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  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
  • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
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  • C07C43/03—Ethers having all ether-oxygen atoms bound to acyclic carbon atoms
  • C07C43/14—Unsaturated ethers
  • C07C43/17—Unsaturated ethers containing halogen
  • C07C43/174—Unsaturated ethers containing halogen containing six-membered aromatic rings
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  • C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
  • C07C45/27—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by oxidation
  • C07C45/29—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by oxidation of hydroxy groups
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  • C07C45/68—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups by isomerisation; by change of size of the carbon skeleton by increase in the number of carbon atoms
  • C07C45/70—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups by isomerisation; by change of size of the carbon skeleton by increase in the number of carbon atoms by reaction with functional groups containing oxygen only in singly bound form
  • C07C45/71—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by reactions not involving the formation of >C = O groups by isomerisation; by change of size of the carbon skeleton by increase in the number of carbon atoms by reaction with functional groups containing oxygen only in singly bound form being hydroxy groups
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  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
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  • C07F9/28—Phosphorus compounds with one or more P—C bonds
  • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
  • C07F9/40—Esters thereof
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  • C07F9/4056—Esters of arylalkanephosphonic acids
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  • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
  • C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
  • C07F9/6551—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a four-membered ring
  • C07F9/65512—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a four-membered ring condensed with carbocyclic rings or carbocyclic ring systems
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  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
  • C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
  • C07F9/65515—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a five-membered ring
  • C07F9/65517—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a five-membered ring condensed with carbocyclic rings or carbocyclic ring systems
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  • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
  • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
  • C07F9/02—Phosphorus compounds
  • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
  • C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
  • C07F9/6552—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a six-membered ring
  • C07F9/65522—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a six-membered ring condensed with carbocyclic rings or carbocyclic ring systems
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  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C2603/00—Systems containing at least three condensed rings
  • C07C2603/56—Ring systems containing bridged rings
  • C07C2603/58—Ring systems containing bridged rings containing three rings
  • C07C2603/70—Ring systems containing bridged rings containing three rings containing only six-membered rings
  • C07C2603/74—Adamantanes

Definitions

• 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.
• 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)
• 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.
• 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
• 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., im unoassays, nucleic acid probe assays, enzyme assays, and the like.
• T, R 3 , Y and Z are defined herein below, from enol ether-type precursors of the general formula:
• Enol ethers have also been prepared by Peterson or Wittig reactions of alkoxy ethylenesilanes or phosphoranes with aldehydes or ketones in basic media [Magnus, P., et al.. Or ⁇ anometallics. 1:553 (1982); Wynberg, H. and Meijer, E.W. , Tetrahedron Lett.. 41:3997 (1979)].
• 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.
• This invention fills this need.
• this invention is concerned with a synthetic route to such 1,2-dioxetanes that employs, for the first time, dialkyl 1-alkoxy-l- arylmethane phosphonate-stabilized carbanion intermediates in the synthesis of key enol ether intermediates for the desired 1,2-dioxetane end products.
• 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.
• 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 trimethyl ⁇ ilyl 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 a inophenyl enol ether or its N-acyl or sulfonamide derivatives.
• AM + the alkali metal cation
• T, R 3 , X 1 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.
• the use of an alkali metal salt of the enol ether rather than the free hydroxy compound results in savings in materials of reaction.
• 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.
• 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.
• 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.
• 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.
• 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
• R 3 represents a C,-C 20 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, 0, or S heteroatom containing group, e.g, 4-hydroxybutyl, methoxyethyl, or polyalkyleneoxyalkyl; an ary
• 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 a ino group, or a nitrogen anion, and particularly an amido anion such as sulfonamido anion.
• a chemically-cleavable group such as a hydroxyl group, an alkanoyloxy or aroyloxy ester group, silyloxy group
• substituents T, R 3 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.
• 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.
• 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.
• an enzyme such as alkaline phosphatase that will cleave a bond in, for example, a Z substituent such as a phosphate mono ester group
• 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.
• 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, l-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, ⁇ -D-galactoside group, / 3-D-galactoside group, ⁇ -D-glucoside group,
• an enzyme-cleavable alkanoyloxy group e.g., an acetate ester group, an oxacarboxylate group, or an oxaalkoxycarbonyl group, l-phospho-2,3- diacylglyceride group, 1-thio-D-glucoside group, adenos
• 0-D-glucoside group ⁇ -D-mannoside group, /3-D-mannoside group, /3-D-fructofuranoside group, jB-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 X 1 groups, these substituents being ortho. meta. or para to each other.
