# US 6,296,787 B1 · Compounds, compositions and methods for generating chemiluminescence with phosphatase enzymes

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- Number: US 6,296,787 B1
- Document ID: US6296787B1
- Status: Granted
- Kind: B1
- Application number: 09/557,726
- Filed: 2000-04-26
- Granted: 2001-10-02
- Earliest claimed: 1996-01-16
- Assignees: Lumigen, Inc.
- Inventors: Hashem Akhavan-Tafti; Zahra Arghavani; Renuka DeSilva
- Web page: https://patentgrep.com/patent/US6296787B1

## CPC

- C07F 9/6541: condensed with carbocyclic rings or {carbocyclic} ring systems
- C07F 9/5765: {condensed with carbocyclic rings or carbocyclic ring systems}
- C07F 9/64: Acridine or hydrogenated acridine ring systems
- C12Q 1/42: involving phosphatase
- C12Q 2334/00: O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
- Y10S 435/968: High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
- C07F: ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM (metal-containing porphyrins C07D487/22)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

Novel heterocyclic compounds which generate chemiluminescence on reaction with a phosphatase enzyme are provided as well as a process for their preparation and intermediates useful therein. The compounds comprise a nitrogen, oxygen or sulfur-containing heterocyclic ring system bearing an exocyclic carbon—carbon double bond. The double bond is further substituted at the distal carbon with a phosphate group and an oxygen or sulfur atom-containing group. Novel compositions further comprising a cationic aromatic compound (CAC) in addition to the heterocyclic phosphate compound are provided. The addition of the CAC in the composition greatly increases the production of chemiluminescence and provides improved detection sensitivity. Compositions further comprising an anionic surfactant and a non-ionic surfactant provide additional improvements in detection sensitivity. The novel chemiluminescent compounds and compositions are useful in methods for producing light and in assays for phosphatase enzymes and enzyme inhibitors and in assays employing enzyme-labeled specific binding pairs.

## Claims

### Claim 1

A compound which is selected from the group consisting of compounds of formula II and compounds of formula III:
wherein R1 is selected from the group consisting of aryl and substituted aryl groups, each of R2 to R5 is a substituent containing from 1 to 50 atoms selected from C, H, N, O, S, P and halogen atoms which permits chemiluminescence to be produced and wherein pairs of adjacent groups can be joined together to form a carbocyclic or heterocyclic ring system comprising at least one 5 or 6-membered ring, wherein Z is selected from the group consisting of O and S atoms, wherein R6 is an organic group which allows chemiluminescence to be produced, wherein each M is independently selected from H and a cationic center and wherein n is a number which satisfies electroneutrality.

### Claim 2 (depends on 1)

The compound of claim 1 wherein R6 contains from 1 to 50 atoms selected from the group consisting of C, H, N, O, S, P and halogen atoms.

### Claim 3 (depends on 1)

The compound of claim 1 wherein R6 is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl and aralkyl groups.

### Claim 4 (depends on 1)

The compound of claim 1 having the formula:
wherein each of R7 to R14 is independently a substituent containing from 1 to 50 atoms selected from C, H, N, O, S, P and halogen atoms and which permits the chemiluminescence to be produced and wherein Ar is an aryl ring group or a substituted aryl group.

### Claim 5 (depends on 4)

The compound of claim 4 wherein at least one of R7 to R14 is an alkoxy group and the remaining of R7 to R14 are hydrogen.

### Claim 6

A reagent composition which comprises in an aqueous solution:
a). a compound selected from the group consisting of compounds of formula II and compounds of formula III:
wherein R1 is selected from the group consisting of aryl and substituted aryl groups, each of R2 to R5 is a substituent containing from 1 to 50 atoms selected from C, H, N, O, S, P and halogen atoms which permits chemiluminescence to be produced and wherein pairs of adjacent groups can be joined together to form a carbocyclic or heterocyclic ring system comprising at least one 5 or 6-membered ring, wherein Z is selected from the group consisting of O and S atoms, wherein R6 is an organic group which allows chemiluminescence to be produced, wherein each M is independently selected from H and a cationic center and wherein n is a number which satisfies electroneutrality, and
b) at least one surfactant enhancer in an amount effective to enhance the chemiluminescence.

### Claim 7 (depends on 6)

The composition of claim 6 in which the compound has the formula:
wherein each of R7 to R14 is independently a substituent containing from 1 to 50 atoms selected from C, H, N, O, S, P and halogen atoms and which permits the chemiluminescence to be produced and wherein Ar is an aryl ring group or a substituted aryl group.

### Claim 8 (depends on 6)

The composition of claim 6 wherein the surfactant enhancer is selected from polymeric quaternary ammonium and phosphonium salts, monomeric quaternary ammonium and phosphonium salts and dicationic quaternary ammonium and phosphonium salts.

### Claim 9 (depends on 6)

The composition of claim 6 wherein the surfactant enhancer is a copolymer of a vinylbenzyltributylphosphonium salt and a vinylbenzyltrioctylphosphonium salt.

