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Title:
ASSAYS UTILIZING IMPROVED CHEMILUMINESCENT ESTERS, THIOESTERS AND AMIDES
Document Type and Number:
WIPO Patent Application WO/1989/006231
Kind Code:
A1
Abstract:
This invention concerns chemiluminescent moieties useful as labels in assays and immunoassays, and compositions and assay kits incorporating the moieties. The chemiluminescent moieties are of the type wherein attack by peroxide or molecular oxygen results in an electronically excited intermediate which decays with emission of light. The moieties are distinguished by being less prone to hydrolysis and thus exhibit greater stability.

Inventors:
Mccapra, Frank Beheshti Iraj Hart Russel C.
Koelling, Harlen Patel Ashokkumar Ramakrishnan Kastooriranganathan
Application Number:
PCT/US1988/004719
Publication Date:
July 13, 1989
Filing Date:
December 30, 1988
Export Citation:
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Assignee:
LONDON DIAGNOSTICS, INC.
International Classes:
G01N33/532; C07D215/50; C07D215/52; C07D219/04; C07D219/06; C07D221/12; C07D403/12; C07D521/00; C07J1/00; C07J5/00; C07J43/00; C09B15/00; C09K11/06; G01N21/76; G01N33/53; G01N33/533; G01N33/536; G01N33/542; G01N33/543; G01N33/58; C07D221/06; C07D227/02; C07D233/56; C07D247/00; C07D249/08; C07D261/02; C07D263/10; C07D275/02; (IPC1-7): C07D211/82; C07D213/00; C07D215/04; C07D217/16; C07D219/02; C07D221/12; C07H17/02; C12Q1/68; G01N33/53; G01N33/533; G01N33/536; G01N33/542; G01N33/543
Foreign References:
US4745181A1988-05-17
US4687747A1987-08-18
EP0082636A11983-06-29
EP0216553A21987-04-01
Other References:
ALEX BURFORD, "Mechanism in Peroxide Chemiluminescence", a PhD Thesis from the School of Molecular Sciences the University of Sussex, Falmer, Brighton, UK, pages 26-36, September 1977.
RUSSELL COLIN HART, "Bacterial Bioluminescence", a PhD Thesis from the School of Molecular Sciences, the University of Sussex, Falmer, Brighton, UK, pages 94-130, September 1975.
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Claims:
We claim : 79
1. In a specific binding assay for the presence of an analyte in a sample wherein (i) said assay utilizes a chemiluminescent conjugate comprising > a chemiluminescent moiety attached to a specific binding material and (ii) the presence of analyte in said sample is proportional to the formation of one or more specific binding reaction products containing said chemiluminescent conjugate, said assay comprising: allowing under suitable conditions substantial formation of said one or more specific binding reaction products containing said chemiluminescent conjugate; and measuring the chemiiuminescence of (i) said one or more specific binding reaction products containing said chemiluminescent conjugate or (ii) the chemiluminescent conjugate not contained in said one or more specific binding reaction products; wherein the improvement comprises selecting as the chemiluminescent moiety an ester, thioester or amide in which the ester, thioester or amide linkage is between: a heterocyclic ring or ring system containing a carbon atom to which said linkage is attached, wherein the heteroatom within said heterocyclic ring or ring system is in an oxidation state which renders said carbon atom susceptible to attack by peroxide or molecular oxygen to form an intermediate which decays to produce chemiiuminescence; and an aryl ring or ring system, said aryl ring or ring system containing at least one substituted sixmember ring, said substituted sixmember ring having two or more substituent groups, where at least two of said two or more substituent groups sterically hinder the hydrolysis of said linkage; and wherein said carbon atom to which said linkage is attached has a secondary substituent of the formula R X, where X is selected from the group consisting of 0 N, S and C, where R is any group, and where n is a number such that X has proper valenc .
2. The specific binding assay of claim 1 wherein said specific binding material is capable 0 of specifically binding with the analyte and said one or more specific binding reaction products is an analyteche iluminescent conjugate complex, said assay further comprising: allowing under suitable conditions substantial 15 formation of said analytechemiluminescent conjugate complex; and measuring the chemiiuminescence of (i) said analytechemiluminescent conjugate complex or (ii) the chemiluminescent conjugate not contained in said 2.0 analytechemiluminescent conjugate complex.
3. The specific binding assay of claim 1 wherein said assay further utilizes a reactant which is capable of specifically binding (i) with said analyte to form an analytereactant complex and (ii) with 25 said specific binding material to form a chemiluminescent co jugatereactant complex, and wherein said one or more specific binding reaction products is said chemiluminescent conjugatereactant complex, said assay further comprising: 30 allowing under suitable conditions substantial formation of said analytereactant complex and said chemiluminescent conjugatereactant complex; and measuring the chemiiuminescence of (i) said chemiluminescent conjugatereactant complex or (ii) the chemiluminescent conjugate not contained in said chemiluminescent conjugatereactant complex.
4. The specific binding assay of claim 1 wherein said specific binding material is capable of specifically binding with said analyte and said assay further utilizes a reactant capable of specifically binding with said analyte to form a reactantanalyte chemiluminescent conjugate complex, and wherein said one or more specific binding reaction products is said reactantanalytechemiluminescent conjugate complex, said assay further comprising: allowing under suitable conditions substantial formation of said reactantanalytechemiluminescent conjugate complex; and measuring the chemiiuminescence of (i) said reactantanalyatechemiluminescent conjugate complex or (ii) the chemiluminescent conjugate not contained in said reactantanalyte chemiluminescent conjugate complex.
5. In a specific binding assay for the presence of an analyte in a sample wherein (i) said assay utilizes a chemiluminescent moiety and (ii) the presence of analyte in said sample is proportional to the formation of one or more specific binding reaction products not containing said chemiluminescent moiety, and wherein said chemiluminescent moiety chemiluminesces in proportion to the formation of said one or more specific binding reaction products, said assay comprising: allowing under suitable conditions substantial formation of said one or more specific binding reaction products; and measuring the chemiiuminescence of said chemiluminescent moiety caused by said formation of said one or more specific binding reaction products; wherein the improvement comprises selecting as the chemiluminescent moiety an ester, thioester or amide in which the ester, thioester or amide linkage is between: : a heterocyclic ring or ring system containing a carbon atom to which said linkage is attached, wherein the heteroatom within said heterocyclic ring or ring system is in an oxidation state which renders said carbon atom susceptible to attack by peroxide or molecular oxygen to form an intermediate which decays to produce chemiiuminescence; and an aryl ring or ring system, said aryl ring or ring system containing at least one substituted sixmember ring, said substituted sixmember ring having two or more substituent groups, where at least two of said two or more substituent groups sterically hinder the hydrolysis of said linkage; and wherein said carbon atom to which said linkage is attached has a secondary substituent of the formula R X, where X is selected from the group consisting of 0, N, S and C, where R is any group, and where n is a number such that X has proper valency.
