium periodate with potassium permanganate catalyst, with ruthenium (III) chloride or ruthenium (VI) dioxide catalyst, (bi
py)H2CrOCl5 and potassium permanganate and silica gel. These systems are further described in Comprehensive Organic Transformations, by Larock, R. C, VCH, 1989, pp595-596.
The oxidation reaction is preferably carried out with the compound of the formula VTII in solution, for instance in water or an organic solvent, or a mixture thereof. Suitable organic solvents are alcohols, dimethyl formamide or glacial acetic acid.
The oxidation reaction may be carried out with cooling. The reaction is generally carried out to completion, using an excess of oxidising agent.
The novel starting material of the formula VIII may be made from known starting materials by various routes. In the synthetic process the zwitterionic group X is introduced into the molecule prior to oxidation of the ethylenic bond(s), using suitable chemistry. The synthesis of sulphobetaine monomer may proceed by the reaction of a precursor having a reactive group and a preformed sulphobetaine group with an ethylenically unsaturated alcohol, amine or carboxylic acid. Alternatively a sulphobetaine may be formed by reaction of a tertiary amine with a 1,3 propane sulfone.
Amino acid type compounds may be made by processes analogous to those described in our earlier application no WO-A-9416749. Where, as in the preferred aspect of the invention, the zwitterionic group is a group of formula V in which W+ is W1 N+ R23 3 which W1 is C2 or C3-alkylene (optionally substituted) and at least two of the groups R23 are methyl, the compound of the formula VIII may be formed by the reaction of a compound of the formula IX
in which the groups R3, R4, R6, R9, R10, R11 have the same meanings as in the compound of the formula Vm, X1 and X2 are as defined in the group of formula V is reacted of the compound with a phospholane reagent of the formula II
11
in which Hal is a halogen atom, preferably chlorine,
R13 is a bond or a group C(R14)R15, each group R14 is selected from hydrogen and CM-alkyl groups; each group R15 is selected from hydrogen and C,.4-alkyl, or two groups R15 may form a Cι.5-alkylene group to produce a phospholane intermediate of the formula X
in which all the groups have the same meanings as in the respective compounds of the general formulae IX and X. Subsequently the intermediate of the formula X is subjected to a ring opening amination with an amine NR23 3 reaction to produce the compound of the formula VIII.
In the phospholane reagent of the formula X, the groups R14 and R15 are preferably all the same, each preferably being hydrogen.
Where R5 is N+R6 3, the ring opening reaction is carried out under anhydrous conditions. Preferably each of the groups R23 is methyl. This basic phospholane ring opening reaction has been described by Thuong and Chabrier in Bull. Soc. Chim. de France (1974) (3-4) 667-670 and in FR-A-2,270,887.
The compound of the formula VII is preferably used as a crosslinking reagent for reacting with substrates having pendant primary amine groups, especially proteinaceous substrates. In this application of the compound of the formula VII, it is preferred for the reaction to be conducted in an aqueous environment. The invention is illustrated in the following examples:
Example 1 - Synthesis of 3-Methoxy-fethyl-2'-(trimethylammonium ethyl) phosphate) - hexan-1.6-dial
1.1: Synthesis of 3-Cvclohexen- 1 -methoxyethyl-2'-(trimethylammonium ethyl) phosphate 1.1 To a solution of +/- 3 -cylcohexen-1 -methanol (2.60g, 0.023mole) in dry acetonitrile (60ml) and triethylamine (4. OOg, 0.039mole) at - 10 ° C, a solution of 2-chloro-
2-oxo-l,3,2-dioxaphospholane (CCP) (4.37g, 0.030mole) in acetonitrile (15ml) was slowly added. The reaction mixture was allowed to warm to RT over a period of 1 h.
The solids were removed by filtration, and the solution added to a flask containing trimethylamine (2.4g, 0.041mole) in acetonitrile (50ml). The resulting mixture was heated at 50 °C overnight. The solvents were removed in vacuo to afford a viscous oil
(7.10g, 0.025mole), contaminated by slight amounts of trimethylamine. hydrochloride, and acetonitrile:
Η NMR (199.5MHZ, CDC13), 1.2 (1H, m, CH), 1.6-2.2 (6Η, m, 3xCH2), 3.43 (9Η, s, NMe3), 3.74 (2H, m, CH2OP), 3.8-4.6 (4Η, m, OCH2-CH2-N), 5.65 (2Η, s, CH=CH).
