EP0441948A1 - Synthese stabiler wasserlöslicher und chemilumineszenter 1,2-dioxetane und zwischenprodukte dafür - Google Patents

Synthese stabiler wasserlöslicher und chemilumineszenter 1,2-dioxetane und zwischenprodukte dafür

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Publication number
EP0441948A1
EP0441948A1 EP90913972A EP90913972A EP0441948A1 EP 0441948 A1 EP0441948 A1 EP 0441948A1 EP 90913972 A EP90913972 A EP 90913972A EP 90913972 A EP90913972 A EP 90913972A EP 0441948 A1 EP0441948 A1 EP 0441948A1
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Prior art keywords
group
formula
aryl
carbon atoms
alkyl
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EP0441948A4 (en
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Brooks Edwards
Rouh-Rong Juo
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Tropix Inc
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Tropix Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C07F9/6561—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C43/00—Ethers; Compounds having groups, groups or groups
    • C07C43/02—Ethers
    • 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
    • C07C43/1747—Unsaturated ethers containing halogen containing six-membered aromatic rings containing six membered aromatic rings and other rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • 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
    • C07C45/292—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 with chromium derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
    • C07C45/61—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
    • C07C45/67—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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C47/00—Compounds having —CHO groups
    • C07C47/52—Compounds having —CHO groups bound to carbon atoms of six—membered aromatic rings
    • C07C47/575—Compounds having —CHO groups bound to carbon atoms of six—membered aromatic rings containing ether groups, groups, groups, or groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D321/00—Heterocyclic compounds containing rings having two oxygen atoms as the only ring hetero atoms, not provided for by groups C07D317/00 - C07D319/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C07F9/28—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
    • C07F9/4003—Esters thereof the acid moiety containing a substituent or a structure which is considered as characteristic
    • C07F9/4056—Esters of arylalkanephosphonic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C07F9/6551—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C07F9/65515—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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
  • 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.
  • 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.
  • 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.
  • 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., immunoassays, 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:
  • McMurry et al. [McMurry, J.E., et al., J. Qr ⁇ .
  • Enol ethers have also been prepared by Peterson or Wittig reactions of alkoxymethylenesilanes or
  • 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-1-arylmethane phosphonate-stabilized carbanion
  • 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
  • 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.
  • ring carbon 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
  • 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,
  • This enol ether can be converted to a Grignard reagent or an organolithium derivative for reaction with
  • 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.
  • an alkali metal salt of the enol ether rather than the free hydroxy compound results in savings in
  • 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.
  • 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.
  • the alkali metal salt need not be obtained by first isolating the free hydroxy compound and then forming the salt in a separate reaction.
  • the thus-obtained alkali metal salts can be separated by precipitation or used in situ as starting materials for the acylation, phosphorylation or
  • 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.
  • 1,2-dioxetane compounds that can be synthesized by the method of this invention.
  • 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,
  • R 3 represents a C 1 -C 20 unbranched or branched, substituted or unsubstituted, saturated or unsaturated alkyl group, e.g., methyl, allyl or
  • 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.
  • fluorophore-forming group capable of absorbing energy to form an excited energy state from which it emits
  • 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.
  • a chemically-cleavable group such as a hydroxyl group, an alkanoyloxy or aroyloxy ester group, silyloxy group
  • One or more of the 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 carboxylic acid e.g., a carboxy methoxy group
  • a sulfonic acid e.g., an aryl sulfonic acid group, or their salts
  • a quaternary amino salt group e.g., trimethyl ammonium
  • 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
  • catalytic antibodies may be used to cleave the Z substituent.
  • Destabilization can also be accomplished by using an enzyme such as an
  • 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,
  • diphosphate analog group adenosine monophosphate analog group, adenosine analog group, ⁇ -D-galactoside group, ⁇ -D-galactoside group, ⁇ -D-glucoside group,
  • ⁇ -D-glucoside group ⁇ -D-mannoside group, ⁇ -D-mannoside group, ⁇ -D-fructofuranoside group, ⁇ -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
  • any Q can be independently 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
  • 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, 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 NHSO 2 R 5 group wherein R 5 is methyl, tolyl, or trifluoromethyl; R 2 can also be a substituted aryl, heteroaryl, ⁇ -styreneyl group containing up to
  • 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-OSiX 3 group wherein the alkyl and X radicals are as defined above, an ether (OR 4 ) or a thioether (SR A ) wherein R 4 is as defined above, an SO 2 R 6 group wherein R 6 is methyl, phenyl, or NHC 6 H 5 , 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
  • 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
  • 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 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
  • 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 OH, 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 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 alcohol, R 3 OH.
  • 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.,
  • 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.,
  • step 4 the phosphonate-stabilized carbanion is used to synthesize olefins by the Homer-Emmons
  • 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.