6. The specific binding assay of claim 5 wherein said assay further utilizes a reactant capable of binding with said analyte to form an analytereactant complex and wherein said chemiluminescent moiety chemiluminesces in proportion to the formation of said analytereactant complex.
7. The specific binding assay of claim 1 or 5 wherein said secondary substituent is selected from the group consisting of CN, OR, R2, NR3 SR, "SR2+, SR, NRC02R, NHNR2, R 2, NHOR, ONR2, CR(CN)2, CR(COR)2, CR2N02, C≡COR, .
8. The specific binding assay of claim 7 wherein each of said substituent groups which sterically hinder hydrolysis of said linkage is selected from the group consisting of ~CH,, OCEU and tpropyl.
9. The specific binding assay of claim 7 wherein at least one of said substituent groups which sterically hinder hydrolysis of said linkage is an electron withdrawing group.
10. The specific binding assay of claim 7 wherein said substituted sixmember ring has at least one additional substituent group, in addition to said substituent groups which sterically hinder hydrolysis of said linkage, and said at least one additional substituent group is an electron withdrawing group.
11. The specific binding assay of claim 10 wherein said at least one additional substituent group is selected from the group consisting of NO.,, S0„C1, Br, N(CH ) + and H.
12. The specific binding assay of claim 11 wherein said electron withdrawing group is H.
13. The specific binding assay of claim 7 wherein said aryl ring or ring system is selected from the group consisting of phenyl, naphthalene and anthracene.
14. The specific binding assay of claim 13 wherein said aryl ring or ring system is phenyl.
15. The specific binding assay of claim 14 wherein the sum of the sigma values for all substituent groups on said phenyl which are electron withdrawing groups is less than or equal to about 1.0.
16. The specific binding assay of claim 7 wherein said heterocyclic ring or ring system is a reduced form of a member selected from the group consisting of acridinium, benz[a]acridinium, benz[b]acridinium, benz[c]acridinium, a 1,2,4triazole cation, an isooxazole cation, an isothioazole cation, a 1,2azole cation, an imidazole cation, a benzimidazole cation, quinolinium, isoquinolinium, quinolizinium, a cyclic substituted quinolinium, pyridinium, pyrimidinium, pyridazinium, pyrazinium, phenanthridinium, and quinoxalinium.
17. The specific binding assay of claim 16 wherein said heterocyclic ring or ring system is a reduced form of acridinium quinolinium or phenanthridinium.
18. The specific binding assay of claim 17 wherein said secondary substituent is selected from the group consisting of 0CH. and 0CH„CH .
19. The specific binding assay of claim 18 wherein said chemiluminescent moiety is (2, 6dimethoxy3chlorosulfonyl)phenylNmethylacridan 9ethoxy9carboxylate.
20. The specific binding assay of claim 18 wherein said chemiluminescent moiety is (2, 6dimethoxy3chlorosulfonyl)phenyl~Nmethylacridan 9methoxy9carboxylate.
21. The specific binding assay of claim 18 wherein said chemiluminescent moiety is (2, 6dimethyl3chlorosulfonyl )phenylNmethylacridan9 methoxy9carboxylate.
22. The specific binding assay of claim 7 wherein said heterocyclic ring or ring system has at least one peri substituent, peri to said carbon atom to which said linkage is attached.
23. The specific binding assay of claim 22 wherein said least one peri substituent is an alkyl or aryl group.
24. The specific binding assay of claim 23 wherein said at least one peri substituent is CH..
25. A composition comprising the chemiluminescent moiety of claims 124.
26. The composition of claim 25 further comprising a specific binding partner.
27. As an article of manufacture, a specific binding assay kit comprising a vial containing at least the composition of claims 25 or 26.
28. In a specific binding assay for the presence of an analyte in a sample wherein (i) said assay utilizes a chemiluminescent conjugate comprising a chemiluminescent moiety attached to a specific binding material and (ii) the presence of analyte in said sample is proportional to the formation of one or more specific binding reaction products containing said chemiluminescent conjugate, said assay comprising: allowing under suitable conditions substantial formation of said one or more specific binding reaction products containing said chemiluminescent conjugate; and measuring the chemiiuminescence of (i) said one or more specific binding reaction products containing said chemiluminescent conjugate or (ii) the chemiluminescent conjugate not contained in said one or more specific binding reaction products; wherein the improvement comprises selecting as the chemiluminescent moiety an ester, thioester or amide in which the ester, thioester or amide linkage is between: a heterocyclic ring or ring system containing a carbon atom to which said linkage is attached, wherein the heteroatom within said heterocyclic ring or ring system is in an oxidation state which renders said carbon atom susceptible to attack by peroxide or molecular oxygen to form an intermediate which decays to produce chemiiuminescence; and a leaving group; and wherein said carbon atom to which said linkage is attached has a secondary substituent of the formula R X, where X is selected from the group consisting of 0, N, S and C, where R is any group, and where n is a number such that X has proper valency.
29. The specific binding assay of claim 28 wherein said specific binding material is capable of specifically binding with the analyte and said one or more specific binding reaction products is an analytechemiluminescent conjugate complex, said assay further comprising: allowing under suitable conditions substantial formation of said analytechemiluminescent conjugate complex; and measuring the chemiiuminescence of .(i) said analytechemiluminescent conjugate complex or (ii) the chemiluminescent conjugate not contained in said analytechemiluminescent conjugate complex.
30. The specific binding assay of claim 28 wherein said assay further utilizes a reactant which is capable of specifically binding (i) with said analyte to form an analytereactant complex and (ii) with said specific binding material to form a chemiluminescent conjugatereactant complex, and wherein said one or more specific binding reaction products is said chemiluminescent conjugatereactant complex, said assay further comprising: allowing under suitable conditions substantial formation of said analytereactant complex and said chemiluminescent conjugatereactant complex; and measuring the chemiiuminescence of (i) said chemiluminescent conjugatereactant complex or (ii) the chemiluminescent conjugate not contained in said chemiluminescent conjugatereactant complex.
31. The specific binding assay of claim 28 wherein said specific binding material is capable of specifically binding with said analyte and said assay further utilizes a reactant capable of specifically binding with said analyte to form a reactantanalyte chemiluminescent conjugate complex, and wherein said one or more specific binding reaction products is said reactantanalytechemiluminescent conjugate complex, said assay further comprising: allowing under suitable conditions substantial formation of said reactantanalytechemiluminescent conjugate complex; and measuring the chemiiuminescence of (i) said reactantanalytechemiluminescent conjugate complex or (ii) the chemiluminescent conjugate not contained in said reactantanalytechemiluminescent conjugate complex.