13C NMR (50.1MHz, D2O), 23(CHCH2CH2CH=), 25(CHCH2CH2CH=), 27(=CHCH2CH), 33(CH), 53(NMe3), 59(POCΗ2), 65(CH2N), 70(CH2O), 125(=CHCH2CH), 127(CHCH2CH2CH=).
An analogous process can be carried out using in place of the 3-cyclohexene-l- methanol, 3 -methyl-2-cyclohexen- 1 methanol or 2-cyclohexen- 1 -ol or 3 , -methyl 5 , 5 tri-2- cyclohexen- 1 -methanol .
1.2: Synthesis of 3-Methoxy-(ethyl-2'-ftrimethylammonium ethyl) phosphate) -hexan-
1.6-dial
1.2.1 - Osmium tetroxide.
To a solution of 3-cyclohexen-l-methoxyethyl-2'-(trimethylammonium ethyl) phosphate (2.771g, 0.0099M) in 1,4-dioxane / water (50ml, 1:1, v/v), a grain of osmium tetroxide ( lmg) was added, and the solution stirred for 30 min. Sodium periodate (6.727g, 0.030mole) was slowly added over 30 min and the reaction stirred for 6 h. The solid material was filtered off, and the solvent removed in vaccuo to give a white solid. The solid was dissolved in ethanol (50ml) and again filtered. The solution was concentrated to give a viscous oil (3.03g, 0.0097mole) still containing some solvent and sodium iodide salts:
ΗNMR (199.5MHz, D2O), 1.3-1.9 (7H, m, CH2CH2CHCH2), 3.23 (9Η, s, NMe3), 3.7-
4.3 (6H, CH2OPOCH2CH2N), 9.7(2Η, CH=O).
13C NMR (50.1MHz, D2O) 30.8, 45.2, 53.4(ref NMe3), 59.0, 60.3, 65.6, 66.7, 206.1.
IR(KBr) 3401, 2946, 1716, 1670, 1479, 1398, 1220, 1040, 971, 875, 765, 666, 502.
1.2.2 -Ozone. Ozone was bubbled through a solution 3-cyclohexen-l-methoxyethyl-2'-
(trimethylammonium ethyl) phosphate (7.52g, 0.027mole) in ethanol (200ml) at -78 °C, for 5 h. Triphenylphosphine (14. Og, 0.053mole) was added to quench the reaction mixture, whereupon the reaction darkened from colourless to yellow, and then a fine white precipitate was observed. The reaction mixture was stirred for 48 h. The reaction mixture was filtered, and the solvents removed in vacuo. Dissolution of the resulting oil proved problematic, and 'Η NMR analysis of the reaction mixture showed a complex mixture of signals indicating that there may be decomposition of the starting material.
Example 2
Synthesis of 3-(Oxyethyl-2'-(trimethylammonium ethyl) phosphate)-pentan-l. 5-dial 2.1-2.2: Synthesis of 3-Cvclopenten-l-ol
2.1 To a stirred solution of cyclopentadiene (57.8g, 0.87mole), and sodium carbonate (400g) in dichloromethane ( 1000ml) at 0 ° C, peracetic acid ( 168ml, 40% in acetic acid), pre-treated with sodium acetate (0.2g) was added slowly, and the reaction allowed to stir for 4 h. The reaction mixture was filtered, and the solvent distilled off. [Η NMR (199.5MHz, CDC13) 2.0-3.0(m, CH-O-CH), 3.8(d, CH2), 6.1(m, CH=CH)]
2.2 The residue was slowly added to a slurry of lithium aluminium hydride (13.0g, 0.36mole) in diethyl ether (400ml) at 0°C, and stirred overnight. The reaction was quenched by slow addition of water (50ml), and then after 10 min dried magnesium sulphate was added (50-70g). The solids were filtered off, and then washed with diethyl ether (2x200ml). The organic layers were combined, and the solvent was removed carefully in vacuo (no heating). 3-Cyclopenten-l-ol (20.38g, 0.243mole) was isolated from the mixture by distillation (27°C, ca 754 mmΗg).
ΗNMR (199.5MHz, CDC13) 1.9(1H, br.s, OH), 2.2-2.8(4Η, qd, CH2CHCH2), 4.5(1Η, br.m, CHOH), 5.7(2H, s, CH=CH).
