WO1991003479A1 - Synthesis of stable, water-soluble chemiluminescent 1,2-dioxetanes and intermediates therefor - Google Patents

Abstract

A novel synthesis of compounds having formula (I), wherein T is a stabilizing spiro-linked polycycloalkylidene group, R3 is a C¿1?-C20 alkyl, aralkyl or heteroatom containing group, Y is an aromatic fluorescent chromophore, and Z is a cleavable group which, when cleaved, induces decomposition of the dioxetane ring and emission of optically detectable light, is disclosed. A tertiary phosphorous acid alkyl ester of the formula (R?1O)¿3P, wherein R1 is a lower alkyl group, is reacted with an aryl dialkyl acetal produced by reacting a corresponding aryl aldehyde with an alcohol of the formula R3OH, wherein R3 is as defined above, to produce a 1-alkoxy-1-aryl-methane phosphonate ester of formula (II), reacting the phosphonate with base to produce a phosphonate-stabilized carbanion, reacting the carbanion with a ketone of formula (III), wherein T is as defined above, to produce an enol ether of formula (IV), then oxygenating the double bond in the enol ether to give the corresponding 1,2-dioxetane compound. Hydroxyaryl enol ether alkali metal salts having formula (V), in which T is a fused, substituted or unsubstituted polycycloalkylidene group, OR3 is an ether group, Y is a light-emitting fluorophore-forming group which will be part of a luminescent substance formed by decommposition of a 1,2-dioxetane subsequently formed from the hydroxyaryl enol ether alkali metal salt, capable of absorbing energy to form an excited state from which it emits optically detectable energy to return to its ground state, and AM+ is an alkali metal cation, processes for the preparation of these intermediate salts and their use as starting materials for acylation, phosphorylation and glycosylation reactions to give intermediates reactable to give stable, water-soluble chemiluminescent 1,2-dioxetanes, particularly ones that are enzymatically cleavable, are also disclosed.

Description

DESCRIPTION

Synthesis of Stable, Water-Soluble Chemiluminescent 1,2-Dioxetanes and Intermediates Therefor

Background of the Invention

Field of the Invention

This invention relates to a novel chemical

synthesis of stable, water-soluble chemiluminescent 1,2-dioxetanes and to novel intermediates obtained in the course of synthesizing such 1,2-dioxetanes.

Description of Related Art

1,2-Dioxetanes, cyclic organic peroxides whose central structure is a four-membered ring containing pairs of contiguous carbon and oxygen atoms (the latter forming a peroxide linkage), are a known, but until recently seldom utilized, class of compounds. Some 1,2-dioxetanes can be made to exhibit chemiluminescent decomposition, e.g., by the action of enzymes, as described in the following copending, commonly-assigned U.S. patent applications: Bronstein, Serial No.

889,823, "Method of Detecting a Substance Using

Enzymatically-Induced Decomposition of Dioxetanes", filed July 24, 1986; Bronstein et al., Serial No.

140,035, "Dioxetanes for Use in Assays", filed December 31, 1987; Edwards, Serial No. 140,197, "Synthesis of 1,2-Dioxetanes and Intermediates Therefor", filed

December 31, 1987; Edwards, et al., Serial No. 213,672, "Novel chemiluminescent Fused polycyclic Ring-Containing 1,2-dioxetanes and Assays in Which They Are Used", filed June 30, 1988; as well as in Bronstein, I.Y. et al., "Novel Enzyme Substrates and Their Application in

Immunoassay", J. Biolum. Chem., 2:186 (1988).

The amount of light emitted during such

chemiluminescence is a measure of the concentration of a luminescent substance which, in turn, is a measure of the concentration of its precursor 1,2-dioxetane. Thus, by measuring the intensity of luminescence, the

concentration of the 1,2-dioxetane, and hence the concentration of a substance being assayed (e.g:, a biological species bound to the 1,2-dioxetane member of a specific binding pair in a bioassay) can be

determined. The appropriate choice of substituents on the 1,2-dioxetane ring allows, her alia, for adjustment of the chemical stability of the molecule which, in turn, affords a means of controlling the onset of chemiluminescence, thereby enhancing the usefulness of such chemiluminescence for practical purposes, e.g., immunoassays, nucleic acid probe assays, enzyme assays, and the like.

The preparation of 1,2-dioxetanes by

photo-oxidation of olefinic double bonds is known.

Mazur, S. et al., J. Am. Chem. Soc., 92:3225 (1970). However a need exists for a facile, general synthesis of substituted 1,2-dioxetanes from olefinically-unsaturated precursors derived from readily available or obtainable starting materials through tractable intermediates. In this connection, a particular need exists for a

commercially useful method for producing 1,2-dioxetanes of the general formula:

wherein T, R³, Y and Z are defined herein below, from enol ether-type precursors of the general formula:

McMurry et al. [McMurry, J.E., et al., J. Qrα.

Chem., 43:3255 (1978)] described titanium-induced

reductive coupling of carbonyl groups to form olefins. Schaap, A.P., EPO 254,051, published January 27, 1988, and Bronstein, I.Y., 1986, disclose the use of this reaction to produce compounds of formula (II) by the following general reaction:

Several problems with aforementioned unsymmetrical McMurry coupling are especially important in the radical based mechanism which operates in the above equation when compared with similar mixed couplings between aliphatic and diaryl ketones where the mechanism is ionic in nature. The need to often use molar excesses of the expensive T = O ketone over ester co-reactants in an attempt to favor the mixed coupling product, while at the same time obtaining low yields at best, makes this approach suitable only for small scale preparations.

Furthermore, the well-known capricious nature of the reaction, the large amounts of TiCl₃/LiAlH₄ required to effect the coupling, and the formation of by-products which are difficult to separate from the desired enol ethers also limit the commercial utility of the process. In addition, certain useful meta-substituted starting materials such as:

cannot be used with the McMurry reagents as such substituent groups would be reduced, hydrolysed, or would take part in reductive coupling with T = O. Thus, the double bond cannot be introduced regiospecifically in every case.

Enol ethers have also been prepared by Peterson or Wittig reactions of alkoxymethylenesilanes or

phosphoranes with aldehydes or ketones in basic media [Magnus, P., et al., Organometallics, 1:553 (1982);

Wynberg, H. and Meijer, E.W., Tetrahedron Lett., 41:3997 (1979)]. Bronstein, 1986, above, describes the

synthesis of an olefin of formula (II) above using a Wittig reaction of a phosphonium ylide with a T = O ketone. A major advantage of the Wittig reaction is that it is an ionic reaction, where the double bond can be introduced regiospecifically in almost every case.

One problem with the Wittig reaction, however, is that the product alkene is difficult to separate from the phosphine oxide by-product because of the similar solubility characteristics of these compounds. Another problem is that the initially-produced phosphonium ylides can be made only from relatively expensive phosphine starting materials [Walker, B.J., in Cadogan. J.I.G., ed., "Organophosphorus Reagents in Organic

Synthesis", Academic Press. N.Y., (1978), pp. 155-205]. Also, as phosphonium ylides are relatively weakly nucleophilic, they will react only with a limited range of carbonyl compounds, and can require relatively harsh reaction conditions to do this [Gushurst, A.J., et al., J. Org. Chem., 53:3397 (1988)]. Finally, side reactions frequently occur in the Wittig reactions, which also contribute to relatively low yields [Homer, L., et al., Chem. Ber., 95:581 (1962)].

Because of the many problems attendant upon both the McMurry and Wittig reactions, particularly when used to synthesize olefinic intermediates for enzyme-cleavable 1,2-dioxetanes on a commercial scale, a more-suitable route to enol ether derivatives useful in the synthesis of stable, water-soluble, enzyme-cleavable chemiluminescent 1,2-dioxetanes was needed.

Summary of the Invention

This invention fills this need. A new synthesis of stable, water-soluble chemiluminescent 1,2-dioxetanes, particularly ones that are enzyme-cleavable, substituted with stabilizing and solubilizing groups and ring-containing fluorophore moieties, that avoids problems inherent in previously-employed reactions, has now been discovered. In particular, this invention is concerned with a synthetic route to such 1,2-dioxetanes that employs, for the first time, dialkyl 1-alkoxy-1-arylmethane phosphonate-stabilized carbanion

intermediates in the synthesis of key enol ether

intermediates for the desired 1,2-dioxetane end

products.

The use of phosphonate-stabilized carbanions in a Horner-Emmons reaction [Homer, L., et al., Chem. Ber., 91:61 (1958); Wadsworth, W.S., J. Am. Chem. Soc.,

83:1733 (1961)] for the production of enol ethers used in the synthesis of stable, water-soluble,

chemiluminescent 1,2-dioxetanes such as those of formula (I) above, has been found to exhibit several advantages over previous methods for synthesizing such enol ether intermediates. These include: regiospecific

introduction of the olefinic double bond in the presence of a wide range of ancillary functional groups;

increased nucleophilicity compared to the phosphonium ylides, which not only increases the variety of ketones with which the phosphonate-stabilized carbanions can react, but also permits this reaction to be carried out under milder conditions; more-readily separable alkene and phosphorous-containing byproducts than can be obtained using the Wittig reaction (the phosphoric acid diester salt by-products produced by practicing this invention are highly water-soluble); facile betaine formation due to enhanced reactivity and stability of the phosphonate carbanions compared to the phosphonium ylides; and starting materials, i.e., trialkylphosphites, that are more cheaply and conveniently prepared than the more-expensive phosphines necessary for the Wittig reaction.

It has also been discovered that, not only does the reaction of an arylaldehyde dialkyl acetal with a trialkylphosphite or a trialkylsilyldialkylphosphite in the presence of a Lewis acid [Burkhouse, D., et al.,

Synthesis, 330 (1984); Oh, D.Y., et al., Syn. Comm., 16(8) 859 (1986)] provide a general and facile route to the phosphonate intermediates for Horner-Emmons

reactions with T = O ketones or diones (O = T = O) than does the previously known route employing the Arbuzov reaction [Arbuzov, A.E., et al., Chem. Ber., 60:291 (1927)] between an alpha alkoxy arylmethyl halide and a trialkylphosphite, but also that the aryl moiety of the thus-employed arylaldehyde dialkyl acetal, which may be open chain or cyclic (e.g., a 1,3-dioxolane or dioxane), can be substituted with electron-donating or

-withdrawing meta-substituents. The resulting

meta-substituted dialkyl 1-alkoxy-1-arylmethane

phosphonates, with one exception not useful in the present invention [Creary, X., et al., J. Org. Chem., 50:2165 (1985)], are unknown in the prior art.

Meta-substituted aryl groups are preferred, as the ultimate production of an electrondonating moiety in this position, relative to the point of attachment of a 1,2-dioxetane group, has been found to maximize the efficiencies for production of singlet excited states from 1,2-dioxetanes such as those of formula (I) above, substituted at the 4-position of the dioxetane ring with a monocyclic or polycyclic aromatic ring-containing, fluorophore-forming group.

And, as disclosed and claimed in copending Edwards, et al., U.S. patent application Serial No. 213,672, when fused polycyclic aromatic ring-containing, substituted dialkyl 1-alkoxy-1-arylmethane phosphonates are used, and the labile substituent, or its precursor, is

attached to the ring at a position so that the total number of ring atoms, e.g., ring carbon atoms, including the carbon atoms at the points of attachment of the methane phosphorate group and said labile substituent, is an odd whole number, preferably 5 or greater, chemiluminescent 1,2-dioxetanes so produced, when decomposed in an appropriate environment, emit

red-shifted light of greater intensity and longer duration than when the rings are otherwise substituted.

Other substituents can be included anywhere on the aromatic ring of these phosphonates, but at least one substituent which can be elaborated to a chemically or enzymatically cleavable moiety preferably is present in a meta, or odd position relative to a "benzylic" carbon atom which is further substituted by an alkoxy,

aralkoxy, or an aryloxy group and the phosphorous atom of the phosphonate ester group.

An example of an elaboratable group is the bromine atom in diethyl 1-methoxy-1(3-bromophenyl)methanephosphonate, which upon Horner-Emmons reaction with a T = O ketone yields an enol ether, e.g.:

This enol ether can be converted to a Grignard reagent or an organolithium derivative for reaction with

elemental sulfur, dimethyl disulfide, or methyl

methylthiomethyl-sulfoxide to furnish the corresponding enol ether thiophenol or its methyl ether. The same organometallic species can be reacted with

trimethylsilyl azide or azidomethyl phenyl sulfide

[Tanaka, N., et al., J.C.S. Chem. Comm., 1322 (1983); Trost, B., et al., J. Am. Chem. Soc., 103:2483 (1981)] to give the meta aminophenyl enol ether or its N-acyl or sulfonamide derivatives.

It has also been discovered that it is often times advantageous to conduct the acylation reaction of Step 7 in the above-described reaction sequence, or the

phosphorylation reaction of Step 8, or the glycosylation reaction of Step 11, using hydroxyaryl enol ether alkali metal salts of the formula:

wherein AM⁺, the alkali metal cation, is lithium sodium or potassium and T, R³, X¹ and Y are as described above, in place of the corresponding free hydroxy compounds depicted as compounds i, the products of Steps 6a and 6b, in this reaction sequence. In certain cases the use of an alkali metal salt of the enol ether rather than the free hydroxy compound results in savings in

materials of reaction. For example, acylation of the alkali metal salt of an enol ether by the method of Step 7 above, or phosphorylation of the alkali metal salt by the method of Step 8, preferably proceeds without using a Lewis base in either case. In other instances there is an actual reduction in reaction steps. Simply employing the reaction conditions described above for Steps 6a and 6b but dispensing with post-reaction protic work-up, for example, will give the enol ether as its alkali metal salt rather than as the free hydroxy compound. Hence, the alkali metal salt need not be obtained by first isolating the free hydroxy compound and then forming the salt in a separate reaction.

Instead, the thus-obtained alkali metal salts can be separated by precipitation or used in situ as starting materials for the acylation, phosphorylation or

glycosylation reactions.

It is thus an object of this invention to provide a facile, inexpensive, high-yield, convergent chemical synthesis of stable, water-soluble, chemically,

thermally and enzymatically decomposable,

chemiluminescent 1,2-dioxetanes such as those of formula (I) above, by a route that employs substituted

arylaldehyde alkyl and cycloalkyl acetals and novel phosphonate derivatives capable of forming

phosphonate-stabilized carbanions as intermediates in the formation of the enol ether precursors of such

1,2-dioxetane end products.

It is a further object of this invention to provide methods for synthesizing the individual substituted arylaldehyde alkyl and cycloalkyl acetals, phosphonate derivatives and enol ether intermediates employed in synthesizing chemiluminescent 1,2-dioxetanes in

accordance with this invention.

It is yet another object of this invention to provide as novel compositions of matter substituted arylaldehyde alkyl and cycloalkyl acetals, phosphonate derivatives and enol ether intermediates useful in the synthesis of chemiluminescent 1,2-dioxetanes.