32. In a specific binding assay for the presence of an analyte in a sample wherein (i) said assay utilizes a chemiluminescent moiety and (ii) the presence of analyte in said sample is proportional to the formation of one or more specific binding reaction products not containing said chemiluminescent moiety, and wherein said chemiluminescent moiety chemiluminesces in proportion to the formation of said one or more specific binding reaction products, said assay comprising: allowing under suitable conditions substantial formation of said one or more specific binding reaction products; and measuring the chemiiuminescence of said chemiluminescent moiety caused by said formation of said one or more specific binding reaction produc s; wherein the improvement comprises selecting as the chemiluminescent moiety an ester, thioester or amide in which the ester, thioester or amide linkage is between: : a heterocyclic ring or ring system containing a carbon atom to which said linkage is attached, wherein the heteroatom within said heterocyclic ring or ring system is in an oxidation state which renders said carbon atom susceptible to attack by peroxide or molecular oxygen to form an intermediate which decays to produce chemiiuminescence; and a leaving group; and wherein said carbon atom to which said linkage is attached has a secondary substituent of the formula R X, where X is selected from the group consisting of 0, N, S and C, where R is any group, and where n is a number such that X has proper valency.
33. The specific binding assay of claim 32 wherein said assay further utilizes a reactant capable of binding with said analyte to form an analytereactant complex and wherein said chemiluminescent moiety chemiluminesces in proportion to the formation of said analytereactant complex.
34. The specific binding assay of claim 28 or 32 wherein wherein said secondary substituent is selected from the group consisting of CN, OR, NR_, NR3+, SR, SR2+, S2R, NRC02R, NH R2, NR R2, NHOR, 0NR2, CR(CN)2, CR(CN)2, CR(C0R)2, CR2 02, C=C0R,.
35. The specific binding assay of claim 34 wherein said leaving group is an aryl ring or ring system.
36. The specific binding assay of claim 35 wherein said aryl ring or ring system is selected from the group consisting of phenyl, naphthalene and anthracene.
37. The specific binding assay of claim 36 wherein said aryl ring or ring system is phenyl.
38. The specific binding assay of claim 37 wherein said phenyl ring is unsubstituted.
39. The specific binding assay of claim 34 wherein said chemiluminescent moiety is an amide and said leaving group is a group of the formula NR' (S02R"Y) , where R* and R" are selected from the group consisting of alkyl, alkylene, aryl, optionally substituted alkyl, optionally substituted alkylene, optionally substituted aryl, alk loxy, aryloxy, halo, optionally protected amino, substituted aminohydroxy, protected hydroxy, oxo, thio, imino, optionally substituted mercapto, a heterocyclic ring, and a heteroalkyl group, and where Y is selected from the group consisting of a hydrogen, carboxy, carbonyl halide, sulfonyl halide, carboalkoxy, carboxamido, carboaryloxy, cyano, carboximido, isocyanato, isothiocyanate, sulfo, Nsuccinimidylcarboxy and Nmaleimide.
40. The specific binding assay of claim 34 wherein said chemiluminescent moiety is a thioester and said leaving group is a group of the formula VR"', where V is an aliphatic or aromatic group and where R"1 is a group which is useful for attaching said moiety to a specific binding partner.
41. The specific binding assay of claim 40 wherein said leaving group is a sulfonamido or sulfocarbonyl group.
42. The specific binding assay of claim 34 wherein said heterocyclic ring or ring system is a reduced form of a member selected from the group consisting of acridinium, benz[a]acridinium, benz[b] cridinium, benz[c] cridinium, a 1, 2, 4triazole cation, an isooxazole cation, an isothioazole cation, a 1,2azole cation, an imidazole cation, a benzimidazole cation, quinolinium, isoquinolinium, quinolizinium, a cyclic substituted quinolinium, pyridinium, pyrimidinium, pyridazinium, pyrazinium, phenanthridinium and quinoxalinium.
43. The specific binding assay of claim 42 wherein said heterocyclic ring or ring system is a reduced form of acridinium, quinolinium or phenanthridinium.
44. The specific binding assay of claim 43 wherein said secondary substituent is selected from the group consisting of OHC, and OCHCH,.
45. The specific binding assay of claim 43 wherein said heterocyclic ring or ring system has at least one peri substituent, peri to said carbon atom to which said linkage is attached.
46. The specific binding assay of claim 45 wherein said least one peri substituent is an alkyl or aryl group.
47. The specific binding assay of claim 46 wherein said at least one peri substituent is CH,.
48. The specific binding assay of claim 47 wherein said chemiluminescent moiety is phenylNmethyl 1, 3dimethyl'acridinium9methoxy9carboxylate.
49. A composition comprising the chemiluminescent moiety of any of claims 2848 and a specific binding partner.
50. As an article of manufacture, a specific binding assay kit comprising the composition of claim 49.
51. In a specific binding assay for the presence of an analyte in a sample wherein (i) said assay utilizes a chemiluminescent conjugate comprising a chemiluminescent moiety attached to a specific binding material and (ii) the presence of analyte in said sample is proportional to the formation of one or more specific binding reaction products containing said chemiluminescent conjugate, said assay comprising: allowing under suitable conditions substantial formation of said one or more specific binding reaction products containing said chemiluminescent conjugate; and measuring the chemiiuminescence of (i) said one or more specific binding reaction products containing said chemiluminescent conjugate or (ii) the chemiluminescent conjugate not contained in said one or more specific binding reaction products; wherein the improvement comprises selecting as the chemiluminescent moiety an ester, thioester or amide in which the ester, thioester or amide linkage is between: a heterocyclic ring or ring system containing a carbon atom to which said linkage is attached, wherein the heteroatom within said heterocyclic ring or ring system is in an oxidation state which renders said carbon atom susceptible to attack by peroxide or molecular oxygen to form an intermediate which decays to produce chemiiuminescence; and an aryl ring or ring system, said aryl ring or ring system containing at least one substituted sixmember ring, said substituted sixmember ring having two or more substituent groups, where at least two of said two or more substituent groups sterically hinder the hydrolysis of said linkage.
52. The specific binding assay of claim 51 wherein said specific binding material is capable of specifically binding with the analyte and said one or more specific binding reaction products is an analytechemiluminescent conjugate complex, said assay further comprising: allowing under suitable conditions substantial formation of said analytechemiluminescent conjugate complex; and measuring the chemiiuminescence of (i) said analytechemiluminescent conjugate complex or (ii) the chemiluminescent conjugate not contained in said analytechemiluminescent conjugate complex.
53. The specific binding assay of claim 51 wherein said assay further utilizes a reactant which is capable of specifically binding (i) with said analyte to form an analytereactant complex and (ii) with said specific binding material to form a chemiluminescent conjugatereactant complex, and wherein said one or more specific binding reaction products is said chemiluminescent conjugatereactant complex, said assay further comprising: allowing under suitable conditions substantial formation of said analytereactant complex and said chemiluminescent conjugatereactant complex; and measuring the chemiiuminescence of (i) said chemiluminescent conjugatereactant complex or (ii) the chemiluminescent conjugate not contained in said chemiluminescent conjugatereactant complex.