### Claim 10 (depends on 6)

The composition of claim 6 wherein copolymer comprises vinylbenzyltributylphosphonium salt and vinylbenzyltrioctylphosphonium salt groups in a ratio of about 3 to 1.

### Claim 11 (depends on 6)

The composition of claim 6 further comprising a magnesium salt in an amount effective to promote the reaction.

### Claim 12

A method for producing chemiluminescence which comprises reacting a phosphatase enzyme with at least one compound selected from the group consisting of compounds of formula II and compounds of formula III;
wherein R1 is selected from aryl and substituted aryl, groups, each of R2 to R5 is a substituent containing from 1 to 50 atoms selected from C, H, N, O, S, P and halogen atoms which permits chemiluminescence to be produced and wherein pairs of adjacent groups can be joined together to form a carbocyclic or heterocyclic ring system comprising at least one 5 or 6-membered ring, wherein R6 is an organic group which allows chemiluminescence to be produced, wherein Z is selected from the group consisting of O and S atoms, wherein each M is independently selected from H and a cationic center and wherein n is a number which satisfies electroneutrality.

### Claim 13 (depends on 12)

The method of claim 12 wherein the compound has the formula:
wherein each of R7 to R14 is independently a substituent which can contain from 1 to 50 atoms selected from C, H, N, O, S, P and halogen atoms and which permits the chemiluminescence to be produced and wherein Ar is an aryl ring group or a substituted aryl group.

### Claim 14 (depends on 13)

The method of claim 13 wherein at least one of R7 to R14 is an alkoxy group and the remaining of R7 to R14 are hydrogen.

### Claim 15 (depends on 12)

The method of claim 12 wherein the phosphatase enzyme is selected from the group consisting of bacterial alkaline phosphatase, mammalian alkaline phosphatase, plant acid phosphatase, mammalian acid phosphatase and alkaline phosphatase conjugates.

### Claim 16 (depends on 15)

The method of claim 15 wherein the alkaline phosphatase conjugate comprises alkaline phosphatase linked to a biological molecule selected from the group consisting of haptens, antibodies, proteins, nucleic acids and oligonucleotides.

### Claim 17

A process for the preparation of a compound selected from compounds of formula II and compounds of formula III:
wherein R1 is selected from aryl and substituted aryl groups, wherein each of R2 to R5 is a substituent containing from 1 to 50 atoms selected from C, H, N, O, S, P and halogen atoms which permits chemiluminescence to be produced and wherein pairs of adjacent groups can be joined together to form a carbocyclic or heterocyclic ring system comprising at least one 5 or 6-membered ring and wherein Z is selected from O and S atoms, wherein R6 is an organic group which contains from 1 to 50 atoms selected from the group consisting of C, H, N, O, S, P and halogen atoms, wherein each X is independently selected from H and a cationic center and wherein n is a number which satisfies electroneutrality comprising the steps of;
(a) reacting an ester or thioester compound having the formula IV or V:
wherein R1-R6 and Z are as defined for compound II or III with a base to form an enolate of IV or V;
(b) reacting the enolate of IV or V with a phosphorylating agent to form a protected enol phosphate VI or VII having the formula:
wherein Y is a protecting group; and
(c) deprotecting the enol phosphate to form the enol phosphate salt compound II or III by reacting VI or VII with at least one deprotecting agent in the presence of a cationic species M if the cationic species is not a part of the deprotecting agent.

### Claim 18 (depends on 17)

The process of claim 17 used to prepare a compound having the formula:
wherein each of R7 to R14 is independently a substituent containing from 1 to 50 atoms selected from C, H, N, O, S, P and halogen atoms and which permits chemiluminescence to be produced and wherein Ar is an aryl ring group or a substituted aryl group.

### Claim 19 (depends on 17)

The process of claim 17 wherein the step of reacting the enolate of compound IV or V with a phosphorylating agent to form a protected enol phosphate comprises the steps of:
(a) reacting the enolate of compound IV or V with a phosphorus oxyhalide compound POW3, where W is a halogen atom selected from F, Cl, Br and I to form an enol dihalophosphate VI′ or VII′ having the formula
; and
(b) reacting compound VI′ or VII′ with at least two equivalents of a hydroxylic compound Y—OH to form the protected enol phosphate VI or VII.

### Claim 20 (depends on 17)

The process of claim 17 wherein the step of reacting the enolate of compound IV or V with a phosphorylating agent to form a protected enol phosphate VI or VII comprises reacting the enolate of compound IV or V with a phosphorylating agent containing the protecting groups Y and having the formula W—PO(OY)2 and wherein W is a halogen atom selected from F, Cl, Br and I.

### Claim 21 (depends on 17)

The process of claim 17 wherein the groups Y are selected from the group consisting of lower alkyl groups, substituted lower alkyl groups, phenyl, substituted phenyl and benzyl groups.

### Claim 22 (depends on 17)

The process of claim 17 wherein the groups Y are connected to form the single group —CH2CH2—.