13C NMR (50.1MHz, CDC13) 42.0 (CH2), 10.8 (CHOH), 122.4(C=Q.
2.3: Synthesis of 3-Cvclopenten-l-oxyethyl-2'-("trimethylammonium ethyl) phosphate
To a solution of 3-cyclopenten-l-ol (8.04g, 0.091mole), and N,N,N,N- tetramethylethylenediamine (6.33g, 0.055mole) in acetonitrile (160ml) at -10°C, a solution of 2-chloro-2-oxo-l,3,2-dioxaphospholane (15.6g, 0.073mole) in acetonitrile (50ml) was added, and the reaction allowed to warm to RT over 1.5 h. The reaction mixture was filtered, and then trimethylamine (12.68g, 0.21 mole) was added. The reaction was heated at 50° C in a closed system for 18 h. The reaction was cooled, and solvents removed in vacuo. The reaction mixture was taken up into water, and washed
with chloroform (2x 100ml) . The aqueous layer was concentrated to yield 3 -cyclopenten- l-oxyethyl-2'-(trimethylammonium ethyl) phosphate (15.3g, O.Oόlmole) as an oil.
1HNMR(199.5MHz,D2O) 2.1-2.4(4H, m, 2x-CH2-), 3.0(9Η, s, NMβj), 3.3-4.1(5H, m, CHOP, OCH2CH2N), 5.5(2Η, s, CH=CH).
13C NMR (50.1MHz, D2O) 39.5(CH2), 53(ref NMe3), 58.5&60.0, 65.0&66.5(OCH2CH2N), 75.5(CHOP), 127.2(C=C).
m/s (FAB+) 336(M+«NMe3 «H2O), 250(M+), 185
Ir (KBr smear) 3401, 2959, 2510, 1653, 1483, 1220, 991, 769, 668, 510.
2.4: Synthesis of 3-(Oxyethyl-2 Wtrimethylammonium ethyl) phosphateVpentan - 1.5-dial (ethoxyacetalderivative)
To a solution of 3-cyclopenten-l-oxyethyl-2'-(trimethylammonium ethyl) phosphate (2.43g, 0.0097mole) in water (100ml), a grain of osmium tetroxide ( lmg) was added, and the reaction stirred at RT for 30 min. Sodium periodate (5.43g, 0.0254mole) was added slowly, and the reaction stirred at RT for 3 h, during which a colour change to dark orange, and then back to colourless was observed. Ethanol
(200ml) was added to quench the reaction, and the precipitate was filtered off. The solvent was removed in vacuo to afford quantitative yield of 1, 1,5,5 -tetraethoxy-3-
(oxyethyl-2'-(trimethylammonium ethyl) phosphate)-pentane (based on 13C NMR), i.e. the ethoxy acetal derivative of the dialdehyde.
1H NMR (199.5MHz,D2O) 1.1-2.0(4H, m, 2xCH2), 3.0(9Η, s, NMe3), 3.3-4.2(7H, m,
CHOP, OCH2CH2N, CH(O(D))2).
1 CNMR(50.1MHz, D2O) 17(CΗ3CΗ2O), 35&37(-CH2-), 53(refNMe3), 57(CH3CH2O), 58&60(CH2OP), 65&66(CH2N), 70(CHOP), 90(CH(OEt)2).
IR (KBr smear) 3401, 2505, 2360, 1653, 1481, 1218, 1085,971,759,513.
Example 3 - Tissue interaction
3.1 Tissue Preparation:
The tissue was stored in phosphate buffer solution (PBS) for 3 hours and then washed in fresh PBS prior to cutting. The tissue was cut into strips of 1 cm by 6 cm.
Eighteen strips was used for the fixation solutions and the rest stored in PBS, in the fridge, as control samples. A small sample of tissue from each of the three solutions was investigated by Differential Scanning Calorimetry (DSC).
3.2 Preparation of Fixation solutions: The fixation solutions of 0.35 %w/v glutaraldehyde and 1.03 %w/v 3 -[(oxy)( 1,1,1 trimethyl ammonium) phosphoranate]- pentan-1, 5-dial (Glut-PC) in PBS was prepared as follows -
A. Glutaraldehyde solution contains 0.35% w/v glutaraldehyde and 154mM sodium chloride and has a pH in the range 7.0 to 7.5. B. Glut-PC solution contains 1.03% w/v Glut PC (equimolar with glutaraldehyde),
154mM sodium chloride and 5.26mM trisodium phosphate for adjusting the pH to be in the range 6.5 to 7.0.