• groups R 2 or X 1 need not be static during the reaction sequences, but may be interconverted under conditions which are compatible with structural considerations at each stage.
• n ese ormu ae any can e n epen en y a halogen, e.g., chlorine or bromine, or OR 1 ;
• R 1 can be independently a trialkylsilyl group or a lower alkyl group having up to 12 carbon atoms such as ethyl, propyl, or butyl;
• R 2 can be a hydroxyl group, an ether (OR 4 ) or a thioether (SR 4 ) group wherein R 4 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;
• R 2 can also be an acyloxy group such as acetoxy, pivaloyloxy, or mesitoyloxy, a halogen atom, e.g., chlorine or bromine, a nitro 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 ethanol, 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, R 3 0H, 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 -or p-phthalaldehydes and the like.
• the R 2 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) .
• 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 X 1 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) .
• R 3 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-(CH 2 ) 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.
• a catalyst such as a Lewis acid, HCl(g), p-toluenesulfonic acid or its polyvinylpyridine salt
• 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 BF 3 etherate or the like to give the corresponding phosphonate, essentially according to Burkhouse, D. , et al.. Synthesis. 330 (1984) .
• a Lewis acid catalyst such as BF 3 etherate or the like
• 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 0 ⁇ C, to produce in almost quantitative yields (see Table 2) the corresponding 1-alkoxy-l-arylmethane phosphonate esters.
• step 4 the phosphonate-stabilized carbanion is used to synthesize olefins by the Homer-Emmons reaction.
• Step 4.1 a phosphonate- stabilized carbanion is produced from a dialkyl
• 1-alkoxy-l-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.
• a base such as sodium hydride, sodium amide, a lithium dialkyl amide such as lithium diisopropylamide (LDA) , a metal alkoxide, or, preferably, n-butyllithium
• 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, dimethylforma ide (DMF) , dimethyl-acetamide, and dimethylsulfoxide, and the like, or mixtures of these solvents.
• 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
• dimethylforma ide (DMF) dimethyl-acetamide
• dimethylsulfoxide and the like, or mixtures of these solvent
• 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 0"C 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.
• Step 5 the enol ether is oxidized.
• Oxidation is preferably accomplished photochemically by treating the enol ether with singlet oxygen ( 1 0 2 ) 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.
• 1 0 2 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) .
• oxygen-linked functional group R 2 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 R substituent (wherein R 7 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 120 ⁇ C to about 150 ⁇ C.
• the cleavage can also be accomplished with soft nucleophiles such as lithium iodide in refluxing pyridine, sodium cyanide in refluxing DMSO, or Na 2 S in refluxing N-methyl-2-pyrrolidone.
• ester cleavage can be accomplished with NaOMe, KOH or K 2 C0 3 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.
• an acid halide or anhydride e.g., acetic anhydride
• Lewis base e.g. , triethylamine
• 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, R 3 and X 1 are as defined above.
• Steps 8, 9 and 10 can be performed separately or in a onepot or two-pot operation.
• a cyclic phosphoroha ⁇ lidate e.g., cyclic phosphorochloridate
• 2-pot operation the phenolic hydroxyl group in the free hydroxyl product produced in Step 6 is reacted with 2-halo-2-oxo-l,3,2-dioxaphospho- lane 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).
• MCN e.g., NaCN
• a base e.g., ammonium hydroxide or NaO J.
• Step 10 a filterable disodium sodium ammonium salt
• phosphate triester formation induced by a Lewis base e.g., a tertiary a ine such as triethyla ine
• a preformed alkali metal salt or the phenolic enolether can be effected with phosphorohalidate ⁇ over a temperature range of about -30° to about 60"C.
• the ring cleavage with alkalicyanide (MCN) in DMF or DMSO can be carried out in a narrow temperature range of between about 15" and about 30°C.
• MCN alkalicyanide
• 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:
• R 8 and R 9 are each independently -CN, -N0 2 , arylsulfonyl, or alkylsulfonyl.
• 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.
• M + is an alkali metal
• R 10 is hydrogen or a 0,-0, alkyl, aralkyl, aryl or heterocyclic group, to give the corresponding arylphosphate monoester disalt via ⁇ -elimination.