### Claim 23 (depends on 17)

The process of claim 17 wherein the deprotecting agent is selected from the group consisting of organic and inorganic bases such as sodium hydroxide, potassium hydroxide, potassium carbonate, sodium methoxide, sodium ethoxide, potassium t-butoxide, ammonium hydroxide, nucleophilic agents such as cyanide ion, fluoride ion.

### Claim 24 (depends on 17)

The process of claim 17 wherein Y is a CH2CH2CN group and the deprotecting agent is selected from sodium hydroxide and sodium carbonate.

### Claim 25 (depends on 19)

The process of claim 19 wherein Y is a CH2CH2CN group and the deprotecting agent is selected from sodium hydroxide and sodium carbonate.

### Claim 26

A compound selected from the group consisting of compounds of formulas:
wherein R1 is selected from aryl and substituted aryl groups, wherein each of R2 to R5 is a substituent containing from 1 to 50 atoms selected from C, H, N, O, S, P and halogen atoms which permits chemiluminescence to be produced and wherein pairs of adjacent groups can be joined together to form a carbocyclic or heterocyclic ring system comprising at least one 5 or 6-membered ring, wherein Z is selected from O and S atoms, wherein R6 contains from 1 to 50 atoms selected from the group consisting of C, H, N, O, S, P and halogen atoms and wherein Y is a protecting group which is removable to form a phosphate salt compound.

### Claim 27 (depends on 26)

The compound of claim 26 having the formula:
wherein each of R7 to R14 is independently a substituent containing from 1 to 50 atoms selected from C, H, N, O, S, P and halogen atoms and which permits the chemiluminescence to be produced and wherein Ar is an aryl ring group or a substituted aryl group.

### Claim 28 (depends on 26)

The compound of claim 26 wherein Y is a CH2CH2CN group.

### Claim 29

A compound selected from the group consisting of compounds of formulas:
wherein R1 is selected from aryl and substituted aryl groups, wherein each of R2 to R5 is a substituent containing from 1 to 50 atoms selected from C, H, N, O, S, P and halogen atoms which permits the chemiluminescence to be produced and wherein pairs of adjacent groups can be joined together to form a carbocyclic or heterocyclic ring system comprising at least one 5 or 6-membered ring, wherein Z is selected from O and S atoms, wherein R6 contains from 1 to 50 atoms selected from the group consisting of C, H, N, O, S, P and halogen atoms and wherein W is a halogen atom selected from F, Cl, Br and I atoms.

### Claim 30 (depends on 29)

The compound of claim 29 having the formula:
wherein each of R7 to R14 is independently a substituent containing from 1 to 50 atoms selected from C, H, N, O, S, P and halogen atoms and which permits the chemiluminescence to be produced and wherein Ar is an aryl ring group.

### Claim 31 (depends on 29)

The compound of claim 29 wherein W is a Cl atom.

### Claim 32

A compound having formula IV or V:
wherein R1 is selected from aryl and substituted aryl groups, wherein each of R2 to R5 is a substituent containing from 1 to 50 atoms selected from C, H, N, O, S, P and halogen atoms which permits chemiluminescence to be produced and wherein pairs of adjacent groups can be joined together to form a carbocyclic or heterocyclic ring system comprising at least one 5 or 6-membered ring, Z is selected from O and S atoms, and R6 is an organic group containing from 1 to 50 atoms selected from C, H, N, O, S, P and halogen atoms.

### Claim 33 (depends on 32)

The compound of claim 32 wherein R6 is selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl and aralkyl groups.

### Claim 34 (depends on 32)

The compound of claim 32 having the formula:
wherein each of R7 to R14 is independently a substituent containing from 1 to 50 atoms selected from C, H, N, O, S, P and halogen atoms and wherein Ar is an aryl group or a substituted aryl group.

### Claim 35 (depends on 34)

The compound of claim 34 wherein the groups R7 to R14 are each independently selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, alkenyl, alkynyl, alkoxy, aryloxy, halogen, amino, substituted amino groups, carboxyl, carboalkoxy, carboxamide, cyano, and sulfonate groups.

### Claim 36 (depends on 34)

The compound of claim 34 wherein at least one of R7 to R14 is an alkoxy group and the remaining of R7 to R14 are hydrogen.

### Claim 37 (depends on 4)

The compound of claim 4 wherein the groups R7 to R14 are each independently selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, alkenyl, alkynyl, alkoxy, aryloxy, halogen, amino, substituted amino groups, carboxyl, carboalkoxy, carboxamide, cyano, and sulfonate groups.

### Claim 38 (depends on 27)

The compound of claim 27 wherein the groups R7 to R14 are each independently selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, alkenyl, alkynyl, alkoxy, aryloxy, halogen, amino, substituted amino groups, carboxyl, carboalkoxy, carboxamide, cyano, and sulfonate groups.

### Claim 39 (depends on 30)

The compound of claim 30 wherein the groups R7 to R14 are each independently selected from hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aralkyl, alkenyl, alkynyl, alkoxy, aryloxy, halogen, amino, substituted amino groups, carboxyl, carboalkoxy, carboxamide, cyano, and sulfonate groups.