Two sets of 9 strips of tissue were placed into vials containing the control fixation solution (45mls) or the test solution. Each vial was then stored inside a brown-glass bottle for 96 hours at 25 °C.
3.3 Method for testing the Cross-linked tissue:
A modified method of testing the shrinkage temperature of leather (BSI 3144: 1968 Sampling and Physical Testing of Leather) was used to test the tissue.
The three types of tissues were suspended in a beaker containing a solution of PB S with a thermometer placed at close proximity to the tissue. The solution of PB S was heated in water at a constant power to provide a temperature rise of approximately 2 °C/min from 40 °C to 90 °C. The change in physical appearances of the samples was recorded prior to testing and recorded in Table 1. Also a thermometer positioned close to the sample was used to measure the temperature rise with time. Inflexion points in the curve were recorded and are reported in Tables 2-4. The results of the DSC test is reported in Table 5.
Results
Physical Appearance prior to testing:
Table 1
Experimental Results: Experiment 1
Table 2 Starting temperature - 40°C Final Temperature - 89°C
Experiment 2
Table 3
Starting temperature - 36.5°C Final temperature - 88.5°C
Experiment 3 :
Table 4 Starting temperature - 36°C Final Temperature - 86°C
DSC Experimentation- Conditions - Sealed pans containing samples.
Heating 25°C - 100°C at 10°C/minute.
Table 5
The results show that the PC group containing compound performs differently to glutaraldehyde. The physical properties of the treated tissue appear to be more similar to those of the untreated tissue, which is believed to be desirable.
Example 4
4.1 Synthesis of 5-|"(oxy)-( 1.1.1 trimethyl ammonium) phosphoranate methyl] -bicyclo f2.2.11 hept-2-ene.
To a solution of 5-norbornene-2-methanol (1.00 g, 0.0079 mol) and N,N,N\N'- tetramethylethylenediamine (0.46 g, 0.0038 mol) in acetonitrile (30 ml) at 0 °C, a solution of 2-chloro- 1 ,3 ,2-dioxaphospholane-2-oxide (1.13 g, 0.0079 mol) in MeCN (10 ml) was slowly added over 20 minutes under N2. The solution was stirred for 4h. The solution was filtered and cooled to 0 °C. Trimethylamine (1.17 g, 0.0198 mol) was added and the solution heated at 50 °C overnight in a closed system. The solution was cooled, degassed, and the solution decanted. The solvent was evaporated and the residue partitioned between H2O (20 ml) and Et2O (20 ml). The aqueous layer was washed with Et2O (20 ml), separated and evaporated. The yellow oil was identified as the product: Η NMR (400 MHz, D2O) δ complex spectra.
13C NMR (50.1 MHz, D2O) δ 27.0 (CH2CH(CH2O)), 38.0 (CHCH2OP), 41.8 (C(CH2)CH), 43.5 (C(CH2)CH2), 47.8 (CH2 bridge), 54.0 (M&N), 58.1 (CH2NMe3), 64.8 (CH2OP), 68.5 (CHCH2OP), 131.1 (CHCH, trans), 135.4 (CHCH, cis), 136.2 (CHCH, cis) 136.7 (CHCH, trans).
4.2 Synthesis of 4-[Yoxy (l.l.l trimethyl ammonium) phosphoranate methyll- cyclopentan-1. 3-dicarbaldehvde. A 5% solution of 5-[(oxy)-(l , 1 , 1 trimethyl ammonium) phosphoranate methyl] -bicyclo
[2.2.1] hept-2-ene (1.00 g, 0.0035 mol) in H2O (20 ml) was subjected to a stream of O3
(0.0017 mol/min, 2 min.) at 5 °C. Triphenyl phosphine (1.00 g, 0.0038 mol) was added and stirred for lh at room temperature. The solution was filtered, frozen and the water removed by freeze-drying.
Η NMR (400 MHz, D2O) δ complex spectra.
13C NMR (50.1 MHz, D2O) δ 29.3 (CH2CH(CH2O)), 41.2 (CHCH-OP), 43.5
(C(CH2)CH), 44.7 (C(CH2)CH2), 51.1 (CH2 bridge), 54.0 (M&N), 59.5 (CH2NMe3),
61.2 (CH2OP), 66.0 (CHCH2OP), 97.4 & 101.4 (CH(OH)2).