It is still another object of this invention to provide variations in the new synthesis of stable, waster-soluble chemiluminescent 1 , 2-dioxetanes disclosed and claimed in our above-mentioned copending U.S. patent application. Another object of this invention is to provide methods for obtaining enol ether alkali metal salt intermediates useful in the acetylation, phosphorylation and glycosylation reactions disclosed and claimed in our above-mentioned copending U.S. patent application.

A further object of this invention is to provide methods for obtaining and using such enol ether alkali metal salt intermediates that result in savings in materials of reaction, reductions in reaction steps, or both.

These and other objects of this invention, as well as a fuller understanding of the advantages thereof, can be had by reference to the following disclosure and the appended claims. Detailed Description of the Invention

The 1,2-dioxetanes, and in particular the

enzymatically-cleavable dioxetanes in which T is a spiro-bonded substituent, a gem carbon of which is also the 3-carbon atom of the dioxetane ring, disclosed and claimed in the aforementioned copending Bronstein,

Bronstein et al., Edwards, and Edwards et al.

applications, and their thermally, chemically and electrochemically cleavable analogs, form one class of water-soluble chemiluminescent 1,2-dioxetane compounds that can be synthesized by the method of this invention. These 1,2-dioxetanes can be represented by formula (I) above, T being a stabilizing group. The most preferred stabilizing group is a fused polycycloalkylidene group bonded to the 3-carbon atom of the dioxetane ring through a spiro linkage and having two or more fused rings, each having from 3 to 12 carbon atoms, inclusive, e.g., an adamant-2-ylidene, which may additionally contain unsaturated bonds or 1,2-fused aromatic rings, or a substituted or unsubstituted alkyl group having from 1 to 12 carbon atoms, inclusive, such as tertiary butyl or 2-cyanoethyl, or an aryl or substituted aryl group such as carboxyphenyl, or a halogen group such as chloro, or heteroatom group which can be a hydroxyl group or a substituted or unsubstituted alkoxy or aryloxy group having from 1 to 12 carbon atoms,

inclusive, such as an ethoxy, hydroxyethoxy,

methoxyethoxy, carboxymethoxy, or polyethyleneoxy group.

The symbol R³ represents a C₁-C₂₀ unbranched or branched, substituted or unsubstituted, saturated or unsaturated alkyl group, e.g., methyl, allyl or

isobutyl; a heteroaralkyl or aralkyl (including

ethylenically unsaturated aralkyl) group, e.g., benzyl or vinylbenzyl; a polynuclear (fused ring) or

heteropolynuclear aralkyl group which may be further substituted, e.g., naphthyl-methyl or 2-benzothiazol-2-yl)ethyl; a saturated or unsaturated cycloalkyl group, e.g., cyclohexyl or cyclohexenyl; a N, O, or S

heteroatom containing group, e.g, 4-hydroxybutyl, methoxyethyl, or polyalkyleneoxyalkyl; an aryl group, any of which may be fused to Y such that the emitting fragment contains a lactone ring, or an enzyme-cleavable group containing a bond cleavable by an enzyme to yield an electron-rich moiety bonded to the dioxetane ring; preferably, X is a methoxy group.

The symbol Y represents a light-emitting

fluorophore-forming group capable of absorbing energy to form an excited energy state from which it emits

optically detectable energy to return to its original energy state. Preferred are phenyl, biphenyl,

9,10-dihydrophenanthryl, naphthyl, anthryl, pyridyl, quinolinyl, isoquinolinyl, phenanthryl, pyrenyl,

coumarinyl, carbostyryl, acridinyl, dibenzosuberyl, phthalyl or derivatives thereof.

The symbol Z represents hydrogen (in which case the dioxetane can be thermally cleaved by a rupture of the oxygen- oxygen bond), a chemically-cleavable group such as a hydroxyl group, an alkanoyloxy or aroyloxy ester group, silyloxy group, or an enzyme-cleavable group containing a bond cleavable by an enzyme to yield an electron-rich moiety bonded to the dioxetane ring, e.g., a bond which, when cleaved, yields a Y-appended oxygen anion, a sulfur anion, an amino or substituted amino group, or a nitrogen anion, and particularly an amido anion such as sulfonamido anion.

One or more of the substituents T, R³ and Z can also include a substituent which enhances the water

solubility of the 1,2-dioxetane, such as a carboxylic acid, e.g., a carboxy methoxy group, a sulfonic acid, e.g., an aryl sulfonic acid group, or their salts, or a quaternary amino salt group, e.g., trimethyl ammonium, with any appropriate counter ion.

When using an enzymatically-cleavable

1,2-dioxetane, cleavage can be accomplished using an enzyme such as alkaline phosphatase that will cleave a bond in, for example, a Z substituent such as a

phosphate mono ester group, to produce a Y oxy-anion of lower oxidation potential that will, in turn,

destabilize the dioxetane and cleave its oxygen-oxygen bond. Alternatively, catalytic antibodies may be used to cleave the Z substituent. Destabilization can also be accomplished by using an enzyme such as an

oxido-reductase enzyme that will cleave the

oxygen-oxygen bond directly; see the aforementioned Bronstein and Bronstein et al. applications.

Besides a phosphate ester group, Z in formula I above can be an enzyme-cleavable alkanoyloxy group, e.g., an acetate ester group, an oxacarboxylate group, or an oxaalkoxycarbonyl group, 1-phospho-2,3- diacylglyceride group, 1-thio-D-glucoside group,

adenosine triphosphate analog group, adenosine

diphosphate analog group, adenosine monophosphate analog group, adenosine analog group, α-D-galactoside group, β-D-galactoside group, α-D-glucoside group,

β-D-glucoside group, α-D-mannoside group, β-D-mannoside group, β-D-fructofuranoside group, β-D-glucosiduronate group, an amide group, p-toluene sulfonyl-L-arginine ester group, or p-toluene sulfonyl-L-arginine amide group.

The method for producing 1,2-dioxetanes according to this invention can be illustrated in part by the following reaction sequences leading to the preparation of 1,2-dioxetanes having both an alkoxy (or aryloxy) and an aryl substituent at the 4-position in which the latter (illustrated here as an aryl Y substituent) is itself substituted by one or more X¹ groups, these substituents being ortho, meta, or para to each other. As will be appreciated by one skilled in the art, groups R² or X¹ need not be static during the reaction

sequences, but may be interconverted under conditions which are compatible with structural considerations at each stage.

In these formulae: any Q can be independently a halogen, e.g., chlorine or bromine, or OR¹; R¹ can be independently a trialkylsilyl group or a lower alkyl group having up to 12 carbon atoms such as ethyl, propyl, or butyl; R² can be a hydroxyl group, an ether (OR⁴) or a thioether (SR⁴) group wherein R⁴ is a

substituted or unsubstituted alkenyl, lower alkyl or aralkyl group having up to 20 carbon atoms such as methyl, allyl, benzyl, or o-nitrobenzyl; R² can also be an acyloxy group such as acetoxy, pivaloyloxy, or mesitoyloxy, a halogen atom, e.g., chlorine or bromine, a nitro group, an amino group, a mono or di (lower) alkyl amino group or its acid salt wherein each lower alkyl substituent contains up to 7 carbon atoms such as methyl, ethyl, or butyl, where any or all of these lower alkyl groups may be bonded to Y generating one or more fused rings, a NHSO₂R⁵ group wherein R⁵ is methyl, tolyl, or trifluoromethyl; R² can also be a substituted aryl, heteroaryl, β-styreneyl group containing up to 20 carbon atoms such as a 4-methoxyphenyl, or 6-methoxybenzthiazol-2-yl group; R³ can be a substituted or unsubstituted lower alkyl, aralkyl, or heteroaralkyl group having up to 20 carbon atoms such as methyl, trifluoroethyl, or benzyl, an aryl or heteroaryl group having up to 14 carbon atoms which may be further substituted, e.g., a 4-chlorophenyl group, a (lower) alkyl-OSiX₃ group wherein the lower alkyl group contains up to 6 carbon atoms such as ethyl, propyl, or hexyl and any X is independently methyl, phenyl, or t-butyl, an alkoxy (lower) alkyl group such as ethoxyethyl, or ethoxypropyl, a hydroxy (lower) alkyl group having up to 6 carbon atoms such as ethyl, butyl, or hexyl, or an amino (lower) alkyl or mono or di (lower) alkylamino alkyl group where each lower alkyl group contains up to 7 carbon atoms such as methyl, ethyl, or benzyl; X¹ can be hydrogen or a substituted or unsubstituted aryl, aralkyl, heteroaryl, or heteroaralkyl group having up to 20 carbon atoms such as 4,5-diphenyloxazol-2-yl,

benzoxazol-2-yl, or 3,6-dimethoxy-9-hydroxyxanthen-9-yl groups, an allyl group, a hydroxy (lower) alkyl group having up to 6 carbon atoms such as hydroxymethyl, hydroxyethyl, or hydroxypropyl, a (lower) alkyl-OSiX₃ group wherein the alkyl and X radicals are as defined above, an ether (OR⁴) or a thioether (SR^A) wherein R⁴ is as defined above, an SO₂R⁶ group wherein R⁶ is methyl, phenyl, or NHC₆H₅, a substituted or unsubstituted alkyl group containing up to 7 carbon atoms such as methyl, trifluoromethyl or t-butyl, a nitro group, a cyano group, an aldehydic function or its oxime or

dimethylhydrazone, an alkyl halide group having up to 6 carbon atoms and the halide group being chlorine or bromine, a halogen group, a hydroxyl group, a carboxyl group or its salt, ester or hydrazide derivatives, a tri-substituted silicon-based group such as a

trimethylsilyl group, or a phosphoryloxy (phosphate monoester) group.

Step 1 of the foregoing reaction sequence involves the formation of a tertiary phosphorous acid alkyl ester from a phosphorous trihalide, e.g., phosphorous

trichloride or dialkylchlorophosphite, and an alcohol, e.g., a short chain alkyl alcohol, preferably one having up to 7 carbon atoms such as methanol, ethanol or butanol, in the presence of a base such as

triethylamine. An alkali metal alcoholate or

trialkylsilanolate can also be used in a direct reaction with the chlorophosphite.

Step 2 involves reacting an aryl aldehyde or heteroarylaldehyde with an alcohol, R³OH, to give the corresponding aryl aldehyde acetal, wherein the aryl aldehyde may be a benzaldehyde, a naphthaldehyde, a anthraldehyde and the like, or aryl dialdehydes such as m-or p-phthalaldehydes and the like. The R² substituent on the aryl aldehyde, which is preferably positioned meta to the point of attachment of the aldehydic group in the benzaldehydes illustrated above, can be an

oxygen-linked functional group, e.g., an ester group such as pivaloyloxy, acetoxy and the like, an ether group such as methoxy, benzyloxy, and the like, a nitro group, a halogen atom, or hydrogen (see Tables 2-6 below). Functional group X¹ in the aryl aldehyde may be located ortho, meta or para to the point of attachment of the aldehydic group to the aryl ring, and can be a lower alkoxy group such as methoxy, ethoxy or the like, hydrogen, or an alkyl group (see Table 2 below). In the alcohol reactant R³OH, R³ can be, for example, a lower alkyl group such as methyl, ethyl and the like, a lower aralkyl group, a lower alkoxy alkyl group, a substituted amino alkyl group, or a substituted siloxy alkyl group (see Tables 2-6). Diols such as ethylene glycol or propylene glycol, e.g., HO-(CH₂)_n-OH, produce cyclic acetals which are within the scope of this invention. The acetalization reaction between the aryl aldehyde and the alcohol or diol is carried out in conventional fashion, preferably in the presence of a catalyst such as a Lewis acid, HCl(g), p-toluenesulfonic acid or its polyvinylpyridine salt, or Amberlyst XN1010 resin, accompanied by removal of water using, e.g.,

trialkylorthoformate, 2,2-dialkoxypropane, anhydrous copper sulfate, or molecular sieves, or by azeotropic distillation in, for example, a Dean-Stark apparatus. In cases in which acetalization may proceed with poor conversion or yield, it is possible to use the Noyori reaction wherein any of the aforementioned alcohols (R³OH) or diols are reacted with the aldehyde as their mono or bis trialkylsilyl ether with trimethylsilyl triflate as catalyst in a chlorinated hydrocarbon solvent.

Step 3 involves reacting the tertiary phosphorous acid alkyl ester (trialkylphosphite) produced in Step 1 with the aryl aldehyde dialkyl or cyclic acetal produced in Step 2, preferably in the presence of at least one equivalent of a Lewis acid catalyst such as BF₃ etherate or the like to give the corresponding phosphonate, essentially according to Burkhouse, D., et al.,

Synthesis, 330 (1984). Aryl aldehyde dialkyl acetals react with between 1 and 1.5 equivalents of a

trialkylphosphite in the presence of a Lewis acid in an organic solvent such as methylene chloride, under an inert atmosphere, e.g., argon, at temperatures below 0ºC, to produce in almost quantitative yields (see Table 2) the corresponding 1-alkoxy-1-arylmethane phosphonate esters. The phosphonates are generally oils that can be used directly or purified by chromatography on silica gel. ¹HNMR spectra will exhibit a doublet near 4.7 ppm (J = 15.5 Hz) due to the benzylic proton, split by the adjacent phosphorous anion; occasionally, two doublets of unequal intensity will be observed.

In step 4, the phosphonate-stabilized carbanion is used to synthesize olefins by the Homer-Emmons

reaction. Specifically, in Step 4.1 a phosphonatestabilized carbanion is produced from a dialkyl

1-alkoxy-1-arylmethane phosphonate in the presence of a base such as sodium hydride, sodium amide, a lithium dialkyl amide such as lithium diisopropylamide (LDA), a metal alkoxide, or, preferably, n-butyllithium, in a suitable solvent, preferably in the presence of a slight excess of base, e.g., about 1.05 equivalents for each ionizable group present. Suitable solvents for the reaction can have an appreciable range of polarities, and include, for example, aliphatic hydrocarbons such as hexanes, aromatic hydrocarbons such as benzene, toluene and xylene, ethers such as tetrahydrofuran (THF) or glymes, alkanols such as ethanol and propanol,

dimethylformamide (DMF), dimethyl-acetamide, and

dimethylsulfoxide, and the like, or mixtures of these solvents. As lithiophosphonates are insoluble in

diethylether, but soluble in ethers such as THF, reactions using LDA or n-butyllithium are preferably run in dry THF/hexane mixtures. It is also preferred to carry out the reaction in an inert atmosphere, e. g., under argon gas. At temperatures below 0ºC the reaction of n-butyllithium with phosphonates proceeds rapidly, as indicated by the instantaneous formation of a dark yellow to burgundy colored solution, depending upon the particular phosphonate used and its concentration.

In Step 4.2, the phosphonate-stabilized carbanion is reacted, preferably in molar excess, with a carbonyl compound T = O or dicarbonyl compound O = T = O. When T = O is a substituted or unsubstituted adamantanone, e.g., adamantanone itself, the reaction begins

immediately upon addition of the ketone, preferably from about 0.8 to about 0.95 equivalents of the ketone, to the stabilized carbanion, and goes to completion under reflux conditions in from about 2 to about 24 hours.

Optimization of the T = O equivalency in each case allows complete conversion of this expensive component.