• Dioxetane formation of the reaction of singlet oxygen ( 1 0 2 ) 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 NaOCH 3 , K 2 C0 3 , or NH 3 gas, in an alcohol such as methanol, first at 0°C and then at 25'C for 1 to 10 hours (Step 12) , leaving a hexosidase-cleavable Dhexopyranosidyl moiety on the aryl ring.
• a base such as NaOCH 3 , K 2 C0 3 , or NH 3 gas
• 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 5*C. 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.
• solid phase sensitizers such as polymerbound Rose Bengal (Sensitox I) or methylene blue on silica gel.
• aryl monoaldehydes 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:
• 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 hetero ⁇ yclic 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
• R 2 is as described above
• W can be OR 3 , wherein R 3 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:
• R 2 is as described above.
• 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 l -spiroadamantane)-4-methoxy-4-(3"-phos- phoryloxy) phenyl-l,2-dioxetane salts represented by the formula:
• M * represents a cation such as an alkali metal, e.g. sodium or potassium, or a C ⁇ c ⁇ alkyl, aralkyl or aromatic quaternary ammonium cation, N(R 10 ) , in which each R 10 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.
• M * represents a cation such as an alkali metal, e.g. sodium or potassium, or a C ⁇ c ⁇ alkyl, aralkyl or aromatic quaternary ammonium cation, N(R 10 )
• each R 10 can be alkyl, e.g., methyl or ethyl, aralkyl, e.g., benzyl,
• T, R 3 , Y and Z are as described herein above. These can then be converted to the corresponding
• 1,2-dioxetane £ 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.
• 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 £ 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 react on 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-l-arylmethane phosphonate:
• Y is an aryl moiety, e.g, a phenyl ring
• R 2 is an acyloxy substituent, preferably in the meta-position on the aryl moiety, e.g., a pivaloyloxy group
• X 1 can be hydrogen or another of the ⁇ ubstituents listed above, is converted to the corresponding phosphonate-stabilized ⁇ -carbanion, preferably in solution at low temperature, -20*C 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 diisopropyl- amide or an alkali metal alkyl compound such as n-butyllithium.
• an alkali metal- containing base e.g., from about 1 to about 1.2 equivalents of the alkali metal-containing
• R 2 esterified aryl enol ether where R 2 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 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.
• an acid chloride or acid anhydride e.g., pivaloyl chloride
• 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 0 ⁇ C and about 50 ⁇ C, over a period of from about 2 to about 24 hours, using thin layer chromatography to monitor the completeness of the reaction.
• R 2 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 ⁇ C), followed by removal of the volatiles from the reaction mixture in vacuo (1 mm Hg) with heating at from about 35°C to about 65*C 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.
• an alkali metal alkoxide e.g., sodium methoxide
• a lower alkanol e.g., methanol or enthanol
• anhydrous conditions i.e., in the
• the free hydroxy enol ether starting material of Example 106 in our copending application Serial No. 402,847 — 3-(methoxy- tricyclo[3.3.1.1 3 ' 7 ]dec-2-ylidene-methyl)phenol — can be replaced with its sodium salt — sodium 3-(methoxytri- cyclo[3.3.1.1 3,7 ]dec-2-ylidenemethyl)phenoxide — in a one pot reaction with between about 1 and 1.2 equiva ⁇ lents of 2-chloro-2-oxo-l,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 3-elimination with sodium hydroxide or ammonium hydroxide to give the phosphate monoester salt.
• 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 3-elimination steps being run in dimethylformamide or dimethylsulfoxide.
• a halogenated solvent e.g., methylene chloride
• a polar solvent e.g., acetonitrile
• an ether or polyether solvent e.g., tetrahydrofuran or diglyme
• 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-l-arylmethane phosphonate, Formula d above, whose aryl moiety (Y) has an acyloxy substituent (R 2 ) 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, -20"C or less, to give the correspond ⁇ ing phosphonate-stabilized ⁇ -carbanion as its lithio salt.
• a lithium alkyl compound e.g., n-butyllithium
• the thus obtained salt can be separated by precipitation at 0 ⁇ C, 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.
• 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.
• 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 /3-human chorionic gonadotropin ( ⁇ 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.
• hormones such as /3-human chorionic gonadotropin ( ⁇ HCG) , thyroid stimulating hormone (TSH) , follicle stimulating hormone (FSH) , luteinizing hormone (LH) or the like
• cancer markers such as alpha fetal protein (AFP) , car
• 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 C V) , Hepatitis B surface antigen (HB C V) , 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.