DNPH test - yellow precipitate formed (positive result).
Proton and carbon nmr are consistent with the following proposed reaction scheme
Analogous methods to those of Example 4.1 may be used to make the following cycloalkene compounds
From these dialdehydes may be formed using techniques analogous to Example
4.2.
Example 5
5.1 Synthesis of 1.6-heptadien-4-ol:
To a suspension of activated magnesium ribbon [activated overnight by stirring in an inert atmosphere] (19.6 g, 0.82 mol) and anhydrous diethyl ether [Et2O] (307 ml), an initiating solution of allyl bromide (10%, 9.0 g, 0.074 mol) in Et2O (13 ml) was added. Once the reaction had started to reflux gently, the remainder of the allyl bromide (81.0 g, 0.646mol) in Et2O( 119 ml) was slowly added maintaining a gentle reflux. The reaction was left refluxing for 2h. The mixture was cooled in an ice bath and a solution of ethyl formate (24.77 g, 0.33 mol) in Et2O (53 ml) was slowly added to the stirred mixture over lh. The reaction was left to warm to room temperature overnight. The reaction was quenched with saturated ammonium chloride solution (260 ml), and H2O then added (250 ml). The aqueous layer was acidified with dilute (0.1 M) hydrochloric acid and the organic layer separated. The organic layer was washed with NaHCO3 (300 ml), H2O (200 ml), andNaCl (200 ml). The organic layer was dried with anhydrous sodium sulphate and the solvent removed. The resulting pale yellow oil was identified as a mixture of the alcohol and the formate ester (approximately 5:2 ratio):
Η NMR (400 MHz, CDC13) δ 1.90 (1H, m, CHOH), 2.20 (2H,m, CHCH2), 2.35 (2H, m, CHCH2), 3.70 (1H, m, CHOH), 5.15 (4H, m, 2xCHCH2), 5.75 (2H, m, 2xCHCH2), 8.05 (1H, s, OC(H)-O-CH). 13C NMR (50.1 MHz, CDC13) δ 37.8 (CH2), 41.2 (CH2), 69.7 (CHOH), 72.4 (CHO- COH), 118.1 (CHCH2), 118.3 (CHCH2), 133.0 (CHCH2), 134.6 (CHCH2), 160.7 (OC(H)-O-CH).
5.2 Synthesis of 4-[foxy)( 1,1.1 trimethyl ammonium) phosphoranate]- 1.6-heptadiene. To a solution of l,6-heptadien-4-ol (10.00 g, 0.089 mol) and N,N,N',N'- tetramethylethylenediamine (5.18 g, 0.045 mol) in acetonitrile [MeCN] (100 ml) at 0 °C, a solution of 2-chloro-l,3,2-dioxaphospholane-2-oxide (12.72 g, 0.089 mol) in MeCN (60 ml) was slowly added over 20 minutes under N2. The solution was stirred for 4h. The solution was filtered and cooled to 0 °C. Trimethylamine (13.17 g, 0.223 mol) was added and the solution heated at 50 °C overnight in a closed system. The solution was cooled, degassed, and the solution decanted. The solvent was evaporated and the residue partitioned between H2O (150 ml) and dichloromethane [DCM] (150 ml). The aqueous
layer was washed with DCM (150 ml), separated and evaporated. The residue was washed with acetone (2x 150 ml) and stored in acetone (100 ml) overnight. The acetone layer was evaporated to leave a solid residue. The solid residue was identified as the product: Η NMR (400 MHz, D2O) δ complex spectra.
13C NMR (50.1 MHz, D2O) δ 43.5 (CH2), 54.0 (M&NCH,), 55.5 (CH2NMe3), 59.8 (CH2OPO), 65.7 (CHOP), 118.1 (CHCH2), 134.2 (CHCH2).
5.3 Synthesis of 3-|Yoxy)(l.l.l trimethyl ammonium) phosphoranate]- pentan-1.5- dial. A 10% solution of 4-[(oxy) (1,1,1 trimethyl ammonium) phosphoranate]- 1,6- heptadiene (2.00 g, 0.0073 mol) in H2O (20 ml) was subjected to a stream of O3 (0.0017 mol min, 10 min.) at 5 °C. Triphenyl phosphine (4.75 g, 0.018 mol) was added and stirred for lh at room temperature. The solution was filtered, frozen and the water and formaldehyde removed by freeze-drying. Η NMR (400 MHz, D2O) δ complex spectra.