54. The specific binding assay of claim 51 wherein said specific binding material is capable of specifically binding with said analyte and said assay further utilizes a reactant capable of specifically binding with said analyte to form a reactantanalyte chemiluminescent conjugate complex, and wherein said one or more specific binding reaction products is said reactantanalytechemiluminescent conjugate complex, said assay further comprising: allowing under suitable conditions substantial formation of said reactantanalytechemiluminescent conjugate complex; and measuring the chemiiuminescence of (i) said reactantanalytechemiluminescent conjugate complex or (ii) the chemiluminescent conjugate not contained in said reactantanalytechemiluminescent conjugate complex.
55. In a specific binding assay for the presence of an analyte in a sample wherein (i) said assay utilizes a chemiluminescent moiety and (ii) the presence of analyte in said sample is proportional to the formation of one or more specific binding reaction products not containing said chemiluminescent moiety, and wherein said chemiluminescent moiety chemiluminesces in proportion to the formation of said one or more specific binding reaction products, said assay comprising: allowing under suitable conditions substantial formation of said one or more specific binding reaction products; and measuring the chemiiuminescence of said chemiluminescent moiety caused by said formation of said one or more specific binding reaction products; wherein the improvement comprises selecting as the chemiluminescent moiety an ester, thioester or amide in which the ester, thioester or amide linkage is between : : 95 a heterocyclic ring or ring system containing a carbon atom to which said linkage is attached, wherein the heteroatom within said heterocyclic ring or ring system is in an oxidatin state which renders said carbon atom susceptible to attack by peroxide or molecular oxygen to form an intermediate which decays to produce chemiiuminescence; and an aryl ring or ring system, said aryl ring or ring system containing at least one substituted sixmember ring, said substituted sixmember ring having two or more substituent groups, where at least two of said two or more substituent groups sterically hinder the hydrolysis of said linkage.
56. The specific binding assay of claim 55 wherein said assay further utilizes a reactant capable of binding with said analyte to form an analytereactant complex and wherein said chemiluminescent moiety chemiluminesces in proportion to the formation of said analytereactant complex.
57. The specific binding assay of claim 51 or 55 wherein each of said substituent groups which sterically hinder hydrolysis of said linkage is selected from the group consisting of CH,, OCH, and ipropyl .
58. The specific binding assay of claim 51 or 55 wherein at least one of said substituent groups which sterically hinder hydrolysis of said linkage is an electron withdrawing group.
59. The specific binding assay of claim 51 or 55 wherein said substituted sixmember ring has at least one additional substituent group, in addition to said substituent groups which sterically hinder hydrolysis of said linkage, and said at least one additional substituent group is an electron withdrawing grou .
60. The specific binding assay of claim 59 wherein said at least one additional substituent group is selected from the group consisting of NO, S0_C1, Br, N(CH3)3+ and H.
61. The specific binding assay of claim 60 wherein said electron withdrawing group is H.
62. The specific binding assay of claim 51 or 55 wherein said aryl ring or ring system is selected from the group consisting of phenyl, naphthalene and anthracene.
63. The specific binding of claim 62 wherein said aryl ring or ring system is phenyl.
64. The specific binding assay of claim 63 wherein the sum of the sigma values for all substituent P groups on said phenyl which are electron withdrawing groups is less than or equal to about 1.0.
65. The specific binding assay of claim 63 wherein said chemiluminescent moiety has the formula:.
66. The specific binding assay of claim 63 wherein said chemiluminescent moiety has the formula:.
67. The specific binding assay of claim 63 wherein said chemiluminescent moiety has the formula:.
68. The specific binding assay of claim 63 wherein said chemiluminescent moiety has the formula:.
69. The specific binding assay of claim 63 wherein said chemiluminescent moiety has the formula:.
70. The specific binding assay of claim 63 wherein said chemiluminescent moiety has the formula:.
71. The specific binding assay of claim 63 wherein said chemiluminescent moiety has the formula:.
72. The specific binding assay of claim 63 wherein said chemiluminescent moiety has the formula:.
73. The specific binding assay of claim 63 wherein said chemiluminescent moiety has the formula:.
74. The specific binding assay of claim 63 wherein said chemiluminescent moiety has the formula:.
75. The specific binding assay of claim 63 wherein said chemiluminescent moiety has the formula: sr .
76. The specific binding assay of claim 63 wherein said chemiluminescent moiety has the formula:.
77. The specific binding assay of claim 51 or 55 wherein said heterocyclic ring or ring system is selected from the group consisting of acridinium, benz[a]acridinium, benz[b]acridinium, benz[c]acridinium, a 1,2,4triazole cation, an isooxazole cation, an isothioazole cation, a 1,2azole cation, an imidazole cation, a benzimidazole cation, quinolinium, isoq inolinium, quinolizinium, a cyclic substituted quinolinium, pyridinium, pyrimidinium, pyridazinium, pyrazinium, phenanthridinium, and quinoxalinium.
78. The specific binding assay of claim 77 wherein said heterocyclic ring or ring system is selected from the group consisting of acridinium, quinolinium and phenanthridinium.
79. The specific binding assay of claim 78 wherein said heterocyclic ring or ring system is acridinium.
80. The specific binding assay of claim 51 or 55 wherein said heterocyclic ring or ring system is a reduced form of a member selected from the group consisting of acridinium, benz[a]acridinium, benz[b]acridinium, benz[c]acridinium, a 1,2,4triazole cation, an isooxazole cation, an isothioazole cation, a 1,2azole cation, an imidazole cation, a benzimidazole 5 cation, quinolinium, isoquinolinium, quinolizinium, a cyclic substituted quinolinium, pyridinium, pyrimidinium, pyridazinium, pyrazinium, phenanthridinium, and quinoxalinium.
81. The specific binding assay of claim 80 10 wherein said heterocyclic ring or ring system is a reduced form of acridinium, quinolinium or phenanthridinium.
82. The specific binding assay of claim 80 wherein said carbon atom has a substituent selected 15 from the group consisting of H, CN, a halogen, OR, NR ~,2 ' NR3+, SR, ~SR2+, and S2R, wherein R= alkyl aryl, an amino acid or a sugar.
83. The specific binding assay of claim 81 wherein said conjugate has the formula:.
84. A composition comprising the chemiluminescent moiety of any of claims 5183.
85. The composition of claim 84 further comprising a specific binding partner linked to said moiety.
86. As an article of manufacture, a specific binding assay kit comprising a vial containing at least the composition of claim 84 or 85.
Description:
ASSAYS UTILIZING IMPROVED CHEMILUMINESCENT ESTERS, THIOESTERS AND AMIDES