In Step 5 the enol ether is oxidized. Oxidation is preferably accomplished photochemically by treating the enol ether with singlet oxygen (¹O₂) wherein oxygen adds across the double bond to create the 1,2-dioxetane ring. Photochemical oxidation is preferably carried out in a halogenated solvent such as methylene chloride or the like. ¹O₂ can be generated using a photosensitizer, such as polymer bound Rose Bengal (Hydron Labs, New

Brunswick, N.J.) and methylene blue or 5, 10, 15,

20-tetraphenyl- 21H,23H-porphine (TPP). Chemical methods of dioxetane formation using triethylsilylhydrotrioxide, phosphite ozonides, or triarylamine radical, radical cation mediated one electron oxidation in the presence of ³O₂ can also be utilized.

When the oxygen-linked functional group R₂ on the aryl ring of the enol ether is an alkoxy group or pivaloyloxy group, it can be converted to an enzyme- cleavable group such as a phosphate, acetoxy, or

O-hexopyranoside group, by carrying out the following additional steps involving the enol ether produced in Step 4 of the foregoing reaction sequence prior to carrying out the oxidation reaction of Step 5, as shown below:

Step 6a. involves phenolic ether cleavage of the R⁷ substituent (wherein R⁷ is preferably methyl, allyl or benzyl), preferably with sodium thioethoxide, in an aprotic solvent such as DMF, NMP, or the like, at temperatures from about 120ºC to about 150ºC. The cleavage can also be accomplished with soft nucleophiles such as lithium iodide in refluxing pyridine, sodium cyanide in refluxing DMSO, or Na₂S in refluxing

N-methyl-2-pyrrolidone. When R⁷ is pivaloyl, ester cleavage can be accomplished with NaOMe, KOH or K₂CO₃ in an alcoholic solvent such as MeOH at temperatures from about 25°C to reflux (Step 6b.).

The acylation of the phenolic hydroxyl group in the thus obtained hydroxy compound is carried out in Step 7 by adding a small equivalent excess of an acid halide or anhydride, e.g., acetic anhydride, or oxalyl chloride with Lewis base, e.g., triethylamine, in an aprotic solvent.

The substituent Q on the cyclic phosphorohalidate used in Step 8 is an electronegative leaving group such as a halogen. The monovalent cation M⁺ of the cyanide used in Step 9 can be a metallic or alkali metal cation such as Na⁺ or K⁺, or a quaternary ammonium cation. The cation B⁺ of the ammonium base of Step 10 is an ammonium cation; however, NaOMe can also be used as the base. T, R³ and X¹ are as defined above.

Steps 8, 9 and 10 can be performed separately or in a onepot or two-pot operation. A cyclic phosphorohalidate, e.g., cyclic phosphorochloridate, is preferred for use in Step 8 not only because of its monofunctionality, chemoselectivity and enol ether-compatible deprotection mode of action, but also because it is 10⁶ times more reactive than the corresponding acyclic compounds. In a 3-step, 2-pot operation, the phenolic hydroxyl group in the free hydroxyl product produced in Step 6 is reacted with 2-halo-2-oxo-1,3,2-dioxaphospholane to yield the cyclic phosphate triester (Step 8). This triester is subjected to ring opening with MCN

(e.g., NaCN) to yield the corresponding 2-cyanoethyl die sr (Step 9). A base, e.g., ammonium hydroxide or NaOMe, then provokes a facile β-elimination reaction, yielding a filterable disodium sodium ammonium salt

(Step 10). In benzene, THF, diethylether or DMF,

phosphate triester formation induced by a Lewis base (e.g., a tertiary amine such as triethylamine) or with a preformed alkali metal salt or the phenolic enolether can be effected with phosphorohalidates over a

temperature range of about -30° to about 60ºC.

Subsequently, if a pure monosodium cyanoethylphosphate ester is desired, the ring cleavage with alkalicyanide (MCN) in DMF or DMSO can be carried out in a narrow temperature range of between about 15º and about 30°C. However, in a one-pot or in situ mode this is not as important, and the temperature range widens to about 60ºC on the high end.

Aryl phosphate disalts can also be made from the aryl alcohol enol ether product of Step 6 (formula IV) using an activated phosphate triester of the general formula:

wherein Q is as described above, and R⁸ and R⁹ are each independently -CN, -NO₂, arylsulfonyl, or alkylsulfonyl. Alternatively, the phosphate triester may contain two trimethyl silyl ester groups, linked to the phosphorous, as shown in the formula above. This reaction can be carried out in the presence of a Lewis base in an aprotic solvent, and yields an aryl phosphate triester. The triester can then be hydrolyzed with a base, M⁺OH. or M⁺ OCH₃, wherein the cation M⁺ is an alkali metal, NR¹⁰ ₄ ⁺ wherein R¹⁰ is hydrogen or a C₁-C₁ alkyl, aralkyl, aryl or heterocyclic group, to give the corresponding

arylphosphate monoester disalt via β-elimination.

Dioxetane formation of the reaction of singlet oxygen (¹O₂) with these enol ether phosphate triesters, followed by similar base-induced deprotection to the dioxetane phosphate monester, may also be carried out.

An alkoxy group on the aryl ring of the enol ether can be converted to a D-sugar molecule linked to the ring via an enzyme cleavable glycosidic linkage by reacting the phenolic precursor in an aprotic organic solvent under an inert atmosphere in the presence of a base such as NaN, with a tetra-O-acetyl-D-hexopyranosyl halide to produce the aryl-O-hexopyranoside tetraacetate (Step 11). The protective acetyl groups can then be hydrolyzed off using a base such as NaOCH₃, K₂CO₃, or NH₃ gas, in an alcohol such as methanol, first at 0°C and then at 25ºC for 1 to 10 hours (Step 12), leaving a hexosidase-cleavable Dhexopyranosidyl moiety on the aryl ring.

When the enol ether aryl phosphates are oxidized to a bisguatemary ammonium or corresponding 1,2-dioxetanes (Step 5 above), ion exchange to a bis-guatemary

ammonium or monopyridinium salt allows the facile photooxygenation of 0.06 M chloroform solutions in the presence of, preferably, methylene blue or TPP, at cold temperatures, e.g., about 5ºC. Slower reaction rates and increased photolytic damage to the product may occur with the use of solid phase sensitizers such as

polymerbound Rose Bengal (Sensitox I) or methylene blue on silica gel.

Aryl monoaldehydes or heteroaryl monoaldehydss other than those having formulas such as:

can also be used as starting materials in carrying out the above described reaction sequences. Included among such aryl monoaldehydes are polycyclic aryl or

heteroaryl monoaldehydes such as those having the formula:

wherein R is as defined above and is preferably

positioned so that the total number of ring carbon atoms separating the ring carbon atom to which it is attached and the ring carbon atom to which the aldehyde group is attached, including the ring carbon atoms at the points of attachment, is an odd whole number, preferably 5 or greater; see Edwards, et al., U.S. patent application Serial No. 213,672.

Fused heteroσyclic acetals or hemiacetals can also be used as starting materials in carrying out the above-described reaction sequences. Included among such fused heterocyclic acetals are those having the

formulae:

and the like, wherein R² is as described above, and W can be OR³, wherein R³ is described above, or OH, and is an integer greater than zero.

Aryl or heteroaryl dialdehydes can also be used as the aldehydic starting material, e.g., ones having the formula:

wherein R² is as described above.

Purification of the thus-obtained water-soluble dioxetanes is best achieved at alkaline pH values, e.g., about 7.5 to about 9.0, using reverse phase HPLC with an acetonitrile-water gradient, followed by lyophilization of the product, according to Edwards et al., U.S. patent application Serial No. 244,006.

Typical enzymatically-cleavable water-soluble chemiluminescent 1,2-dioxetanes for use in bioassays which can be prepared by the method of this invention are the 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy) phenyl-1,2-dioxetane salts represented by the formulaa

wherein M⁺ represents a cation such as an alkali metal, e.g. sodium or potassium, or a C₁-C₁₈ alkyl, aralkyl or aromatic quaternary ammonium cation, N(R¹⁰)₄ ⁺, in which each R¹⁰ can be alkyl, e.g., methyl or ethyl, aralkyl, e.g., benzyl, or form part of a heterocyclic ring system, e.g., N-methylpyridinium, a fluorescent onium cation, and particularly the disodium salt. A more systematic name for the latter is 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo[3.3.1.1^3,7]decan]4-yl)phenylphosphate disodium salt.

The availability of the herein described

Horner-Emmons methodology and a pool of reactants containing the particular aforementioned class of mono and bis-phosphonate esters along with T = O ketones or O = T = O diones such as 2,6-adamantanedione, allows the synthesis of three different enol ether product types. (formula VI)

wherein T, R³, Y and Z are as described herein above. These can then be converted to the corresponding

1,2-dioxetanes shown below in formula (VII) with singlet oxygen as described herein above.

In the case of 1,2-dioxetane B of formula (VII), one T group serves to stabilize two dioxetane rings;

however, each ring must be destabilized individually by chemical or enzymatic means at each Z group. In

1,2-dioxetane C, one Z group can activate the

decomposition of two dioxetane rings, especially if all groups appended to aromatic ring Y are disposed in a meta or odd-pattern relationship with one another as described above.

The bis-enol ether phenol of formula (VIII) below is synthesized by sodium ethane thiolate cleavage of the aromatic methoxy group (Step 6 of the flow chart (III)) of the compound described in Examples 62 and 105 below. The product can be converted to any one of the enzyme cleavable groups described above, e.g., a phosphate mono ester. As such it represents a pivotal intermediate for the synthesis of 1,2-dioxetanes of type C of formula (VII) as shown above.

A modified method of providing the enol ether alkali metal salts of this invention involves modificat on of the step in the above-described reaction followed by modification of the subsequent ester

cleavage step, Step 6b. Specifically, and as described above, in the first part of this modified procedure a dialkyl 1-alkoxy-1-arylmethane phosphonate:

preferably one in which Y is an aryl moiety, e.g, a phenyl ring, R² is an acyloxy substituent, preferably in the meta-position on the aryl moiety, e.g., a

pivaloyloxy group, and X¹ can be hydrogen or another of the substituents listed above, is converted to the corresponding phosphonate-stabilized α-carbanion, preferably in solution at low temperature, -20ºC or less, under an inert atmosphere, using an alkali metal-containing base, e.g., from about 1 to about 1.2

equivalents of the alkali metal-containing base, and preferably slightly more than one equivalent of an alkali metal alkylamide such as lithium diisopropylamide or an alkali metal alkyl compound such as

n-butyllithium.

Once the α-carbanion is formed the polycyclic ketone T = O is added to the reaction mixture at low temperature, preferably in slightly less than molar excess, and reacted under reflux conditions for from about 2 to about 24 hours to give a reaction mixture which can include, inter alia, the dialkyl 1-alkoxy-1- arylmethane phosphonate starting material as its anion, its R² deesterified dianion, or its decomposition products, the hydroxyaryl enol ether alkali metal salt, and the R² esterified aryl enol ether, the latter particularly being present when the phosphonate starting material includes an aryloxy-substituted aryl moiety (Y - R²) whose acyloxy substituent (R²) has an acyl group that is a good hydroxy protecting group that remains substantially intact during this reaction, e.g., a pivaloyl group (R² = pivaloxyloxy). It has been found, in fact, that when the phosphonate starting material's Y - R² substituents constitute a pivaloyloxyphenyl group, only about 10-20 percent of the total enol ether product obtained is present as the deesterified enol ether alkali metal salt.

Mild protic work-up of this reaction mixture to separate the desired R² esterified aryl enol ether (as described, e.g., in Example 7 of our copending

application Serial No. 402,847) is complicated by the presence of several other useful components, all which should, if possible, be recovered in some fashion to reduce costs. The R² esterified aryl enol ether where R² is a pivaloyloxy group, for example, is a high R_f, early eluting product when subjected to column chromatography, while the corresponding hydroxyaryl (deesterified) compound, which is produced during protic work-up to from the hydroxyaryl enol ether lithium salt, and the phosphonate starting material and its decomposition products, are somewhat lower R_f materials, making for a difficultly separable mixture which yields somewhat impure fractions on a large synthetic scale.

Reesterification of the crude, post-reflux Horner-Emmons reaction mixture, however, to substantially esterify the hydroxyaryl enol ether alkali metal salt, preferably using an acid chloride or acid anhydride, e.g., pivaloyl chloride, in at least a molar equivalent amount to the total amount of all aryloxide alkali metal salt present, permits facile separation of the

esterified aryl enol ether in near quantitative yield without the above-mentioned complications during

chromatography because the hydroxyaryl enol ether is absent after protic workup.

The minimum quantity of acid halide or anhydride to consume the hydroxyaryl alkali metal salt is added in several aliquots to the crude reaction mixture, at a temperature between about 0ºC and about 50ºC, over a period of from about 2 to about 24 hours, using thin layer chromatography to monitor the completeness of the reaction. Where R² is a pivaloyloxy group one gets a much cleaner product, isolated from the reesterified mixture as a crystalline solid using standard

techniques, such as recrystallization from hexanes. The mother liquors, uncontaminated with free hydroxyaryl enol ether, are easily plug chromatographed on a large scale, again due to the absence of hydroxyaryl enol ether byproduct.

The final reaction in this preferred method of providing enol ether alkali metal salts involves

carrying out ester cleavage to give, instead of the free hydroxy aryl enol ether obtained as in Step 6b of the reaction sequence set out supra. the corresponding alkali metal salt. The salt-forming reaction is

preferably carried out using about one molar equivalent of an alkali metal alkoxide, e.g., sodium methoxide, in a lower alkanol, e.g., methanol or enthanol, under anhydrous conditions, i.e., in the presence of as low an amount of moisture as can practicably be achieved, for from about 1 to about 4 hours at room temperature (about 25 ºC), followed by removal of the volatiles from the reaction mixture in vacuo (1 mm Hg) with heating at from about 35°C to about 65ºC for about 24 hours to give the hydroxyaryl enol ether alkali metal salt as a dry solid, directly usable in an acylation, phosphorylation or glycosylation reaction. For example, the free hydroxy enol ether starting material of Example 106 in our copending application Serial No. 402 , 847 - - 3- (methoxytricyclo[3.3.1.1^3,7]dec-2-ylidene-methyl)phenol - - can be replaced with its sodium salt - - sodium 3-(methoxytricyclo[3.3.1.1^3,7]dec-2-ylidenemethyl)phenoxide - - in a one pot reaction with between about 1 and 1.2 equivalents of 2-chloro-2-oxo-1,3,2-dioxaphospho-lane in anhydrous dimethylform-amide or dimethylsulfoxide to give the corresponding cyclic triester. This triester readily undergoes ring opening with sodium methoxide, and β-elimination with sodium hydroxide or ammonium hydroxide to give the phosphate monoester salt.