13C NMR (50.1 MHz, D2O) δ 43.8 (CH2), 54.0 (M&NCH,), 55.6 (CH2NMe3), 60.0
(CH2OPO), 65.8 (CHOP), 91.8 & 92.7 (CH(OH)2).
DNPH test - orange precipitate formed. [Formaldehyde produces a bright yellow precipitate] The reaction is believed to take place according to the following scheme:
6.1 Synthesis of 3. 5 -dimethyl- 1.6-heptadien-4-ol:
To a suspension of activated magnesium ribbon [activated overnight by stirring in an inert atmosphere] (6.22 g, 0.26 mol) and anhydrous tetrahydrofuran [THF] (110 ml), an initiating solution of 3 chloro-1-butene (10%, 2.0 g, 0.02 mol) in THF (6 ml) and a crystal of iodine were added. Once the reaction had started to reflux gently, the remainder of the 3 chloro- 1 -butene ( 18.0 g, 0.199 mol) in THF (51 ml) was slowly added maintaining a gentle reflux. The reaction was left refluxing for 2h and heated further at 70 °C for 1 h. The mixture was cooled to room temperature and a solution of ethyl formate (7.38 g, 0.10 mol) in THF (20 ml) was slowly added to the stirred mixture over lh. The reaction was left overnight. The reaction was quenched with saturated ammonium chloride solution (270 ml). The organic layer was separated and washed with NaHCO3 (275 ml) and brine solution (250 ml). Ethyl acetate (300 ml) was added and organic layer separated. The organic layer was treated with charcoal, filtered with celite and the solvent removed. The resulting yellow oil was believed to be a mixture of the three types of alcohol and their ester derivatives:
Η NMR (400 MHz, CDC13) complex spectra, δ 0.9 - 1.10 (3H, m, CHMe), 1.60 (3H, d, MeCH=), 2.20 (2H,m, CHCH2), 2.35 - 2.55 (2H, m, CHCH2), 3.25 - 3.70 (1H, m, CHOH), 5.00 - 5.15 (4H, m, 2xCHCH2), 5.65 - 5.85 (2H, m, 2xCHCH2 and MeCHCH), 8.05 - 8.15 (1H, s, OC(H)-O-CH).
13C NMR (50.1 MHz, CDC13) complex spectra, δ 15 - 20 (CH3), 38.0 - 41.5 (CH2 and CHMe), 75 - 80 (CHOH and CHO-COH), 114 - 118 (CHCH2 and CHCHMe), 138 - 141 (CHCH2 and CHCHMe), 160.7 (OC(H)-O-CH).
6.2 Synthesis of 4-[(oxy)-( 1.1.1 trimethyl ammonium) phosphoranate] -3. 5-dimethyl- 1.6-heptadiene.
To a solution of 3, 5-dimethyl-l,6-heptadien-4-ol (2.00 g, 0.014 mol) and N,N,N',N' -tetramethylethylenediamine (0.83 g, 0.007 mol) in acetonitrile [MeCN] (20 ml) at 0 °C, a solution of 2-chloro-l,3,2-dioxaphospholane-2-oxide (2.04 g, 0.14 mol) in MeCN (25 ml) was slowly added over 20 minutes under N2. The solution was stirred for 4h. The solution was filtered and cooled to 0 °C. Trimethylamine (3.10 g, 0.053 mol) was added and the solution heated at 50 °C overnight in a closed system. The solution was cooled, degassed and the solution decanted. The solvent was evaporated and the
residue partitioned between H2O (125 ml) and ether [Et2O] (130 ml). The aqueous layer washed with Et2O (130 ml), separated and evaporated. The residue was identified as a phosphorous species, but without the dialkene. Η NMR (400 MHz, D2O) δ complex spectra. I3C NMR (50.1 MHz, D2O) δ 54.0 (M&NCH,), 55.0 (CH2NMe3), 62.2 (CH2OPO).
The product contained several isomers and was not further treated. The product could be separated into the individual isomers and be subjected to ozonolysis to form analogous dialdehydes to those of example 5, for instance having the formula
Analogous methods may be used to make the following PC dialdehyde compounds