FTET-T) OF THE INVENTION This invention relates to assays and immunoassays utilizing chemiluminescent conjugates as the label.

BACKGROUND OF THE INVENTION Certain compounds give off light or "chemiiuminesce" upon treatment with peroxide or molecular oxygen at high pH. Light is produced by the decay of a chemical intermediate which is formed by the attack of peroxide or molecular oxygen at an sp2 or sρ3 hybridized carbon center. The carbon center which is attacked can be part of a chain or a ring or ring system.

The characteristics and behavior of some of these chemiluminescent compounds is more fully described in McCapra,

"Chemiluminescence of Organic Compounds," in Progress in rganic

Chem istry, vol. 8, Carruthers and Sutherland ed. (Wiley & Sons 1973) which is incorporated herein by reference.

Chemiluminescent compounds have been used as labels in prototype assays including immunoassays for many years. Examples of

such use can be found in U.S. Patent Nos. 4,383,031, 4,380,580, 4,226,993 and many others. In general, however, these compounds have not been used in commercial assays, because of their lack of stability in aqueous solutions. This lack of stability is especially important for the subclass of chemiluminescent compounds comprising certain esters, thioesters and amides. These compounds react according to the general reaction illustrated below for esters:

where A- an aryl ring or ring system and B- a heterocyclic ring or ring system. The compounds of this subclass tend to "lose their chemiiuminescence" due to the premature hydrolysis of the ester, thioester or amide linkage. Once the linkage is hydrolyzed the compound no longer produces chemiiuminescence efficiently. If the compound is being used as a label in an assay, the label will gradually lose its capacity to chemiluminesce, thus producing unreliable assay results.

It is therefore desirable to provide chemiluminescent ester, thioester and amide compounds for use in assays which are not as susceptible to hydrolysis and thus show increased stability in aqueous solution.

SUMMARY OF THE TNVFNTTON In accordance with the present invention, specific binding assays are disclosed which utilize an chemiluminescent compound, i.e., moiety, which has increased stability in aqueous solution. The chemiluminescent moiety is an ester, thioester or amide in which the ester, thioester or amide linkage is between ( 1 ) a heterocyclic ring or ring system containing a carbon atom to which the linkage is attached, wherein the heteroatom within the heterocyclic ring or ring system is in an oxidation state which renders such carbon atom susceptible to attack by peroxide or

molecular oxygen to form an intermediate which decays to produce chemiiuminescence, and (2) an aryl ring or ring system. The aryl ring or ring system contains at least one substituted six-member ring. The substituted six-member ring has two or more substituent groups, where at least two of said two or more substituent groups sterically hinder hydrolysis of said linkage. One or more of the substituent groups which sterically hinder hydrolysis of said linkage may be an electron withdrawing group. The substituted six-member ring may have one or more additional substituent groups in addition to the substituent groups which sterically hinder hydrolysis of the linkage. Such additional substituent groups may also be an electron withdrawing group.

The carbon atom in the heterocyclic ring or ring system, to which the linkage is attached, may also have a secondary substituent of the formula nX-. where X is selected from the group consisting of 0, N, S and C, where R is any group, and where n is a number such that X has proper valency. Other chemiluminescent moieties are disclosed which are characterized by a heterocyclic ring or ring system and a secondary substituent of the formula RnX-, with the ester, thioester or amide linkage being between the heterocyclic ring or ring system and a leaving group. The disclosed chemiluminescent moieties can also include substituents at peri positions within the heterocyclic ring or ring system.

Also in accordance with the present invention, compositions including and assay kits incorporating such chemiluminescent moieties are disclosed.

BRTEF DESCRIPTION OF THE FIGURES

The following abbreviations are used throughout the Figures to identify various moieties:

"P" represents a phenyl acridinium ester; "DM" represents a (2,6-dimethyl)phenyl acridinium ester;

"DMC" represents a ( 2,6-dimethoxy-3-chiorosuifonyl)phenyl acridinium ester;

"DMS" represents a ( 2,6-dimethyl-3 chiorosulfonyDphenyl acridinium ester; "DMN" represents a (2,6-dimethyl-4-nitro)phenyl acridinium ester;

"Quat" represents a (2,6-dimethyl-4-trimethylammonio)phenyl acridinium ester;

"DMB" represents a (2,6-dimethyl-4-bromo)phenyl acridinium ester;

" C 2 " o r " C 2 N H S " r e p r e s e n t s a 4 - ( 2 - succini idylcarboxyethyDphenyl acridinium ester; "DME"represents a (2,6-dimethoιy-3-chlorosulfonyi)phenyl-9- ethoxy-acridan ester; and

"RT" represents room temperature.

Fig.l is a graph of the comparative stability data of various N- methyl acridinium esters in phosphate buffer at pH 6.3 at 35 * C.

Fig. 2 is a graph of the comparative stability data of various N- methyl acridinium esters in phosphate buffer at pH 6.3 at 45 * C.

Fig. 3 is a graph of the comparative stability data of various N- methyl acridinium esters in phosphate buffer at pH 8.0 at 35 * C. Fig. 4 is a graph of the comparative stability data of various N- methyl acridinium esters in phosphate buffer at pH 8.0 at 45 * C.

Fig. 5 is a graph of the comparative stability data of DMN, DMC and C2NHS ester IgG conjugates in Standard Phosphate Buffer at pH 6.3 at 4 * C. Fig. 6 is a graph of the comparative stability data of DMN, DMC and C2NHS ester IgG conjugates in Standard Phosphate Buffer at pH 7.3 at 4 * C. Fig. 7 is a graph of the comparative stability data of DMN, DMC and C2NHS ester IgG conjugates in Standard Phosphate Buffer at pH 8.0 at 4 * C. Fig. 8 is a graph of the comparative stability data of DMN, DMC and C2NHS ester IgG conjugates in Standard Phosphate Buffer without sheep IgG at pH 6.3 at 4'C.