Alternatively, the same reaction can be carried out in a halogenated solvent, e.g., methylene chloride, a polar solvent, e.g., acetonitrile, or an ether or polyether solvent, e.g., tetrahydrofuran or diglyme, in the presence, if desired, of hexamethylphosphoramide or a phase transfer catalyst such as tetrabutylammonium bisulfate, with the remaining ring opening and

β-elimination steps being run in dimethylformamide or dimethylsulfoxide. These same procedures can also be used when reacting the enol ether alkali metal salt with the other phosphorylating agents listed above, except that the β-elimination or hydrolysis reactions can be run immediately following triester formation.

The enol ether alkali metal salts of this invention can be obtained by yet another modification in the above-described reaction sequence, this time to Step 4 alone. A dialkyl 1-alkoxy-1-arylmethane phosphonate, Formula d above, whose aryl moiety (Y) has an acyloxy substituent (R²) the acyl group of which is a poor hydroxy protecting group, i.e., one that will be

substantially cleaved during this reaction, such as an acetyl group or the like, can be reacted with three equivalents of a lithium alkyl compound, e.g.,

n-butyllithium, in solution under an inert atmosphere at low temperature, -20ºC or less, to give the corresponding phosphonate-stabilized α-carbanion as its lithio salt. Addition of the polycyclic ketone T = O,

preferably in less than a molar equivalent quantity, to the reaction mixture, followed by refluxing for from about 2 to about 24 hours, gives the lithio salt of the hydroxyaryl enol ether directly. Similarly, phenolic ether or thioether cleavage of the R⁷ substituent exactly as described for Step 6a in the above-described reaction sequence, using an alkali metal-containing reagent, initially yields the

corresponding hydroxyaryl or mercaptoraryl alkali metal salt. Instead of subjecting the thus-obtained reaction mixture to protic work-up, the thus obtained salt can be separated by precipitation at 0ºC, preferably in the presence of a nonsolvent such as an ether, e.g., diethyl ether, or used in situ to accomplish direct acylation, phosphorylation or glycosylation in the manner described in Steps 7, 8 and 11 of the above-described reaction sequence.

The conditions under which the hydroxyaryl enol ether alkali metal salts of this invention can be subjected to acylation, phosphorylation or glycosylation are as described in our copending application Serial No. 402,847, except that any of the solvents mentioned above, e.g., dimethylformamide or tetrahydrofuran, or mixtures of these solvents, are used for the reaction with the acylating, phosphorylating or glycosylating reagent over a temperature range of about 0ºC to about 60ºC, preferably in the absence of a Lewis base, with any remaining process steps being identical to those in our copending application.

Such chemiluminescent water-soluble dioxetanes and their derivatives can be used in a variety of detection techniques, such as ligand binding assays and enzyme assays. Immunoassays and nucleic acid probe assays are examples of ligand binding techniques, in which a member of a specific binding pair is, for example, an

antigenantibody pair, or a nucleic acid target paired with a probe complementary to and capable of binding to all and or a portion of the nucleic acid. The ligand: an antibody and a nucleic acid probe, can be labeled with an enzyme and a chemiluminescent water-soluble dioxetane used as a substrate, or a chemiluminescent dioxetane can be used as a label directly and conjugated to a ligand and activated to emit light with heat, suitable chemical agents, and enzymes. Such assays include immunoassays to detect hormones, such as β-human chorionic gonadotropin (β HCG), thyroid stimulating hormone (TSH), follicle stimulating hormone (FSH), luteinizing hormone (LH) or the like, cancer markers, such as alpha fetal protein (AFP), carcinoembryonic antigen, cancer antigen CA 19-9 for pancreatic cancer, cancer antigen CA125 for ovarian cancer, haptens, such as digoxin, thyroxines prostaglandins, and enzymes such as phosphatases, esterases, kinases, galactosidases, or the like, and cell surface receptors. These assays can be performed in an array of formats, such as solution, both as a two-antibody (sandwich) assay or as a

competitive assay, in solid support such as membranes (including Western blots), and on surfaces of latex beads, magnetic beads, derivatized polystyrene tubes, microtiter wells, and the like. Nucleic acid assays can be used to detect viruses e.g. Herpes Simplex Viruses, HIV or HTLV I and III, cytomegalovirus (CNV), human papilloma virus (HPV), hepatitis C core virus antigen (HB_cV) , Hepatitis B surface antigen (HB_cV), Rotavirus, or bacteria, e.g., campylobacter jejuni/coli, E. coli, ETEC heat labile and stable, plasmodium falciparum, or oncogenes, or in forensic applications using human finger-printing probes, mono and multi loci. The nucleic acid detections can be performed for both DNA and RNA in a variety of formats, e.g., solution, derivatized tubes or microtiter plates, membranes (dot, slot, Southern and Northern blots) and directly in tissues and cells via in-situ hybridization. DNA and RNA can also be detected in sequencing techniques and histocompatibility assays using chemiluminescent

dioxetanes. Such chemiluminescent water-soluble

dioxetanes can also be used in biosensors where the ligand-binding reaction occurs on a surface of a semiconductor layer which detects chemiluminescence as photocurrent.

Furthermore, these dioxetanes can be used in in vivo applications both for diagnostics, such as imaging tumor sites when coupled to a tumor site-specific monoclonals and other ligands, or as a therapeutic, such as in photodynamic therapy to photosensitive

hematoporphyrins to generate singlet oxygen - the cytotoxic agent. In addition, enol ethers - the

precursors to 1,2-dioxetanes can be used as singlet oxygen scavengers both in vivo and in vitro, to monitor and/or inactivate this very reactive species.

In order that those skilled in the art can more fully understand this invention, the following examples are set forth. These examples are given solely for purposes of illustration, and should not be considered as expressing limitations unless so set forth in the appended claims.

Example 1

5-Methoxyisophthaldehyde

3 , 5-Bishydroxymethylanisole was synthesized

according to the procedure of V. Boekelheide and R.W. Griffin, Jr., J. Org. Chem., 34, 1960 (1969). This diol (366 mg., 2.17 mmol) was added as a solid to a stirred slurry of 3 g. crushed 3A molecular sieves and 2.5 g. pyridinium dichromate (6.65 mmol) in 20 ml dichloromethane. After 3 hours at room temperature, the mixture was diluted with 40 ml ether and filtered through celite, and washed with 2:1 ether-dichloromethane. The orange filtrate was concentrated to a solid which was boiled with 3 × 30 ml hexanes, decanting the supernate each time from a gummy residue. As the combined hexane fractions cooled to room temperature, fine white needles developed in the colorless mother liquor. Filtration and drying provided 150 mg (42%) of the dialdehyde which exhibited a melting point of 110-112ºC. NNR and IR data are listed in Tables 3 and 7. TLC showed one spot (K5F, 10% ethyl acetate: dichloromethane; Rf = 0.75). These data support the structure:

Example 2

4-Ethoxy-3-methoxybenzaldehyde

Vanillin (10 g., 66 mmol) in acetonitrile (100 ml) was treated with finely-powdered, anhydrous potassium carbonate (12 g., 87 mmol) with vigorous stirring to yield a mobile suspension. Diethyl sulfate (11 ml, 84 mmol) was added at room temperature. The suspension was brought to reflux, becoming quite thick after 10

minutes, but thinning again after 20 minutes. Refluxing was continued for 48 hours, at which point water (5 ml) was added. After an additional 2 hours of reflux, the mixture was cooled and treated with 500 ml ice water. Stirring at 0º for several hours produced a granular precipitate which was filtered off and washed with water. Air drying afforded 11.5 g. of the product (97%) as an off-white solid melting at 61-62.5ºC. NMR and IR data are listed in Tables 3 and 7.

Example 3

3-Methoxy-2-methylbenzaldehyde

This compound was synthesized according to the method of Kende, A.S., et al., J. Am. Chem. Soc.,

101:1860 (1979). As seen in Tables 3 and 7, NMR and IR data are identical to those reported. TLC showed the title compound to be homogeneous (K5F, 20% CH₂CL₂:

hexanes; Rf = 0.17). The major by-product in this reaction was 2-methoxybenzylphenysulfide (Rf = .38 under the same conditions).

Example 4

m-Methoxybenzaldehyde dimethyl acetal m-Anisaldehyde (204.3 g, 1.5 mol) was placed in a 1 litre flask under an argon atmosphere. Trimethyl orthoformate (191 g, 1.8 mol) was added quickly,

followed by 150 ml anhydrous methanol. Amberlyst

XN-1010 resin (2.1 g, Aldrich Chemical Co.), which had been previously boiled with methanol was added. The mixture was stirred at room temperature for 22 hours with the exclusion of moisture. Sodium bicarbonate (1.5 g) was added with stirring. After 20 minutes the mixture was filtered under vacuum into a 2 litre flask which was placed on the rotory evaporator with the water bath temperature at 40°C. Over 30 minutes the bath was heated to 80º to produce a clear, colorless oil. With magnetic stirring, the oil was pumped at 65° under vacuum (2mm Hg) for 30 minutes. The resulting product weighted 272.5 g (99.8%). I.R. (near, cm^-1): 2935, 2824, 1598, 1584, 1350, 1258, 1100, 1050, 984, 772. 1HNMR (400 MHZ, CDCl₃): δ 3.33 (6H, s, OCH₃); 3.81 (3H, s, ArOCH₃); 5.35 (1H, s, ArCH(OCH₃)₂; 6.87 (1H, br d, 8.1 Hz); 7.00 - 7.03 (2H, m); 7.27 (1H, t, 8.1Hz). These data indicated that the product was pure enough for use in the next step and were consistent with the following structure:

Example 5

Diethyl 1-methoxy-1-(3-methoxynhenyl)methane phosphonate m-Methoxybenzaldehyde dimethyl acetal from Example 4 (271.4 g, 1.49 mol), triethyl phosphite (250.3 g, 1.51 mol), and methylene chloride (600 ml) were charged into a 3 litre 3-necked flask which was outfitted with a dropping funnel, an argon inlet, and an argon outlet. The flask was flushed with argon and the funnel was capped with a septum. The mixture was stirred and cooled to -40° in a liquid nitrogen-acetone bath. Soron trifluoride etherate (198.1 ml, 1.61 mol) was then added dropwise from the funnel over a 25 minute period. The mixture was allowed to slowly warm up to 5° over 3 hours. Stirring was then continued at room temperature for another 15 hours. The light yellow solution was then stirred rapidly as 500 ml saturated sodium

bicarbonate solution was added. After 1 hour the mixture was transferred to a separatory funnel. The organic layer was isolated and washed with 500 ml water, 2 × 300 ml saturated sodium bisulfite, and 300 ml saturated bicarbonate solution. Drying was accomplished over 30 g anhydrous sodium sulfate just before

decolorizing carbon (3g) was added to the solution, and the whole was filtered under vacuum through celite.

Concentration on the rotory evaporator and high vacuum pumping to a final pressure of 0.15 mm Hg at 100°C provided a light yellow oil weighing 380 g (90%). I.R. (neat, cm^-1): 2974, 1596, 1582, 1480, 1255 (P = O), 1098, 1050, 1020, 965, ¹HNMR (400 MHz, CDCl₃) : δ 1.21 and 1.25 (6H, two t, 7Hz, OCH₂CH₃); 3.37 (3H, s,

ArCHOCH₃); 3.80 (3H, s, ArOCH₃); 3.90 - 4.10 (4H, m, OCH₂CH₃); 4.46 (1H, d, 15.6Hz, ArCHPO); 6.85 (1H, m); 7.00 (2H, m) , 7.26 (1H, m). This product was

sufficiently pure for use in a Horner-Emmons reaction. However, further purification to remove a trace of a non-polar fluorescent impurity may be accomplished with silica gel chromatography using dichloromethane to elute the impurity and subsequent elution with 20% ethyl acetate in dichloromethane to elute the phosphonate.

Example 6

α-2-Adamantylidene-α-methoxy-m-methoxytoluene

One hundred grams of the phosphonate ester from Example 5 (0.347 mol) were dissolved in 650 ml HPLC grade THF (no special precautions to dry the solvent were taken). The solution was placed in a dry 2 litre, 3-necked flask which was outfitted with an addition funnel connected to an argon outlet, an argon inlet, and a septum.

After purging with argon, the flask was lowered into a dry ice/acetone bath at -78° and magnetic

stirring was initiated. After stirring for 10 minutes, a solution of n-butyllithium in hexane (217 ml of a 1.6 M solution, 0.347 mol) was added by syringe in several portions over 10 minutes. The resulting deep red solution was stirred at -78° for another 45 minutes. A solution of 2-adamantanone (49.47 g, 0.33 mol) in 200 ml THF was then added in a thin stream from the funnel over 5 minutes.

The cooling bath was removed and the stirred mixture was slowly allowed to warm to approximately 0° over 1.5 hours. At this point the slightly cloudy red solution was heated to reflux for 4 hours whereupon a clear, light red solution was obtained after gas

evolution ceased.

During cooling to room temperature, the solution became light yellow-brown after exposure to the

atmosphere. The mixture was carefully rotovapped

(foaming) to remove 750 ml of the solvent. Hexane (500 ml) was added, and the resulting slurry was extracted with 500 ml water. The aqueous layer was back extracted with hexane (250 ml) and the combined organics were extracted with saturated brine (2 × 250 ml). The hexane solution was dried over anhydrous potassium carbonate and treated with 1 g. decolorizing carbon. Filtration through celite, followed by evaporation produced a light yellow viscous oil which was pumped at 90° with stirring under high vacuum to remove a small amount of residual adamantanone.

The final weight of the crude product was 94 g. The infrared spectrum showed no carbonyl absorption due to adamantanone (1705 cm^-1) or the corresponding

adamantyl methoxyphenyl ketone (1670 cm^-1). Although this product was sufficiently pure for subsequent reaction, it was found that an identical procedure using

46.8 g. 2-adamantanone (0.9 equivalents) provided an oil, which when passed through a silica gel column (15 cm × 3 .5 cm) and eluting with 2% ethyl acetate in hexanes, gave an oil which solidified in the cold.

Recrystallization from a minimal amount of hexanes yielded a waxy, white solid melting at 34-37.C. Anal.

Calcd for C₁₉H₂₄O₂: C, 80.24; H, 8.51. Found: C, 81.23;

H, 8.49.

Both the crude oil and the waxy solid gave

identical I.R. and NMR spectra:

I.R. (neat, cm^-1); 2900, 2838, 2655, 2640, 2620,

1655, 1600, 1592, 1580, 1574, 1444, 1282, 1240, 1202,

1095, 1078.

1HNMR (400 MHz, CDCl₃) : δ 1.75 - 2.05 (12H, m, adamantyl); 2.66 (1H, br s, Hα,) ; 3.27 (1H, br s, Hα₂);

3.31 (3H, s, OCH₃); 3.83 (3H, s, ArOCH₃); 6.82 - 6.94 (3H, m); 7.23 - 7.30 (1H, m).

Example 7

3-Pivaloyloxybenzaldehyde

3-Hydroxybenzaldehyde (2.04 g., 16.7 mmol) in 25 ml dichloromethane under an argon atmosphere was treated with triethylamine (3.5 ml, 25.1 mmol). The solution was cooled to 0° in an ice bath. Trimethylacetyl chloride (2.3 ml, 18.4 mmol) was added dropwise via syringe with magnetic stirring. After ten minutes, the ice bath was removed and the mixture was stirred

overnight at room temperature. The reaction was quenched with 100 ml saturated sodium bicarbonate solution. The organic layer was separated and the aqueous layer extracted again with dichloromethane (2 × 30 ml). The combined organics were dried over anhydrous sodium sulfate and concentrated in vacuo to an orange residue which was passed through a short silica gel plug with dichloromethane as eluent.