• CNV cytomegalovirus
• HPV human papilloma virus
• HB C V hepatitis C core virus antigen
• HB C V Hepatitis B surface antigen
• Rotavirus or bacteria, e.g., campylobacter jejuni/coli, E. coli, ETEC heat labile and stable, plasmodium falciparum, or oncogen
• 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.
• 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.
• 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.
• 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.
• 3,5-Bishydroxymethylanisole was synthesized according to the procedure of V. Boekelheide and R.W. Griffin, Jr., J. Or ⁇ . 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 dichloro- methane. 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.
• 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 0* 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 61-62.5"C. NMR and IR data are listed in Tables 3 and 7.
• Example 4 m-Methoxybenzaldehvde dimethyl acetal -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 ethanol. 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.
• Example 5 Diethyl 1-methoxy-l-f3-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 ⁇ eparatory funnel. The organic layer was isolated and washed with 500 ml water, 2 x 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.
• Example 6 ⁇ -2-Adamantylidene- ⁇ -methox ⁇ -m-methoxytoluene
• 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 ada antyl methoxyphenyl ketone (1670 cm “1 ) . Although this product was sufficiently pure for subsequent reaction, it was found that an identical procedure using
• 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- l-(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) .
• De-acylation of the mixture was completed in 2.5 hours by refluxing the mixture of crude products, 16.5 g of K 2 C0 3 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 H 2 0 and then scratched vigorously with a spatula to afford a filterable material. The solid was filtered and washed thoroughly with 1.5 L of H 2 0. After removal of most of the moisture under vacuum, the slightly yellow solid was redissolved in 600 ml of CH 2 C1 2 (with gentle heating if necessary) and dried over Na 2 S0 4 . The solution was filtered on a Buchner funnel, packed with 40 g of silica gel.
• 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 dimethylamino- pyridine (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 x 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 l-methoxy-l-(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-l-methoxy-l-f3-hvdroxyphenyl methanephosphonate Diethyl-l-methoxy-1-(3-acetoxyphenyl)methanephos- phonate 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 x 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:
• 6-Methoxynaphthalene-l-carboxaldehvde dimethyl acetal 6-Methoxynaphthalene-l-carbonitrile was synthesized from 6-methoxy-l-tetralone by the method of Harvey, R.G., et al.. J Or ⁇ . Chem.. 48:5134 (1983).
• the nitrile 354.6 mg. , 1.94 mmol
• 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.
• 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 NaNC0 3 solution and dried over Na 2 SO A . After decanting and evaporating the solution, the resultant oil was dissolved in minimal CH 2 C1 2 , followed by addition of hexanes until the solution clouded.
• Example 11B 6-Methoxy-2-naphthaldehvde dimethyl acetal
• the 6-methoxy-2-naphthyldimethyI acetal was synthesized in 61% yield (m.p. 27°) according to the procedure described in Example 4.
• Example 11C Diethyl 1-Methoxy-l-f6-methoxynapth-2-yl)methane phosphate The corresponding phosphonate was synthesized in 60% yield (oil) as described in Example 5.
• IR (CNC1 3 , cm *1 ): 1687 (C 0) , 1601, 1460, 1389, 1331, 1266, 1175, 1115, 1030, 842.
• Example HE 7-Methoxy-2-naphthaldehvde dimethyl acetal The corresponding dimethyl acetal was synthesized in 86% yield (oil) , following the conditions described in Example 4.
• Example R3 90 -CHaCHs 2975. 2925. 2900. 1595. 1580. 14B5, 1435. 1365. 1315. 1255. (P-0) . 1100. 1040 (br) , 965. B70. 695
• 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 0 2 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) .
• 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 13 C spectrum (D 2 0/CD 3 OD) ; 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.
• 2-adamantanone (24.8 g, 0.165 mol.). The solution was stirred to homogeneity and set aside.
• 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.
• 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) ethylene 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 acetate- hexanes.
• 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.
• Example 109 A flame-dried flask was charged with ' methoxy(3-pivaloyloxyphenyl) ethane 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).
• TLC Whatman K5F; 10% ethyl acetate-hexanes
• phenolate salt did not exhibit a melting point below 280°, but did darken somewhat beginning at 170A It was kept dry during all subsequent manipulations, and stored in a dessicator over Drierite. IR (nujol mull): 1572, 1405, 1310, 1285, 1198,
• 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-l,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.

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