Fig. 9 is a graph of the comparative stability data of DMN, DMC and C2NHS ester IgG conjugates in Standard Phosphate Buffer without sheep IgG at pH 7.3 at 4 * C.

Fig. 10 is a graph of the comparative stability data of DMN, DMC and C2NHS ester IgG conjugates in Standard Phosphate Buffer without sheep IgG at pH 8.0 at 4'C.

Fig. 11 is a graph of the comparative stability data of DMN, DMC and C2NHS ester IgG conjugates in Standard Phosphate Buffer without sheep IgG and with 0.1 % bovine serum albumin (instead of human seru m. albumin) at pH 6.3 at 4 * C.

Fig. 12 is a graph of the comparative stability data of DMN, DMC and C2NHS ester IgG conjugates in Standard Phosphate Buffer without sheep

IgG and with 0.1 % bovine serum albumin (instead of human serum albumin) at pH 7.3 at 4'C.

Fig. 13 is a graph of the comparative stability data of DMN, DMC and C2NHS ester IgG conjugates in Standard Phosphate Buffer without sheep IgG and with 0.1 % bovine serum albumin (instead of human serum albumin) at pH 8.0 at 4'C.

Fig. 14 is a graph of the comparative stability data of the C2 HS ester IgG conjugate in Standard Phosphate Buffer at room temperature at pH 6.3. 7.3 and 8.0. Fig. 15 is a graph of the comparative stability data, of DMN IgG conjugate in Standard Phosphate Buffer at room temperature at pH 6.3, 7.3 and 8.0.

Fig. 16 is a graph of the comparative stability data of DMC IgG conjugate in Standard Phosphate Buffer at room temperature at pH 6.3, 7.3 and 8.0.

Fig. 17 is a graph of the comparative stability data of C2NHS ester

IgG conjugate in Standard Phosphate Buffer at 37 * C at pH 6.3, 7.3 and 8.0.

Fig. .18 is a graph of the comparative stability data of DMC IgG conjugate in Standard Phosphate Buffer at 37 * C at pH 6.3, 7.3 and 8.0. Fig. 19 is a graph of the comparative stability data of DMN IgG conjugate (made with 20x mole excess label) in Standard Phosphate Buffer at 37 * C at pH 6.3.7.3 and 8.0.

Fig. 20 is a graph of the comparative stability data of DMN IgG conjugate (made with 5x mole excess label) in Standard Phosphate Buffer at 37 * C at pH 6.3. 7.3 and 8.0.

Fig. 21 is a graph of the comparative stability data of DMN, DMC and C2NHS ester IgG conjugates in azide buffer at pH 6.9 at 37'C.

Fig. 22 is a graph of the comparative stability data of DMN, DMC and C2NHS ester IgG conjugates in azide buffer at pH 7.0 at 37'C. Fig. 23 is a graph of the comparative stability data of DMN, DMC and

C2NHS ester IgG conjugates in azide buffer at pH 7.3 at 37'C.

Fig. 24 is a graph of the comparative stability data of DMN, DMC and C2NHS ester IgG conjugates in azide buffer at pH 8.0 at 37'C.

Fig. 25 is a graph of the comparative stability data of DMN, DMC and C2NHS ester IgG conjugates in azide buffer at pH 6.3 at 37'C.

Fig. 26 is a graph of the comparative stability data of DMN and C2NHS ester IgG conjugates in azide buffer at pH 5.9 at room temperature and 4 * C.

Fig. 27 is a graph of the comparative stability data of phenyl-, (2,6- dimethyDphenyl- and (2,6-dimethyl-4-nitro)phenyl- N-methyl-acridan esters in phosphate buffer at pH 6.3 at 35 * C.

Fig. 28 is a graph of phenyl-, (2,6-dimethyl)phenyl- and (2,6- dimethyl-4-nitro)phenyl- N-methyl-acridan esters in phosphate buffer at pH 6.3 at 45 * C. Fig. 29 is a graph of phenyl-, (2,6-dimethyl)ρhenyl- and (2,6- dimethyl-4-nitro)phenyl- N-methyl-acridan esters in phosphate b uffer at pH δ.O at 35 * C.

Fig. 30 is a graph of phenyl-, (2,6-dimethyi)phenyl- and (2,6- dimethyl-4-nitro)phenyl- N-methyl-acridan esters in phosphate buffer at pH δ.O at 45 * C.

Fig. 3 1 is a UV-Vis spectrum of (2,6 -dimethoxy-3 - chlorosulfonyl)phenyl-N-methyl-acridan-9-ethoxy-9-carboxylat e.

Fig. 32 is a fast atom bombardment mass spectrum of (2,6 - dimethoxy-3-chlorosulfonyl)phenyl-N-methyi-acridan-9-ethoxy- 9- carboxylate.

Fig. 33 is a 300 MHz proton NMR spectrum of (2.6-dimethoxy-3- chlorosulfonyl)phenyl-N-methyl-acridan-9-methoxy-9 -carboxylate.

Fig. 34 is a 300 MHz proton NMR spectrum of (2,6-dimethyi-3- chlorosulfonyl)phenyl-N-methyl-acridan-9-methoxy-9 -carboxylate. Fig. 35 is a graph of an assay of TSH standards using (2,6- dimethoxy-3-chlorosulfonyl)phenyl-N-methyl-acridan-9-ethoxy- 9- carboiylate as a label.

Fig. 36 is a graph of the comparative stability data of DMC and DME in bicarbonate buffer (0.1 M bicarbonate. 0.00025% Thimerosai, 0.1 % bovine serum albumin) at pH 9.6 at 25 * C.

Fig. 37 is a graph of the comparative stability data of DMC and DME TSH conjugates in TSH antibody diluent buffer ( l OOmM phosphate, 150mM NaCl, 0.001 % Thimerosai, 0.4% Bovine serum albumin, 0.1 mg/ml murine gamma globulin, 0.1 mg/ml goat gamma globulin) at pH 6.0 at 25 * C. Fig. 38 is a graph of the comparative stability data of DMC and DME

TSH conjugates in TSH antibody diluent bufferat pH 6.0 at 30'C.

Fig. 39 is a graph of the comparative stability data of DMC and DME TSH conjugates in TSH antibody diluent bufferat pH 6.0 at 35 * C.

Fig. 40 is a 300 MHz proton NMR spectrum of phenyl- 1 ,3- dimethyl- N-methyl-acridan-9-methoxy-9 -carboxylate.

DESCRIPTION OF THE PREFERRED AND ALTERNATIVE EMBODIMENTS The chemiluminescent moieties of the present invention have either of the two following schematic formulae:

I.