The solvent was evaporated from the silica gel eluate to yield 3.40 g. (quant.) of the title compound as a light yellow oil which was homogeneous according to TLC (K5F, 20% ethylacetate: hexanes). See Tables 3 and 7 for NMR and IR data.

The pivaloyl ester group is not deacylated under the acidic conditions required for acetal and

phosphonate synthesis which are described in Examples 4 and 5 for the 3-methoxy derivatives, but they also serve as general procedures. The resulting diethyl 1-methoxy¬1-(3-pivaloyloxyphenyl)methane phosphonate is used as follows to procure methoxy (3-hydroxyphenyl)methylene adamantane.

Lithium diisopropylamide (LDA) solution was freshly prepared in the following manner. A dry, three-necked, 2 L, round bottomed flask was equipped with a magnetic stirring bar, a reflux condenser, a gas-inlet and a 500-ml dropping funnel. The flask and dropping funnel were flamed in a stream of argon. To the flask was added 78 ml (0.56 mole) of diisopropylamine and followed by 500 ml of dry THF (Baker, reagent grade). The solution was stirred and cooled to -78º in an acetone dry ice bath, while 202 ml (0.51 mole) solution of 2.5 M nbutyllithium in hexane (Aldrich) was transferred from the bottle to a dropping funnel via a double-tipped needle (3 ft., 16 gauge, Aldrich) and then added

dropwise to the solution over 20 min. After another 20 min. of stirring at -78°, the 500-ml dropping funnel was rapidly replaced with a 250-ml dropping funnel

containing a solution of 151.4 g (0.42 mole) of

phosphonate in 120 ml THF. The addition of phosphonate to LDA solution at -78° caused a color change

immediately. After addition was completed (over 15 minutes), the resulting deep red mixture was stirred at -78° for 1 hour longer. Then 49.1 g (0.33 mole) of 2-adamantanone was added. The mixture was stirred at -78° for 10 minutes and allowed to warm to room

temperature in ca. 1.5 hour, and finally brought to reflux for 1.5 hour. Vigorous gas evolution was noticed during refluxing. The cooled reaction mixture was treated with 0.5 L of saturated NaHCO3 solution for 10 minutes and poured into a 4 L separatory funnel

containing 2 L of water. The aqueous phase was extracted three times with 10% EtOAc in hexane (3 × 250 ml). The combined organic phase was washed with 1.5 L of water, then with 1.5 L of brine and dried over Na₂SO₄. Removal of solvent gave 135.5 g of viscous brown oil. The crude product was diluted with 100 ml of 10% EtOAc in hexane and loaded onto a column (O.D.-4.5 cm., length-40 cm.), packed with 80 g of silica gel (60-200 mesh, Baker). Elution with 10 to 20% EtOAc-hexanes gave five

fractions; 118 g of orange oil was recovered after concentration, which was a mixture of the pivaloyloxy enol ether and the phenolic enol ether (Rf values are 0.62 and 0.22, respectively, in 10% EtOAc-hexanes) along with impurities. The oily pivaloyloxy enol ether was isolated by further chromatography to provide an

analytical sample, characterized by IR and ¹HNMR (see Tables 6 and 10).

De-acylation of the mixture was completed in 2.5 hours by refluxing the mixture of crude products, 16.5 g of K₂CO₃ and 300 ml of MeOH. After removal of solvents on a rotavap, an orange muddy solid was obtained. The solid was treated with 200 ml of H₂O and then scratched vigorously with a spatula to afford a filterable

material. The solid was filtered and washed thoroughly with 1.5 L of H₂O. After removal of most of the moisture under vacuum, the slightly yellow solid was redissolved in 600 ml of CH₂Cl₂ (with gentle heating if necessary) and dried over Na₂SO₄. The solution was filtered on a Buchner funnel, packed with 40 g of silica gel. Upon concentration to the half volume, a white solid began to fall out of the solution. Recyrstallization in a mixture of 1:1 CH₂Cl₂ and hexane gave 58.79 g (67%) of white phenol enol ether (mp: 131-133). Another 20-22 g of product could be collected from the mother liquor after chromatography.

Example 8

3-Acetoxybenzaldehyde

3-Hydroxybenzaldehyde (10 g., 81.88 mmol) was dissolved in 150 ml dichloromethane under argon.

Triethylamine (17.12 ml, 0.123 mol) and dimethylaminopyridine (5 mg.) were added, and the resulting stirred solution was treated with acetic anhydride (8.5 ml, 90 mmol). After stirring for fifteen hours, the reaction mixture was transferred to a separatory funnel using an additional 50 ml dichloromethane. The organic layer was washed with water (2 × 100 ml) and concentrated to give a light brown oil weighing 14.85 g. Plug filtration through silica gel using dichloromethane furnished

13.3 g (quant.) of a light orange oil which was shown by NMR and IR to be pure enough for use in subsequent reactions (see Tables 3 and 7).

The aldehyde was converted to the corresponding dimethyl acetal by way of the general procedure in

Example 4. The oily product, which was homogeneous according to TLC, was obtained in good yield. The structure was confirmed by proton NMR and IR spectra (see Tables 4 and 8). Conversion of the acetal to diethyl 1-methoxy-1-(3-acetoxyphenyl) methane

phosphonate was carried out as in Example 5. NMR and IR spectral data confirmed the structure (see Tables 5 and 9) and indicated that the crude product (oil) was pure enough for subsequent use. Example 9

Diethyl-1-methoxy-1-(3-hydroxyphenyl)methanephosphonate Diethyl-1-methoxy-1-(3-acetoxyphenyl)methanephosphonate from Example 8 (10.29 g., 32.56 mmol) was dissolved in methanol (35 ml). Water (5 ml), and sodium bicarbonate (5 g, 60 mmol) were then added with

stirring. After 48 hours at room temperature, the reaction mixture was concentrated in vacuo to remove methanol. The residue was treated with 150 ml

dichloromethane and washed with water (2 × 50 ml). The organic layer was rotory evaporated and pumped at high vacuum to yield 8.21 g. (93%) of the product as a light yellow, viscous oil. Spectral data (Tables 5 and 9) are in accordance with the structure:

Example 10A

6-Methoxynaphthalene-1-carboxaldehyde dimethyl acetal

6-Methoxynaphthalene-1-carbonitrile was synthesized from 6-methoxy-1-tetralone by the method of Harvey, R.G., et al., J Org. Chem., 48:5134 (1983). The nitrile (354.6 mg., 1.94 mmol) was dissolved in 10 ml dry toluene under argon. The solution was cooled to -78° in a dry ice/acetone bath. A toluene solution of DIBAL (1.3 ml of a 1.5 M solution, 1.95 mmol) was added dropwise by syringe with stirring. After 10 minutes the mixture was warned slowly to room temperature and partitioned between 3N HCl and dichloromethane (25 ml of each). The organic layer was washed with two additional portions of 3N HCl. The combined aqueous layers were back-extracted several times with 10 ml portions of dichloromethane. The combined organics were dried over Na₂SO₄ and concentrated to yield yellow crystals of the aldehyde which were immediately dissolved in methanol (10 ml) and trimethyl orthofornate (0.25 ml, 2.29 mmol). Several crystals of p-toluenesul-fonic acid were added, and the solution was stored for 3 days in the

refrigerator. A small amount of NaHCO3 was added and the solvents were stripped. The residue was taken up in minimal dichloromethane and chromatographed on a silica gel column using hexanes as the eluant. The appropriate fractions were evaporated to furnish 395 mg. of the title compound (88% yield for 2 steps) as a light yellow oil which was homogeneous on TLC and exhibited no carbonyl absorption in the infrared spectrum. NMR and IR spectral data are consistent with the structural assignment.

IR (CNCl₃, cm^-1): 2995, 2822, 1622, 1598, 1509, 1465, 1430, 1370, 1250, 1109, 1050, 841.

NMR (CDCl3, ppm): 3.36 (6N, s); 3.92 (3N, s); 5.85 (IN, s); 7.16 (IN, d); 7.18 (IN, dd); 7.42 (IN, t); 7.56 (IN, d, J=7.08 Nz); 7.72 (1H, d, J=8.11 Hz); 8.19 (1H, d, J=9.09).

Example 10B

Diethyl 1-methoxy-1-(6-methoxynaphth-1-yl)methane phosphonate

The title phosphorate was synthesized according to the general procedure described in Example 5. Spectral data confirm the product structure.

IR (CNCl₃, cm^-1): 2994, 1619, 1594, 1504, 1458, 1429, 1372, 1242 (P=O), 1050 (br), 968, 845, 810.

NMR (CDCl₃, ppm): 3.38 (3H, s); 3.92 (3H, s); 3.9 4.06 (4H, m); 5.25 (1H, d, J=16.4 Hz); 7.15 (1H, d, J=2.2 Hz); 7.18 (1H, dd, J=9.3, 2.85 Hz); 7.72 (1H, d, J=8.05 HZ); 8.12 (1H, d, J=9.28 Hz) . Example 11A

6-Methoxy-2-naphthaldehyde

6-Methoxy-2-naphthaldehyde was synthesized, using a Bouveault reaction [E.A. Evans, J. Chem. Soc., 4691 (1956); P.T., Szzo, et al., J. Org. Chem., 24:701

(1959); D.C. Owsley, et al., J. Org. Chem., 38:901

(1973)], by lithiating 5.08 g. (21.4 mmol) of 6-methoxy-2-bromonaphthalene (dissolved in 50 ml dry THF) with n-butylithium (13.7 ml, 21.8 mmol, 1.6 M) at -78° and quenching the arylilthium with dropwise addition of sieve-dried dimethylformide (1.8 ml, 23.2 mmol). After allowing the reaction to warm slowly to 0°, the

intermediate aryl hydroxytamine was acidified with 3N NCI at 0°C, facilitating amine elimination to the desired aldehyde. The solution was partitioned between EtOAc and 3N NCI, washing the aqueous layer 3 times with EtOAc to recover all the aldehyde, and then the combined EtOAc solutions were washed with saturated NaNCO₃ solution and dried over Na₂SO_A. After decanting and evaporating the solution, the resultant oil was

dissolved in minimal CH₂Cl₂, followed by addition of hexanes until the solution clouded. Refrigeration for 48 hours afforded 2.292 g (73%) of white crystals upon filtration, which melted at 47-48°.

IR (CNCl₃, cm^-1): 1685 (C=O), 1618, 1475, 1389, 1263, 1190, 1168, 1027, 895, 856, 839.

1H NMR (CDCl₃, ppm): 3.94 (3H, s); 7.16 (1H, d. J=2.44 Hz); 7.21 (1H, dd, J=8.88, 2.44 Nz); 7.79 (1H, d, J=8.55 Nz); 7.87 (1H, d, J=8.85 Hz); 7.90 (1H, d, J=8.55 Hz); 7.87 (1H, d, J=8.85 Hz); 7.90 (1H, dd, J=8.54, 1.52 Hz); 8.23 (1H, s); 10.07 (1H, s).

Example 11B

6-Methoxy-2-naphthaldehyde dimethyl acetal The 6-methoxy-2-naphthyldimethyI acetal was

synthesized in 61% yield (m.p. 27°) according to the procedure descrieed in Example 4. IR (CNCl₃, cm^-1): 2930, 2825, 1629, 1604, 1480, 1260, 1190, 1167, 1098, 1046, 890, 850.

¹H NMR (CDCl₃, ppm): 3.38 (6H, s); 3.93 (3H, s); 5.54 (1H, s); 7.15 - 7.18 (2H, m); 7.52 (1H, dd, J=8.55, 1.51 HZ); 7.75 (1H, d, J=8.55 Hz); 7.76 (1H, d, J=8.55 Hz); 7.87 (1H, s).

Example 11C

Diethyl 1-Methoxy-1-(6-methoxynapth-2-yl)methane

phosphate

The corresponding phosphonate was synthesized in 60% yield (oil) as described in Example 5.

IR (CNCl₃, cm^-1): 2998, 1619, 1603, 1480, 1390, 1258 (P=O), 1161, 1094, 1050 (br), 970, 852.

¹H NMR (CDCl₃, ppm): 1.21 (3H, t, J=7.16 Hz); 1.26 (3H, t, J=7.16 Hz); 3.41 (3H, s); 3.92 (3H, s); 4.04 - 4.11 (4H, m); 4.64 (1H, d, J=15.41 Hz); 7.14 - 7.17 (2H, m); 7.54 (1H, d, J=8.68 Hz); 7.75 (1H, d, J=8/86 Hz); 7.76 (1H, d, J=8.47 Hz); 7.82 (1H, s).

Example 11D

7-Methoxy-2-naphthaldehyde

7-Methoxy-2-naphthaldehyde was synthesized in 48% yield (oil) using the Bouveault reaction as described above.

IR (CNCl₃, cm^-1): 1687 (C=O), 1601, 1460, 1389, 1331, 1266, 1175, 1115, 1030, 842.

¹H NMR (CDCl₃, ppm): 3.97 (3H, s); 7.28 - 7.33 (2H, m); 7.80 - 7.89 (3H, m); 8.25 (1H, s); 10.15 (1H, s).

Example HE

7-Methoxy-2-naphthaldehyde dimethyl acetal The corresponding dimethyl acetal was synthesized in 86% yield (oil), following the conditions described in Example 4.

¹H NMR (CDCl₃, ppm): 3.38 (6H, s); 3.93 (3H, s) ; 5.55 (1N,S) 7.15 - 7.18 (2N, m); 7.42 (IN, dd, J=8.03, 1.95 Hz) 7.75 (1H, d, J=9.77 Nz) ; 7.78 (1H, d, J=8.36 Hz) ; 7.85 (1H, s) .

Example 11F

Diethyl 1-methoxγ-1-(7-methoxynaphth-2-yl)methane phosphonate

Diethyl 1-methoxy-1-(7-methoxynaphth-2-yl)methane phosphonate was synthesized in 65% yield (oil) following the general procedure outlined in Example 5.

IR (CNCl₃, cm^-1): 2295, 1630, 1603, 1460, 1390, 1256 (P=O), 1092, 1050 (br), 1027 (br), 970, 908, 840.

1H NMR (CDCl₃, ppm): 1.22 (3H, t, J=7 Hz); 1.27 (3NH t, J=7 Hz) 3.43 (3H, s) 3.93 (3H, s) ; 4.04 - 4.11 (4H, m) 4.66 (1H, d, J=15.6 Hz); 7.15 - 7.17 (2H, m); 7.43 (1H, d, J=7.93 Hz); 7.73 (1H, d); 7.78 (1H, d, J=8.46 Nz); 7.82 (1H, s).