  • 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,
  • 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
  • reaction 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.
  • Step 5 the enol ether is oxidized.
  • Oxidation is preferably accomplished photochemically by treating the enol ether with singlet oxygen ( 1 O 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 O 2 can be generated using a photosensitizer, such as polymer bound Rose Bengal (Hydron Labs, New
  • TPP 20-tetraphenyl- 21H,23H-porphine
  • 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
  • Step 6a involves phenolic ether cleavage of the R 7 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 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 Na 2 S in refluxing
  • ester cleavage can be accomplished with NaOMe, KOH or K 2 CO 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 phosphorohalidate 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-1,3,2-dioxaphospholane to yield the cyclic phosphate triester (Step 8). This triester is subjected to ring opening with MCN
  • Step 9 A base, e.g., ammonium hydroxide or NaOMe, then provokes a facile ⁇ -elimination reaction, yielding a filterable disodium sodium ammonium salt
  • phosphate triester formation induced by a Lewis base e.g., a tertiary amine such as triethylamine
  • a preformed alkali metal salt or the phenolic enolether can be effected with phosphorohalidates over a Lewis base
  • 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:
  • R 8 and R 9 are each independently -CN, -NO 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, NR 10 4 + wherein R 10 is hydrogen or a C 1 -C 1 alkyl, aralkyl, aryl or heterocyclic group, to give the corresponding
  • Dioxetane formation of the reaction of singlet oxygen ( 1 O 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 CO 3 , or NH 3 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.
  • a base such as NaOCH 3 , K 2 CO 3 , or NH 3 gas
  • 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
  • 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
  • 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
  • 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'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy) phenyl-1,2-dioxetane salts represented by the formulaa
  • M + represents a cation such as an alkali metal, e.g. sodium or potassium, or a C 1 -C 18 alkyl, aralkyl or aromatic quaternary ammonium cation, N(R 10 ) 4 + , 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.
  • 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.
  • T, R 3 , Y and Z are as described herein above. These can then be converted to the corresponding
  • one T group serves to stabilize two dioxetane rings
  • each ring must be destabilized individually by chemical or enzymatic means at each Z group.
  • 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
  • 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
  • X 1 can be hydrogen or another of the substituents listed above, is converted to the corresponding phosphonate-stabilized ⁇ -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
  • 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
  • R 2 esterified aryl enol ether where R 2 is a pivaloyloxy group 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.
  • 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.
  • R 2 is a pivaloyloxy group one gets a much cleaner product, isolated from the reesterified mixture as a crystalline solid using standard
  • an alkali metal alkoxide e.g., sodium methoxide
  • a lower alkanol e.g., methanol or enthanol
  • 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.
  • an alkali metal alkoxide e.g., sodium methoxide
  • a lower alkanol e.g., methanol or enthanol
  • anhydrous conditions i.e., in the presence of as low an amount of moisture as can practicably be achieved
  • 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
  • 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.
  • 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.,
  • 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.
  • 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.
  • 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
  • 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 ⁇ -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 ⁇ -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 pan
  • 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.
  • 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.
  • 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 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
  • 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.
  • 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
  • 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.
  • 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
  • decolorizing carbon (3g) was added to the solution, and the whole was filtered under vacuum through celite.
  • 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
  • I.R. (neat, cm -1 ); 2900, 2838, 2655, 2640, 2620,
  • the pivaloyl ester group is not deacylated under the acidic conditions required for acetal and
  • 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).
  • 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
  • Example 9 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
  • 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
  • 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.
  • IR (CNCl 3 , cm -1 ): 2995, 2822, 1622, 1598, 1509, 1465, 1430, 1370, 1250, 1109, 1050, 841.
  • 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
  • IR (CNCl 3 , cm -1 ): 1687 (C O), 1601, 1460, 1389, 1331, 1266, 1175, 1115, 1030, 842.
  • 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 O 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 O/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
  • the thus-obtained reaction mixture was treated with several aliquots of pivaloyl chloride, with stirring for several hours at room temperature between additions.

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US6251581B1 (en) 1991-05-22 2001-06-26 Dade Behring Marburg Gmbh Assay method utilizing induced luminescence
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US5578498A (en) 1991-05-22 1996-11-26 Behringwerke Ag Metal chelate containing compositions for use in chemiluminescent assays
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US5731445A (en) * 1995-12-04 1998-03-24 Fujirebio Inc. 1,2- Dioxetane derivatives
US11312873B2 (en) * 2019-09-04 2022-04-26 Eastman Chemical Company Aromatic enol ether paint additives
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US4956477A (en) * 1987-12-31 1990-09-11 Tropix, Inc. Synthesis of 1,2-dioxetanes
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See also references of WO9103479A1 *
SYNTHESIS. no. 4, April 1984, STUTTGART DE pages 330 - 332; DAVID BURKHOUSE ET AL: 'Novel synthesis of 1-alkoxy-1-arylmethanephosphonic acid esters' *

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