Within the schematic formulae, the dashed box labelled "L" contains the ester, thioester or amide "linkage" which is between two substituted rings or ring systems represented by the circle labelled "0" and the solid box labelled "R3". Whether the linkage L is an ester, thioester or amide is determined by R4 being -0-, -S- or -N(Sθ2CF3)-, respectively. Preferably, the linkage is an ester linkage.

0 is a heterocyclic ring or ring system to which the ester, thioester or amide linkage L is attached at a carbon atom within the heterocyclic ring or ring system which ( 1 ) is either sp 2 or sp3 hybridized, and (2) 'is susceptible to attack by peroxide or molecular oxygen to form an intermediate that decays to produce chemiiuminescence. Whether the carbon atom is rendered susceptible to such attack is determined by the oxidation state of the heteroatom within the heterocyclic ring or ring system. If the carbon to which the linkage is attached is sp 2 hybridized, the heteroatom must be in a positive oxidation state (i.e., have a positive charge; for example, as obtained by N-alkylation or N-oxidation). If the carbon to which the linkage is attached is sp3 hybridized, the heteroatom must be in a neutral oxidation state (i.e., uncharged). When the heteroatom is nitrogen, proper oxidation states can only be achieved if the nitrogen is sub stituted with an alkyl grou p (including a functionalized group), an aryl group including a functionalized group), - 0" (if the nitrogen is in a positive oxidation state) or -OH (if the nitrogen is in a neutral oxidation state). When the heteroatom is in these "proper" oxidation states, the carbon atom will be susceptible to attack by peroxide or molecular oxygen to produce the chemiluminescent intermediate. Heterocyclic rings or ring systems in which the heteroatom is in a positive oxidation state include without limitation acridiniu m, benzlalacridinium, benz[b] acridinium, benz.clacridinium, a 1 ,2,4-triazole cation, an isooxazole cation, an isothioazoie cation, a 1,2-azole cation, an imidazole cation, a benzimidazole cation, quinolinium, isoquinolinium. quinoliziniu m, a cyclic sub stituted quinolinium, pyridiniu m, pyrimidininium, pyridazinium, pyrazininium, phenanthridinium, and quinoxalinium. Rings or ring systems in which the heteroatom is in a neutral oxidation state include the reduced forms of the foregoing. These rings or ring systems are derived from the following rings or ring systems:

Aaαle Series

Acridine Series

1. 2, 4-Triaxola

Isoαxaxnl-s

Be-αx[a]acr--dix--9 -----jtl-iθB-EθI-9

1, 2-Axolβ

Bemfblacridiiiβ

R--m-r.ττ.ir-,-rτ---1,.

SUBSTITUTE SHEET

uino--ine Series

Q-^n-ιHτ.-->

Iao--ι iι-------------β

φτ-τ-r.l..H-----.ττ- f rHnm«

Cyclic C3, C4, C5-Substitut-sd Qu-txιαl-i-Q-53

PyriHirtff/ rmidine Scries isceUaneαus

fyrun--d------e

^rax-De-i

The heterocyclic ring or ring system may be free of optional substitutions not shown in the schematic formulae. Alternatively, the heterocyclic ring or ring system may be optionally substituted at any position, including the heteroatom, with groups not shown in the schematic formula. Such rings and ring systems, whether optionally substituted or free of such optional substitutions, are considered herein to be within the meaning of "heterocyclic ring or ring system."

Possible optional substitutions may be functional or non¬ functional. Functional optional substitutions include those for the purposes of producing peri-interactions around the linkage i, increasing the solubility of the moiety, increasing the chemiluminescent efficiency of the moiety and, preferably, attaching the moiety to protein and other material. Groups useful for attaching the moiety to protein and other material include but are not limited to optionally functionalized alkyl. alkenyl, alkynyi, alkylamino, oxoalkyl, thioalkyl or alkyloxocarbonyl chains (branched or unbranched) or optionally functionalized aryl groups. The aryl groups can be joined to the heterocyclic ring or ring system by an alkyl, alkenyl, alkynyi, alkylamino. oxoalkyl. thioalkyl or alkyloxocarbonyl chain. Other chains, in addition to th'ose listed, are well known in the art. Optional functionalities include without limitation any of the following:

- CO2R, where R- a hydrogen, alkyl or aryl Θ

C = NH 2 OR

, where R- a residue of a functional alcohol

- SO2CI - NCS

- N(CH3)(CH2)m c l. where m is greater than or equal to 1 - N3 and other photolabile functionalities

- NH2. or ions, sugars, polyamines and polyoxy-co m pounds (e.g., poiyoxyethyiene, poloxyb utylene, etc.). Other chains, groups and functionalities useful for attaching compounds of the present invention to protein are discussed in Ji, "Bifunctional Reagents," Meth. Enzymology 1 :580 ( 1983), which is incorporated herein by reference. Methods of joining such attaching groups to protein and other materials utilize both covalent bonding and weak chemical forces and are well known in the art. Peri substituents, which can cause peri-interactions, include any group which can cause steric hindrance with respect to the carbon to which the ester, thioester or amide linkage is attached and/or with respect to the carbon within the ester, thioester of amide linkage. Preferred peri substituents include short alkyl groups (e.g., methyl, ethyl) and aryl groups (e.g., phenyl). The peri substituents, if present, are located on carbon atoms within the heterocyclic ring or ring system which are "adjacent to" the carbon to which the ester, thioester or amide linkage is attached. Moieties can include more than one peri substituent. For example, peri substituents can be placed in the following positions: (a) in acridiniums and acridans: on Cl and C8;

(b) in phenanthridiniums and reduced phenanthridiniums: on C7; and

(c) in quinoliniums and reduced quinoliniums: on C3.

The aryl ring or ring system, represented by R3, includes at least one substituted six-member ring. The ester, amide or thioester linkage is directly attached to such six-member ring. R3 may include but is not limited to phenyl, naphthalene and anthracene, which have the following structures:

NaphthalffTiw

Phe-o -L

R3 may be substituted at any position including but not limited to the substitutions for Rl, R2 and R2' described below. A substitution, including Rl , R2 and R2', can also be used to attach the moiety to protein or other material. Appropriate groups for such purpose are discussed above. R2 and R2' are bulky groups which are located on R3 so as to sterically hinder the hydrolysis of the linkage L between R3 and the heterocyclic ring or ring system 0. Preferably, where R3 is a phenyl ring with the ester linkage being attached at position 1 , R2 and R2' are located at the 2 and 6 (ortho) positions. R2 and R2' may be identical or differ from one another, and either may include: an alkyl or optionally functionalized alkyl group an aryl or optionally functionalized aryl group

- OR, where R- alkyl or aryl

- NR2, where R- alkyl or aryl - NR3 + , where R- alkyl or aryl

- coo-

- COOH

- COOR, where R- alkyl or aryl

- COR, where R- alkyl or aryl - (CF2 ) n CF3 , where n-0 to 10

- CN

- NH 3 + - N02

- N(CH 3 ) 3 + - SR, where R- alkyl or aryl

- SR2 + , where R- alkyl or aryl

- SO2 , where R- alkyl or aryl a halogen.