Example 106

Phosphorylation of Enol Ether Phenol

The enol ether phenol a (R³ = methyl, T = adamant-2-ylidene,from Example 7) was reacted with 2-oxo-1,3,2-dioxaphospholane according to Thuong, N.T., et al.,

Bull. Soc. Chim. France, 2083 (1975) to give a cyclic phosphate triester, which underwent ring opening with NaCN to yield the 2-cyanoethyl diester salt. Ammonium hydroxide then induced a facile β-elimination reaction to a filterable sodium ammonium salt of b (75% yield from a), which was ion exchanged to the disodium salt of b for ¹HNMR (D₂O, 400 MHz); δ 1.6 - 7.9 (12H, m); 2.44 (1H, s); 2.97 (1H, s); 3.22 (3H, s); 6.98 (1H, m, 7.52 Hz); 6.96 (1H, s); 7.05 (1H, m); 7.18 (1H, dd, 7.62, 8.06 Hz). This same salt was obtained directly using sodium methoxide to induce β-elimination.

Example 107

Photooxygenation of an Enol Ether Phosphate

wherein T = adamantane

a b The sodium ammonium salt a was ion exchanged to the monopyridinium salt. A 0.06 M solution of the latter salt was photooxygenated in the presence of O₂ and TPP at 5°C. (Slower reaction rates and increased photolytic damage to the product were experienced with the use of solid phase sensitizers such as Sensitox S or methylene blue on silica gel). Purification on a reversed phase NPLC column at pN 8.6 (Na₂CO₃) and using an acetonitrile-water gradient, followed by lyophilization, provided 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoroyloxy)phenyl-1,2-dioxetane(b) as a faintly yellow solid, in 80% yield.

¹HNMR (D₂O, 400 MHz): 0.85 (1H, d); 1.13 (1H, d); 1.40 1.67 (10H, m); 2.13 (1H, s); 2.75 (1H, s); 3.10 (3H, s); 7.15 (2H, broad, featureless); 7.20 (1H, d, 7.81 HZ); 7.28 (1H, dd, 7.81, 8.09 Hz).

The upfield doublets are characteristic of the beta adamantane ring protons in the dioxetane, which are more shielded by the proximate aromatic ring than in the enol ether. The coalescence of the two aromatic proton resonances into a broad peak at 7.15 ppm mirrors similar behavior in the ¹³C spectrum (D₂O/CD₃OD); two aromatic carbon resonances at 120.95 ppm and 122.10 ppm are broad, low intensity peaks at 0°C, which sharpen and become more intense at 40°C. This indicates restricted rotation of the aromatic substituent, which may

introduce a conforma-tional component into the rate of electron transfer decomposition of the anion to the excited state ester.

Example 108

Diethyl 1-methoxy-1-(3-pivaloyloxyphenyl)methane phosphonate (65.8 g, 0.184 mol.), prepared as described in our copending application Serial No. 402,847, was placed in a dry 1 liter flask under argon. Dry tetrahydrofuran (165 ml.) was added, followed by

2-adamantanone (24.8 g, 0.165 mol.). The solution was stirred to homogeneity and set aside. In a separate 500 ml. flask, n-butyllithium (81 ml. of a 2.5 M

solution in hexanes) was added from a dropping funnel to a solution of diisopropylamine (30 ml., 0.214 mol.) in 200 ml. of tetrahydrofuran, which had been cooled in a dry ice-acetone bath to -78° C under an argon atmosphere. The resulting solution of lithium diisopropylamide was stirred at low temperature for another 25 minutes and then cannulated with a double tipped needle into the solution of phosphonate and 2-adamantanone which had also been cooled to -78ºC. Lithium diisopropylamide was then added dropwise, with vigorous stirring, over a 1.5 hour period. The clear, light brown reaction mixture was then stirred for an additional 30 minutes at low temperature, warmed to room temperature, and then refluxed for 2.5 hours under argon and cooled to room temperature. Thin layer chromatography (TLC) of the crude reaction mixture (Whatman K5F; 10% ethyl acetate hexanes) displayed three U.V. absorbing spots; one at the origin, one at R_f.28, and the major spot at R_f.70.

The thus-obtained reaction mixture was treated with several aliquots of pivaloyl chloride, with stirring for several hours at room temperature between additions.

After a total of 4.75 ml. (38.5 mmol.) of the acid chloride had been added, TLC showed that the spot at R_f.28 had completely disappeared. Thus, the lithium salt of methoxy (3-hydroxyphenyl)methylene adamantane present in the reaction mixture had been converted to the

correspond-ing pivaloate ester at R_f.70. Tetrahydrofuran was then partially removed by distillation at

atmospheric pressure to obtain a thick slurry, which was then partitioned between water and 10% ethyl acetatehexanes. The aqueous layer was separated and washed again three times with the same solvent. The combined organics were then washed several times with a saturated aqueous solution of sodium bicarbonate, dried over sodium sulfate, and filtered to remove any particulates. Concentration of the solution on a rotory evaporator gave a thick slurry of crystalline product. The slurry was diluted with hexanes, cooled to -20°, and filtered. The filter cake was washed under argon with hexanes which had been cooled in a dry ice-acetone bath. The orange-brown filtrate was concentrated to an oil, which was dissolved in minimal hexanes, seeded with crop 1 and cooled to yield a second crop of the product. The mother liquors from this operation were then plug chromatographed on 74 g. of silica gel, eluting with hexanes to leave the origin material (residual

phosphonate ester and its decomposition products) behind. A third crop of product could then be obtained upon concentration of the eluant. The total yield of methoxy (3-pivaloyloxyphenyl) methylene adamantane was 54.67 g (79%), melting point 83-85A Spectral data (IR, and ¹HNMR) were identical to those previously reported in our copending application Serial No. 402,847; see

Example 59.

Example 109

A flame-dried flask was charged with methoxy(3-pivaloyloxyphenyl)methane phosphonate (5.01 g, 14.1 mmol.). Anhydrous methanol (40 ml.) was added under argon. The resulting suspension was stirred vigorously during the dropwise addition of 4.37 M sodium methoxide in methanol (3.25 ml., 14.2 mmol.). Tne suspended solid dissolved during this operation. After stirring the mixture for one hour at room temperature, TLC (Whatman K5F; 10% ethyl acetate-hexanes) showed that a very faint trace of the starting material remained (R_f.70). One drop of the sodium methoxide solution was added to the clear solution, which was then concentrated on a rotory evaporator (bath temperature 35º) and then pumped in vacuo (1.0 mm. Hg) at 40° for 24 hours. The resulting dry, white solid, sodium 3-(methoxytricyclo[3.3.1.1^3,7]dec-2-ylidenemethyl) phenox-ide, weighed 4.1 g. (quantitative yield). It was insoluble in dichloromethane, and TLC of the supernate showed no evidence for the presence of any phenolic impurities. A nujol mull of the product displayed an infrared spectrum which was devoid of OH stretch absorbances between 3500 and 3300 cm^-1. The phenolate salt did not exhibit a melting point below 280°, but did darken somewhat beginning at 170º. It was kept dry during all

subsequent manipulations, and stored in a dessicator over Drierite.

IR (nujol mull): 1572, 1405, 1310, 1285, 1198,

1175, 1150, 1090, 988, 870, 800, 777 cm^-1.

Example 110

Sodium 3-(methoxytricyclo[3.3.1.1^3,7]dec-2- ylidenemethyl)phenoxide (1.74 g., 6.0 mmol.) was added under argon to 10 ml. of scrupulously dried dimethyl formamide containing several drops of triethylamine. The resulting slurry was vigorously swirled during the addition of 2-chloro-2-oxo-1,3,2-dioxaphospholane (0.580 ml., 6.3 mmol.) over 25 minutes. The mixture thinned considerably during this addition and over an additional 3.5 hours of vigorous stirring at room temperature. Dry sodium cyanide (0.325 g. 6.6 mmol.) was then added, with exclusion of moisture, and stirring was continued overnight at room temperature to give an orange, cloudy solution. The solvent was removed in vacuo (1.0 mm Hg) at 50° and the residue was rinsed twice with o-xylenes to further eliminate DMF.

The resulting brown foam was dissolved in 10 ml. of methanol prior to the dropwise addition of 4.37 M sodium methoxide in methanol (1.30 ml., 5.7 mmol.).

After 30 minutes, the solvent was removed on the rotory evaporator and the residue was slurred in 5% water/ acetone (v/v) and filtered. The solid filter cake was dissolved in water and subjected to reverse phase chromatography (PLRP polystyrene preparative HPLC column, using a water-acetonitrile gradient) to

conveniently isolate disodium 3-(methoxytricyclo[3.3.1.1^3,7]dec-2-ylidenemethyl)phenyl phosphate in good yield as a white fluffy solid after lyophilization of the appropriate fractions. The ¹HNMR spectral data for the product were identical to those reported in copending application Serial No. 402,847.

The above discussion of this invention is directed primarily to preferred embodiments and practices thereof. It will be readily apparent to those skilled in this art that further changes and modifications in the actual implementation of the concepts described herein can easily be made without departing from the spirit and scope of the invention as defined by the following claims.

Claims

Claims

1. A process for producing a 1,2-dioxetane

compound having the formula:

wherein T is a spiro-bound substituted or unsubstituted polycycloalkylidene stabilizing group that prevents the dioxetane compound from decomposing before the labile bond between Y and Z is intentionally cleaved; Y is a mono- or poly-substituted aromatic, light-emitting fluorophore-forming chromophobe group capable of

absorbing energy to form an excited energy state from which it emits optically-detectable energy to return to its ground state; Z is hydrogen, a chemically cleavable or enzymecleavable group; R³ is a C₁-C₂₀ branched or unbranched, substituted or unsubstituted, saturated or unsaturated alkyl, heteroalkyl, cycloalkyl,

cycloalkenyl, aryl, aralkyl or aralkenyl group, or a cycloalkenyl, cycloalkyl, aryl, aralkyl or aralkenyl group fused to Y such that the emitting fragment

contains a lactone ring, or an N, O, or S heteroatom- containing group, or enzyme-cleavable group, with one or more of T, Y, Z or R³ being unsubstituted or substituted with a group that enhances the water solubility of the 1,2-dioxetane, comprising alkylating a phosphorus halide of the formula:

PQ₃ wherein any Q is halogen or 1 wherein R¹ is as defined below, with a lower alkanol, or its alkali metal salt, of the formula: R¹OH or R¹O- M⁺ wherein R¹ is a lower alkyl group, or a trialkylsilyl group, and M⁺ is an alkali metal cation, to produce a tertiary phosphorus acid alkyl ester of the formula:

(R¹O)₃P reacting the phosphite ester and a his acid with an aryl dialkyl or cyclic acetal of the formulae:

wherein n is an integer of 2 or more, produced by reacting the corresponding aryl aldehyde with a lower alkanol or diol of the formula:

R³OH or HO-(CH₂)_n-OH to produce a 1-alkoxy-1-arylmethane-phosphonate ester of the formula:

reacting the 1-alkoxy-1-arylmethane-phosphonate ester with base to produce a phosphonatestabilized carbanion of the formula:

reacting the phosphonate-stabilized carbanion with a carbonyl compound of the formula:

T == or O == T == to produce a mono- or di-enol ether of the formula:

and oxygenating the double bond in the enol ether to give the corresponding 1,2-dioxetane compound.

2. A process of claim 1, wherein the phosphorous halide is reacted with R¹OH in the presence of a base.

3. A process of claim 2, wherein the base is triethylamine, pyridine, dimethylaniline, or magnesium oxide.

4. A process of claim 1, wherein the aryl dialkyl or cycloalkylacetal is produced by reacting the

corresponding aryl aldehyde and the lower alkanol or diol in the presence of a catalyst and removing water.

5. A process of claim 4, wherein the Lewis or mineral acidic catalyst is p-toluenesulfonic acid. Amberlyst XN1010 resin, HCl(g), anhydrous copper

sulfate, methane sulfonic acid, or BF₃ etherate.

6. A process of claim 4, wherein water is removed by a water scavenger selected from a group consisting of trialkylorthoformate, 2,2-dimethoxypropane,

trialkylortho-acetate, or dialkylsulfite or molecular sieves.

7. A process of claim 4, wherein water is removed by azeotropic distillation.

8. A process of claim 1, wherein the arylaldehyde alkyl or cycloalkyl acetal is reacted with the tertiary phosphorous acid alkyl ester in an aprotic organic solvent under an inert atmosphere at a temperature at or below about 0ºC.

9. A process of claim 1, wherein the Lewis acid is BF₃, etherate, SNCl₄, TiCl₄ or trimethylsilyl

triflate.

10. A process of claim 1, wherein the aprotic organic solvent is methylene chloride, chloroform, benzene, or toluene.

11. A process of claim 1, wherein the

1-alkoxy-1-aryl methane phosphonate ester is reacted in an aprotic organic solvent under an inert atmosphere at temperatures at or below about 0ºC.

12. A process of claim 1, wherein the base is sodium hydride, sodium amide, a metal alkoxide, or n-alkyl lithium, or a lithium amide.

13. A process of claim 11, wherein the aprotic organic solvent is an aliphatic hydrocarbon, an aromatic hydrocarbon, diglyme, a cyclic ether, an alkanol, an alkylated formamide Or acetamide, an alkylated

sulfoxide, or mixtures of these solvents.

14. A process of claim 1, wherein the phosphonatestabilized carbanion is reacted with a mono- or dicarbonyl compound at a temperature up to and including reflux.

15. A process of claim 14, wherein the mono- or di-carbonyl compound is a substituted or unsubstituted polycyclic alkyl ketone containing from 6 to 30 carbon atoms, inclusive.

16. A process of claim 1, wherein oxygenation is carried out using singlet oxygen in a halogenated

organic solvent at a temperature at or below about 5ºC.

17. A process of claim 1, wherein the aryl

aldehyde is a monocyclic, polycyclic, heterocyclic or polyhetero-cyclic aromatic mono- or polyaldehyde.

18. A process of claim 17, wherein the aryl or heteroaryl aldehyde contains a phenyl, biphenyl, 9,10- dihydrophenanthryl, naphthyl, anthryl, pyridyl,

quinolinyl, isoquinolinyl, phenanthryl, pyrenyl,

coumarinyl, carbostyryl, acridinyl, dibenzosuberyl, or phthalyl, or derivatives thereof.

19. A process of claim 17, wherein the aryl or heteroaryl aldehyde is substituted at a point meta to the point of attachment of the aldehyde side chain to the aryl group with a functional R² group, wherein the R² group is an alkyl ether, halogen, hydroxyl group, an amino group, di (lower alkyl) amino group or its salt, a sulfonamido group, or an acyloxy group.

20. A process of claim 19, wherein the R² group is an alkoxy group or a pivaloyloxy group.

21. A process of claim 19, wherein the aryl aldehyde group is further substituted with a functional group X¹ located ortho, meta. or para to the point of attachment of the aldehyde side chain to the aryl group, wherein X¹ is hydrogen, heteroaryl, aryl, allyl,

hydroxyalkyl, alkylthioether, alkylamino, dialkylamino, alkyl, alkoxy, aldehyde, halo, nitro, cyano, sulfone, alkylhalo, trifluoromenthly a carboxylic acid salt or ester group, an amido sulfonyl group or an SO₂R⁶ group wherein R⁶ is menthyl, Bhenyl or NHC₆H₆.

22. A process of claim 1, wherein R³ is an alkyl, aryl, aralkyl, alkoxy, alkylsiloxyalkyl, aminoalkyl or dialkylamino alkyl group.

23. A process of claim 19, wherein the R² group on the aryl ring of the enol ether is converted to a free hydroxy group by a process further comprising the steps of:

(a) cleaving an alkoxygroup to a hydroxyl group with sodium thioethoxide in an aprotic solvent, lithium iodide in refluxing pyridine, sodium cyanide in refluxing dimethylsulfoxide, or sodium sulfide in refluxing N-methyl-2- pyrrolidone, or,

(b) cleaving a pivaloyloxy group with

potassium carbonate or sodium methoxide in an alkanol solvent.

24. A process of claim 23, wherein the free aryl hydroxy group obtained by cleaving the alkoxy or pivaloyloxy group is converted to an enzyme-cleavable carboxylic ester group by a process further comprising reacting the hydroxy group with a carboxylic acid anhydride or chloride in the presence of a base.

25. A process of claim 23, wherein the free aryl hydroxy group obtained by cleaving the alkoxy or

pivaloyloxy group is converted to an enzyme-cleavable phosphate group by a process comprising the steps of:

(a) reacting the aryl hydroxyl group with a cyclic phosphate ester of the formula:

wherein Q is an electronegative leaving group, with a Lewis or inorganic base in an aprotic organic solvent to yield the corresponding cyclic phosphate triester;

(b) reacting the thus-obtained cyclic

phosphate triester with a cyanide salt to open the cyclic phosphate triesterIs ring and give the

corresponding 2-cyano ethyl diester monosalt; and,

(c) subjecting the monosalt to p-elimination with a base to give the corresponding monophosphate ester salt.

26. A process of claim 25, wherein Q is a halogen.

27. A process of claim 25, wherein the base is a tertiaryamine, magnesium oxide, or molecular sieves.

28. A process of claim 25, wherein the aprotic organic solvent is an aromatic hydrocarbon, a cyclic ether, an alkyl substituted formamide or acetamide, a dialkyl sulfoxide, a dialkyl ether, hexane, or a mixture of these solvents.

29. A process of claim 25, wherein the cyanide salt is an alkali metal salt, quaternary ammonium salt or a polymeric quaternary ammonium salt.

30. A process of claim 25, wherein the base inducing β-elimination of the 2-cyano ethyl diester monosalt is ammonium hydroxide, sodium methoxide, or tetramethyl ammonium hydroxide.

31. A process of claim 23, wherein the free aryl hydroxyl group obtained by cleaving the alkoxy or pivaloyloxy group is converted to an enzyme-cleavable phosphate group by a process comprising the steps of:

(a) reacting the aryl hydroxy group with a compound having the formula:

wherein Q is an electronegative leaving group, and R4 and R5 are each independently cyano, nitrophenyl, dinitrophenyl, arylsulfonyl, alkylsulfonyl, or

trimethylsilyl, with a Lewis base in an aprotic organic solvent, to give the corresponding phosphate triester having the formula:

and; (b) reacting this phosphate triester with a base MF, MOH or MOCN₃, wherein the cation M⁺ is an alkali metal, ammonium, or C,-C₇ alkyl, aralkyl,or aromatic quaternary ammonium cation, (NR¹⁰ ₄)⁺ wherein R¹⁰ is an alkyl or aralkyl group or forms part of a heterocyclic ring system, to give a corresponding phosphate monoester disalt having the formula:

32. A process of claim 31 further comprising the exchange of M⁺ in the enol ether phosphate ester disalt to mono-, di- or trialkyl ammonium cation or pyridinium (H⁺) cation by ion exchange.

33. A process of claim 31, wherein Q is a halogen.

34. A process of claim 23, wherein the free aryl hydroxyl group obtained by cleaving the alkoxy group is converted to an enzyme-cleavable glycoside by a process further comprising the steps of:

(a) reacting the aryl hydroxyl group with a tetra-O-acetyl-D-hexopyranosyl halide with a base in an aprotic organic solvent under an inert atmosphere to produce an aryl-O-D-hexopyranoside tetraacetate; and

(b) hydrolyzing the protective acetyl groups of the hexopyranoside tetraacetate to produce a

hexopyranosidyl moiety.

35. A process of claim 34, wherein the

D-hexopyranoside is D-glucose, D-galactose, D-fructose, or D-mannose.

36. A process of claim 1, wherein T is a polycycloalkylidene group, Y is a phenyl, naphthyl, phenanthryl, biphenyl, pyridyl, anthryl, phthalyl or an unsaturated heterocyclic ring fused to one or more aryl rings, R³ is methyl or ethyl, and Z is acetoxy, phosphate monoester disalt, or O-D-hexopyranoside.

37. A process of claim 36, wherein T is adamant-2- ylidene.

38. A process of claim 17, wherein the aryl group of the aryl aldehyde is substituted with a functional group R2, which can be an alkyl ether, alkyl thioether, hydroxyl, acyloxy, sulfonamido, or substituted or unsubstituted amino group, the point of attachment of the group R² to the aryl group being such that-the total number of ring carbon atoms separating the ring carbon atom to which the group R² is attached and the ring carbon atom to which the aldehyde group of the aryl aldehyde is attached, including the ring carbon atoms at the points of attachment, is an odd whole number.

39. A process of claim 38, wherein the odd whole number is 5 or greater.

40. A process for producing a 1-alkoxy-1- arylmethane-phosphonate ester having the formula

wherein R¹ is a lower alkyl group; R³ is a C₁-C₂₀

unbranched or branched, substituted or unsubstituted, saturated or unsaturated alkyl, heteroalkyl, cycloalkyl, cycloalkenyl, aryl or aralkyl group, fused ring cycloalkenyl, cycloalkyl, aryl, aralkyl or aralkenyl group anyone of which may be fused to Y such that the emitting fragment contains a lactone ring, or an N, O or S heteroatom-containing group, or an enzyme-cleavable group; Y is an aromatic, or heteroaromatic ring; Z is hydrogen, a chemicallycleavable group or an enzyme- cleavable group; n is an integer of 2 or more; with one or more of T, Y, Z or R³ being substituted with a group that enhances the water solubility of the 1,2- dioxetane or unsubstituted with such a group, comprising reacting an aryl dialkyl or cyclic acetal of formulae:

with a tertiary phosphorous acid alkyl ester of the formula

(R¹O)₃P with a Lewis acid in an aprotic solvent under an inert atmosphere at temperatures at or below above 0ºC.

41. A process of claim 40, wherein the acetal is an unsymmetrical aryl or hetero aldehyde cyclic acetal produced by the bonding of one of the R³ groups to the aromatic Y ring, the other R³ being different than the one fused to the Y ring, of the formula:

wherein n is an interger of 2 or more such that the phosphonate ester produced is a 5 or 7-R²-substituted isochhrroman-1-yl, a 4 or 6-R²-substituted 1,3-dihydroisobenzofuran-1-yl, a 5 or 8-R²-substituted 1H, 3H-2-oxaphenalen-1-yl, a 7 or 10-R²-substituted 3-oxa-1,2,3,4-tetrahydrophenanthry-4-yl, a 6,8 or 9-R²- substituted 2-oxa-1,2,3,4-tetrahydrophenanthr-1-yl, or an R²

substituted dihydroisonaphthofuran-1 or 3-yl phosphonate ester, wherein R² is an alkyl ether, halogen, hydroxyl group, an amino group, di (lower alkyl) amino group or its salt, a sulfonamido group, or an acyloxy group.

42. A process of either claim 40 or claim 41, wherein the Lewis acid is BF₃, etherate, SnCl₄, TiCl₄, and trimethylsilyl triflate.

43. A process of either claim 40 or claim 41, wherein the aprotic organic solvent is methylene

chloride, chloroform, benzene, or toluene.

44. A process of either claim 40 or claim 41, wherein Y is phenyl, biphenyl, 9,10-dihydrophenanthryl, naphthyl, anthryl, pyridyl, quinolinyl, isoquinolinyl, phenanthryl, pyrenyl, coumarinyl, carbostyryl,

acridinyl, dibenzosuberyl, phthalyl or derivatives thereof, R³ is methyl or ethyl, and Z is an acetoxy, a phosphate monoester disalt, or a glycoside.

45. A process of claim 44, wherein aryl group Y is further substituted with an X¹ group located ortho, meta or para to the Z group, wherein X¹ is hydrogen, or an aryl, allyl, hydroxyalkyl, alkylthioether, amino, alkylammo, dialkylamino, alkyl, alkoxy, B-9 aldehyde, halo, nitro, cyano, trialkylsilyl, alkylhalo,

trifluoromethryl, heteroaryl, heteroaralkly, alkoxy carbonyl, or an SO₂R⁶ group wherein R⁶ is methyl, phenyl or NHC₆H₅ group.

46. A process for producing an enol ether of the formula:

wherein T is a substituted or unsubstituted

polycycloalkyl group, R³ is C₁-C₂₀ unbranched or branched, substituted or unsubstituted, saturated or unsaturated alkyl, heteroalkyl, cycloalkyl, cycloalkenyl, aryl or aralkyl group, fused ring cycloalkenyl, cycloalkyl, aryl, aralkyl or aralkenyl group anyone of which may be fused to Y such that the emitting fragment contains a lactone ring, N, O or S heteroatom-containing group, or an enzymecleavable group; Y is an aromatic or

heteraromatic ring such that the dioxetane which is formed by adding singlet oxygen to the double bond will be activatable to yield a light-emitting, fluorescent chromophore capable of absorbing energy to form an excited energy state from which it emits optically detectable energy to return to its ground energy state; Z is hydrogen, a chemically-cleavable group or an enzymatically cleavable group, with one or more of T, Y, Z or R³ being substituted or unsubstituted with a group that enhances the water solubility of the compound, comprising reacting a phosphonate-stabilized carbanion of the formula:

wherein R³, Y and Z are defined above, R¹ is a lower alkyl group, and n is an integer of 2 or more, produced by treating the corresponding 1-alkoxy-1-aryl methane phosphonate ester with a metal-containing base in an aprotic organic solvent under an inert atmosphere at temperatures at or below about DC, with a ketone of the formula

T = O or O = T = O wherein T is as defined above, at temperatures up to and including reflux conditions.

47. A process of claim 46, wherein the base is sodium amide, a metal alkoxide, n-alkyl lithium or lithium dialkyl amide.

48. A process of claim 46 wherein the aprotic organic solvent is an aliphatic hydrocarbon, an aromatic hydrocarbon, diglyme, a cyclic ether, an alkanol, an alkylated formamide or acetamide, an alkylated

sulfoxide, or a mixture of these solvents.

49. A process of claim 46, wherein the carbonyl compound is a substituted or unsubstituted polycyclic alkyl ketone containing 6 to 30 carbon atoms.

50. A process of claim 46, wherein the Y group is a phenyl, biphenyl, 9,10-dihydrophenanthryl, naphthyl, anthryl, pyridyl, guinolinyl, isoquinolinyl,

phenanthryl, pyrenyl, coumarinyl, carbostyryl,

acridinyl, dibenzosuberyl, phthalyl or derivatives thereof.

51. A process of any one o cla ms 46-50,

inclusive, wherein T is a polycycloalkylidene group, Y is phenyl, naphthyl, coumarinyl or phenanthryl, R³ is methyl or ethyl, and Z is hydrogen or a carboxylic acid ester, a phosphate monoester salt or a glycoside.

52. A process of claim 46, wherein R³ and Y form a heterocyclic ring.

53. A process of claim 52, wherein the

heterocyclic ring is 5 or 7-R³-substituted

isochroman-1-yl, a 4 or 6-R²-substituted 1,3-dihydroisobenzofuran-1-yl, a 5 or 8-R²-substituted 1H,

3N-2-oxaphenalen-1-yl, a 7 or 10-R²-substituted

3-oxa-1,2,3,4- tetrahydrophenanthry-4-yl, a 6,8 or g-R²-substituted 2-oxa-1,2,3,4-tetrahydrophen-anthr-1-yl, or an R²-substituted dihydroisonaphthofuran-1 or 3-yl phosphonate ester, where R² is an alkyl ether, halogen, hydroxyl group, an amino group, di (lower alkyl) amino group or its salt, a sulfonamido group, or an acyloxy group.

54. A compound having the formula

wherein R¹ is a trialkylsilyl group or a lower alkyl group having up to 12 carbon atoms; R² is a hydroxyl group, an ether (OR⁴) or a thioether (SR⁴) group wherein R⁴ is a substituted or unsubstituted alkenyl, lower alkyl or aralkyl group having up to 20 carbon atoms, an acyloxy group, a halogen atom, a nitro group, an amino group, a mono or di (lower) alkyl group or its acid salt wherein each lower alkyl group has up to 7 carbon atoms and wherein each lower alkyl group may be bonded to the Y group forming one or more fused rings, a NHSO₂R⁵ group wherein R⁵ is a methyl, tolyl, or trifluoromethyl group, a substituted aryl, heteroaryl, or β-styrenyl having up to 20 carbon atoms; R³ is a substituted or unsubstituted lower alkyl, aralkyl or heteroaralkyl group having up to 20 carbon atoms, an aryl or heteroaryl group having up to 14 carbon atoms which can be further substituted, a (lower) alkyl-OSiX³ group wherein the lower alkyl group contains up to 6 carbon atoms and X is independently methyl, phenyl or t-butyl, an alkoxy (lower) alkyl group having up to 6 carbon atoms, or an amino (lower) alkyl or mono or di (lower) alkylamino group wherein each lower alkyl group contains up to 7 carbon atoms; X¹ is hydrogen or a substituted or unsubstituted aryl, aralkyl, heteroalkyl, or heteroalkyl group having up to 20 carbon atoms, an allyl group, a hydroxy (lower) alkyl group having up to 6 carbon atoms, a (lower) alkyl-OSiX₃ group wherein the alkyl and X radicals are as defined above, an ether (OR⁴) or a thioether (SR⁴) wherein R⁴ is as defined above, an SO₂R⁶ group wherein R⁶ is methyl, phenyl or NHC6H5, a substituted or unsubstituted alkyl group having up to 7 carbon atoms, a nitro group, a CN group, an aldehyde or its oxime or dimethyl hydrazone, an alkyl halide group having up to 6 carbon atoms, a halogen group, a carboxylic acid salt, ester or

hydrazide, a trialkyl silicon-based group, or a

phosphoryloxy group; and Y is phenyl, biphenyl,

9,10-dihydrophenanthryl, naphthyl, anthryl, pyridyl, guinolinyl, isoquinolinyl, phenanthryl, pyrenyl, coumarinyl, carbostyryl, acridinyl, dibenzosuberyl, phthalyl or derivatives thereof.

55. A compound as recited m claim 54 wherein R² is meta to the point of attachment of the phosphonate ester-containing side chain to the Y group, and X¹ is ortho, meta or para to the R² group.

56. A compound having the formula:

wherein R² which is meta to the point of attachment of the phosphonate-containing side chain to Y, is an ether (OR⁴) or a thioether (SR⁴) wherein R⁴ is a substituted or unsubstituted, saturated or unsaturated lower alkyl group or aralkyl having up to 20 carbon atoms, a halogen atom, a nitro qroun. a hvdroxvl group, an acetoxy group, a pivaloyloxy group, an amino group, a di (lower alkyl) amino group wherein each lower alkyl substituent

contains up to 7 carbon atoms, or an NHSO₂R⁵ group wherein R⁵ is trifluoromethyl or tolyl, and wherein X¹ is hydrogen, or 2-Me, 4-OEt, 4-OMe, 5-CHO, 5-CH(OMe)₂, 5-Br, 5-hydroxyl, 5-OMe or 6-OMe.

57. A compound as recited in claim 56, wherein R² is 3-O-Me or 3-pivaloyloxy.

58. A compound as recited in claim 56, wherein R² is 3-O-Me or 3-pivaloyloxy and X¹ is 5-CHO.

59. A compound having the formul:a:

60. A compound having the formula:

61. A compound having the formula:

62. A compound having the formula:

63. A compound having the formula:

64. A compound having the formula.

wherein R², which is meta to the point of attachment of the side chain to Y, is an ether (OR⁴) or a thioether (SR⁴) wherein R⁴ is a lower alkyl group having up to 20 carbon atoms, and X¹ is hydrogen, 2-Me, 4-OEt, 4-OMe, 5-CHO, 5-CH(OMe)₂, 5-Br, 5C1, 5-OMe or 6-OMe.

65. A compound having the formula:

wherein R², which is meta to the point of attachment of the side chain to Y, is an ether (OR⁴) or a thioether (SR⁴) wherein R⁴ is a lower alkyl substituent having up to 20 carbon atoms, a halogen atom, a nitro group, a hydroxyl group, an amino group, a di (lower alkyl) amino group wherein each lower alkyl substituent contains up to 7 carbon atoms, or an NHSO₂R⁵ group wherein R⁵ is methyl or tolyl, and X¹ is hydrogen, 2-Me, 4-OEt, 4-OMe, 5-CHO, 5-CH(OMe)₂, 5-Br, 5C1, 5-OMe or 6-OMe.

66. A compound having the formula:

wherein R², which is meta to the point of attachment of the phosphonate ester-containing side chain to Y, is an ether (OR⁴) or a thioether (SR⁴) wherein R⁴ is a lower alkyl or aralkyl substituent having up to 20 carbon atoms, an acetoxy or pivaloyloxy, a halogen atom, a nitro group, a hydroxyl group, an amino group, a di(lower alkyl) amino group wherein each lower alkyl substituent contains up to 7 carbon atoms, or an NHSO₂r⁵ group wherein R⁵ is tolyl or trifluoromethyl, and X is hydrogen, 2-Me, 4-OEt, 4-OMe, 5-CHO, 5-CH(OMe)₂, 5-Br, 5-C1, 5-OMe or 6-OMe.

67. A compound having the formula:

wherein R² is an ether (OR⁴) or a thioether (SR⁴) wherein R⁴ is a lower alkyl or aralkyl group having up to 20 carbon atoms, an acetoxy or pivaloyloxy group, a halogen atom, a nitro group, a hydroxyl group, an amino group, a di (lower alkyl) amino group wherein each lower alkyl substituent contains up to 7 carbon atoms, or an NHSO₂R⁵ group wherein R⁵ is methyl or tolyl, and is preferably positioned so that the total number of ring carbon atoms separating the ring carbon to which it is attached and the ring carbon atom to which the aldehyde group is attached, including the ring carbon atoms at the point of attachment, is an odd whole number.

68. A compound having the formula:

wherein R² is an ether (OR⁴) or a thioether (SR⁴) wherein R⁴ is a lower alkyl or aralkyl substituent having up to 20 carbon atoms, an acetoxy or pivaloyloxy group, a halogenatom, a nitro group, a hydroxyl group, an amino group, a di(lower alkyl) amino group wherein each lower alkyl substituent contains up to 7 carbon atoms, or an NHSO₂R⁵ group wherein R⁵ is tolyl or trifluoromethyl.

69. A compound having the formula

wherein R² is an ether (OR⁴) or a thioether (SR⁴) wherein R⁴ is a lower alkyl or alkyl group having up to 20 carbon atoms, an acetoxy or pivaloyloxy group, a halogen atom, a nitro group, a hydroxyl group, an amino group, a di (lower alkyl) amino group wherein each lower alkyl substituent contains up to 7 carbon atoms, or an NHSO₂R⁵ group wherein R⁵ is tolyl or methyl.

70. A compound having the formula:

wherein R² is an ether (OR⁴) or a thioether (SR⁴) wherein R⁴ is a lower alkyl or aralkyl group having up to 20 carbon atoms, an acetoxy or pivaloyloxy group, a halogen atom, a nitro group, a hydroxyl group, an amino group, a di (lower alkyl) amino group wherein each lower alkyl substituent contains up to 7 carbon atoms, or an NHSO₂R⁵ group wherein R⁵ is tolyl.

71. A compound having the formula:

R² is an ether (OR⁴) or a thioether (SR⁴) wherein R⁴ is a lower alkyl or aralkyl group having up to 20 carbon atoms, a halogen atom, a nitro group, a hydroxyl group, an amino group, a di (lower alkyl) amino group wherein each lower alkyl substituent contains up to 77 carbon atoms, or an NHSO₂R⁵ group wherein R⁵ is tolyl or methyl.

72. A compound having the formula:

73. A compound having the formula:

74. A compound having the formula:

75. A compound having the formula:

76. A compound having the formula:

wherein X¹ is hydrogen, 2-Me, 4-OEt, 4-OMe, 5-CHO ,

5-CH (OMe) ₂, 5-Br, 5-Cl , 5-OMe or 6-OMe .

77. A compound having the formula:

wherein R¹ is a lower alkyl group having up to 12 carbon atoms; R² is an ether (OR⁴) or a thioether (SR⁴) wherein R⁴ is a lower alkyl or aralkyl substituent having up to 20 carbon atoms, an acetoxy or pivaloyloxy group, a halogen atom, a nitro group, a hydroxyl group, an amino group, a di(lower alkyl) amino group wherein each lower alkyl substituent contains up to 7 carbon atoms, or an NHSO₂R⁵ group wherein R⁵ is tolyl or trifluoromethyl; A3 is a lower alkyl, aralkyl, aryl or heteroaralkyl group having up to 20 carbon atoms a (lower alkyl) -O-Si X₃ group wherein the lower alkyl moiety contains up to 7 carbon atoms, and any X is methyl, benzyl, or t-butyl, a hydroxy(lower)alkyl group having up to 6 carbon atoms, or an amino (lower) alkyl or di(lower)alkylammo group wherein each lower alkyl group contains up to 7 carbon atoms.

78. A compound having the formula:

R², which is meta to the point of attachment of each phosphonate ester-containing side chain to the aryl ring, is an ether (OR⁴) or a thioether (SR⁴) wherein R⁴ is a lower alkyl or aralkyl substituent having up to 20 carbon atoms, an acetoxy or pivaloyloxy group, a halogen atom, a nitro group, a hydroxyl group, an amino group, a di(lower alkyl) amino group wherein each lower alkyl substituent contains up to 6 carbon atoms, or an NHSO₂R⁵ group wherein R⁵ is tolyl or trifluoromethyl.

79. A compound having the formula:

wherein R² is an ether (OR⁴) or a thioether (SR⁴) wherein R⁴ is a lower alkyl or aralkyl substituent having up to 20 carbon atoms, an acetoxy or pivaloyloxy group, a halogen atom, a nitro group, a hydroxyl group, an amino group, a di (lower alkyl) amino group wherein each lower alkyl substituent contains up to 7 carbon atoms, or an NHSO₂R⁵ group wherein R⁵ is tolyl or trifluoromethyl.

80. A compound having the formula:

81. A hydroxyaryl enol ether alkali metal salt having the formula:

in which T is a fused, substituted or unsubstituted polycycloalkylidene group, OR³ is an ether group, Y is a light-emitting fluorophore-forming group which will be part of a luminescent substance formed by decomposition of a 1,2-dioxetane subsequently formed from the

hydroxyaryl enol ether alkali metal salt, capable of absorbing energy to form an excited state from which it emits optically detectable energy to return to its ground state, and AM⁺ is an alkali metal cation.

82. A hydroxyaryl enol ether alkali metal salt having the formula:

in which T is a fused, substituted or unsubstituted polycycloalkylidene group, OR³ is methyl, ethyl, benzyl or ethoxyethyl, Y is a light-emitting fluorophoreforming group that will be part of a luminescent substance formed by decomposition of a 1,2-dioxetane subsequently formed from the hydroxyaryl enol ether alkali metal salt, capable of absorbing energy to form an excited state from which it emits optically

detectable energy to return to its ground state, X¹ is hydrogen, alkyl, hydroxyalkyl, halo, alkoxy, cyano, methanesfulfonyl or trifluoromethyl, and AM⁺ is an metal cation.

83. A hydroxyaryl enol ether alkali metal salt of claim 81 or claim 2 wherein T is an adamant-2-ylidene group.

84. Sodium 3-(methoxytricyclo[3.3.1.1^3,7]dec-2-ylidenemethyl)phenoxide.

85. Lithium 3-(methoxytricyclo[3.3.1.1^3,7]dec-2-ylidenemethyl)phenoxide.

86, A process for preparing a hydroxyaryl enol ether alkali metal salt having the formula:

in which T is a fused, substituted or unsubstituted polycycloalkylidene group, OR³ is an ether group, Y is a light-emitting fluorophore-forming group which will be part of a luminescent substance formed by decomposition of a 1,2-dioxetane subsequently formed from the

hydroxyaryl enol ether alkali metal salt, capable of absorbing energy to form an excited state from which it emits optically detectable energy to return to its ground state, and AM⁺ is an alkali metal cation, which comprises the steps of: (1) reacting a corresponding dialkyl 1-alkoxy-1-arylmethane phosphonate having the formula:

in which R¹ is an alkyl group and R² is a hydroxy protecting acyl group that remains substantially intact during the reaction, with an alkali metal-containing base in solution at low temperature under an inert atmosphere, (2) reacting the resulting phosphonatestabilized α-carbanion with a ketone having the formula T = O to give a mixture comprising dialkyl 1-alkoxy-1- arylmethane phosphonate starting material as its anion, the corresponding R² deesterified dianion or its decomposition products, the corresponding hydroxyaryl enol ether alkali metal salt, and the R² esterified aryl enol ether, (3) reesterifying said mixture to

substantially esterify the hydroxyaryl enol ether alkali metal salt from the reesterified mixture, (4) separating the R² reesterified aryl enol ether and (5) carrying out ester cleavage on the separated R² reesterified R² esterified aryl enol ether under anhydrous conditions to give the hydroxyaryl enol ether alkali metal salt.

87. A process as described in claim 86 wherein step (1) is carried out at a temperature of -20 °C or less using from about 1 to about 1.2 equivalents of the alkali metal-containing base, step (2) is carried out from low temperature to reflux using slightly less than a molar excess of the ketone T = O, step (3) is carried out at a temperature of from about 0ºC to about 50°C using an amount of the acid chloride which is at least a molar equivalent of the total amount of all aryloxide alkali metal salt present, step (4) is carried out by recrystallization, and step (5) is carried out at room temperature using about one equivalent of the alkali metal alkoxide in a lower alkanol.

88. A process as described in claim 87 wherein R² is a pivaloyl group, the alkali metal-containing base used in step (1) is lithium diisopropylamide or

n-butyllithium, the acid chloride used in step (3) is pivaloyl chloride, and the alkali metal alkoxide and lower alkanol used in step (5) are sodium methoxide and methanol, respectively.

89. A process as described in claim 88 wherein the dialkyl 1-alkoxy-1-arylmethane phosphonate is diethyl 1-methoxy-1-(3-pivaloyloxyphenyl)methane phosphonate.

90. A process as described in claim 88 or 89 wherein the ketone T = O is 2-adamantanone.

91. A process for preparing a hydroxyaryl enol ether lithio salt having the formula:

in which T is a fused, substituted or unsubstituted polycycloalkylidene group, OR³ is an ether group, and Y is a light-emitting fluorophore-forming group which will be part of a luminescent substance formed by

decomposition of a 1,2-dioxetane subsequently formed from the hydroxyaryl enol ether alkali metal salt, capable of absorbing energy to form an excited state from which it emits optically detectable energy to return to its ground state, which comprises (1) reacting a corresponding dialkyl 1-alkoxy-1-arylmethane

phosphonate having the formula:

in which R₁is an alkyl group and R₂ is a hydroxy

protecting acyl group that will be substantially cleaved during the reaction, with three equivalents of a lithium alkyl compound in solution at low temperature under an inert atmosphere, and (2) adding to the resulting mixture containing the corresponding phosphonatestabilized α-carbanion a ketone having the formula T = O and reacting to give the hydroxyaryl enol ether lithio salt.

92. A process as described in claim 91 wherein step (1) is carried out at a temperature of -20°C or less and step (2) is carried out from low temperature to reflux using slightly less than a molar excess of the ketone T = O.

93. A process as described in claim 92 wherein R² is an acetyl group and the lithium alkyl compound used in step (1) is n-butyllithium.

94. A process as described in claim 92 wherein the dialkyl 1-alkoxy-1-arylmethane phosphonate is diethyl

1-methoxy-1-(3-acetoxyphenyl)methane phosphonate.

95. A process as described in claim 93 or 94 wherein the ketone T = O is 2-adamantanone.

96. A process for preparing a hydroxyaryl enol ether alkali metal salt having the formula:

in which T is a fused, substituted or unsubstituted polycycloalkylidene group, OR³ is an ether group, Y is a light-emitting fluorophore-forming group which will be part of a luminescent substance formed by decomposition of a 1,2-dioxetane subsequently formed from the

hydroxyaryl enol ether alkali metal salt, capable of absorbing energy to form an excited state from which it emits optically detectable energy to return to its ground state, E is oxygen or sulfur and AM⁺ is an alkali metal cation, which comprises subjecting the

corresponding etherified or thioetherified hydroxyaryl enol ether having the formula:

in which R⁷ is a substituted or unsubstituted alkyl, alkenyl or aralkyl group, to either cleavage with an alkali metal-containing reagent to give the hydroxyaryl enol ether alkali metal salt or mercaptoaryl enol ether alkali metal salt.

97. A process as described in claim 96 wherein the alkali metal-containing reagent is sodium thioethoxide, lithium iodide, sodium cyanide or sodium monosulfide.

98. A process as described in claim 97 wherein R⁷ is methyl, E is oxygen, and the hydroxyaryl enol ether alkali metal salt is recovered by

precipitation at 0ºC using an ether nonsolvent.

99. A process as described in claim 98 wherein R³ is pivaloyl and Y is phenyl.

100. A process as described in claim 98 or claim 99 wherein T is adamant-2-ylidene.