The required steric hinderance can also be provided by other rings within a multi-ring R3 which are "adjacent" to the six-member ring to which the ester linkage is attached. For example, if R3- naphthalene and an ester linkage is attached at the 1 position, R2 could be a methyl group at the 2 position and R2" is the "adjacent" ring containing carbons 7- 10. In such cases, the adjacent ring is considered herein to be a substitution (on the six-member ring within R3) which sterically hinders the hydrolysis of the linkage.

R l may be any electron withdrawing group on R3, b ut is preferably selected from -NO2 , -SO2CI, -Br, -N(CH3 )3 + and -H. As used throughout this specification and the appended claims, an "electron withdrawing group" shall mean any group which has a sigma p value greater than or equal to 0.0. Tables of sigmap values for various groups can be found in Hansch et al, J. Med. Chem. 16( 1 1 ):1209- 12 13 ( 1977) and Hansch and Leo, "Substituent Constants for Correlation Analysis in Chemistry and Biology," Ch. 6, pp. 49-52 (John Wiley & Sons, New York 1979), both of which are incorporated herein by reference. In some moieties, Rl and one or both of R2 and R2'. are electron withdrawing groups (which may be identical or differ from one another). If more than one group among Rl , R2 and R2' are electron withdrawing groups, it is preferred that the additive sigma p value for only the groups which are electron withdrawing groups (i.e., excluding those groups with sigma p < 0.0) be less than or equal to about 1.0. Some moieties having an additive sigmap value greater than 1.0 are unstable.

The moieties of the present invention may also be attached to protein and other material through substitutions on R3. Some electron withdrawing groups can be used as a means of attachment. Of the preferred electron withdrawing groups, -SO2CI can be used to attach a moiety to protein or other material. Alternatively, any of the groups listed above for attachment to the heterocyclic ring or ring system 0 can also be used if they can be attached as substitutions to R3. Methods of joining such attaching groups to protein and other materials utilize both covalent bonding and weak chemical forces and are well known in the art.

In schematic formula II above, Z is a secondary substituent group attached to the carbon atom to which the ester, thioester or amide linkage is attached when such carbon atom is sp3 hybridized. Z may include but is not limited to:

- H a halogen - CN - OR - NR 2

- NR 3 + - SR

Generally, in addition to a hydrogen and a halogen. Z can be any nucleophilic group of the general formula R n X, where X- 0, N, S or C (with appropriate changes in n to accommodate valency changes in X) and where R- any substituent (preferably, alkyl, aryl, an amino acid or a sugar, optionally substituted) and where multiple R groups can optionally form cyclic structures (e.g., in the Z groups:

R and R n X can also be ( or be derived from) a drug or steroid molecule.

In compounds in which Z* R n X, Z serves as a "blocking" moiety which prevents or deters hydrolysis of the ester, thioester or amide linkage, thus decreasing the likelihood that such compounds will be unstable. The blocking effect is caused by ( 1 ) the steric bulk of the R n X group which physically blocks attack by chemical species which would induce or increase hydrolysis of the ester, thioester or amide linkage (e.g., species which would form a pseudo base at carbon 9 of an acridinium compound), and (2) electronic effects produced by the change of the carbon atom from sp 2 to sρ3 hybridization (which cause the ester, thioester or amide linkage to behave more like an aliphatic ester, thioester or amide). When compounds including an R n X group are triggered to produce chemiiuminescence they must first be treated with

acid which cleaves the R n X group, thus allowing induction of the chemiluminescent reaction upon the addition of peroiide, base or other triggering agent (acid treatment is not required for compounds where 1- -H or a halogen). The character and structure of the R n X group is limited by only two factors: ( 1 ) the size of the R n X group must be such that the group can be added to compounds of the present invention during synthesis as described below or by other synthetic procedures (i.e., the R n XH species, from which the R n X group derives during synthesis, cannot be so large that steric effects make it impossible to synthesize a compound of the present invention having such R n X group), and (2) the R n X group must be of a steric and chemical character such that it can be cleaved from the molecule with acid before triggering the chemiluminescent reaction.

Other moieties of the present invention have the following schematic formula:

III.

where 0, Z, L and R4 are as described above and R5 is any leaving group. In addition to the moieties wherein R5- R l+R2+R2'+R3 (as previously described as in schematic formula II), preferred cmoieties of the include moieties where R5- an aryl ring or ring system which is substituted or unsubstituted. These moieties can also include peri substituents as previously described. Phenyl-N-methyl- l ,3-dimethyl-acridan-9 - methoιy-9-carboιylate, which has the following formula:

is a preferred moiety which has an Z/RnX group (CH3O- on C9), has a peri substituent (CH3- on Cl) and is an unsubstituted phenyl ester.

Another group of moieties of thepresent invention have the following schematic formula:

IV.

where Sθ2~R"-Y is a leaving group, where 0, Z, L and R4 are as described above, where R" and R" are selected from the group consisting of alkyl. alkylene, aryl, optionally substituted alkyl. optionally substituted alkylene, optionally substituted aryl, alkyioxy, aryloxy, halo, optionally protected amino, substituted aminohydroxy, protected hydroxy, oxo, thio, imino, optionally substituted mercapt, a heterocyclic ring, and a heteroaikyl group, and where Y is selected from the group consisting of a hydrogen, carboxy, carbonyl halide, sulfonyl halide, carboalkoxy. carboxamido, carb oaryloxy, cyano, carboximido, isocyanato, isothiocyanate, sulfo, N-succinimidylcarboxy and N-maleimide. Such moieties can also include peri sybstituents as previously described.

Still another group of moieties of the present invention have the following schematic formula:

V.

where V-R'" is a leaving group, where 0. Z and L are as described above, where V is an aliphatic or aromatic group, where R"' is a group which is useful for attaching the moiety to protein or other specific binding partners (as previously described). Such moieties can also include peri substituents as previously described.

One further group of moieties of the present invention have the following schematic formula:

VI.

where 0, Z and L are as described above, where W is a leaving group and where SW is a sulfonamido or sulfocarbonyl group. Such moieties can also include peri substituents as previously described.

The above-described improved chemiluminescent moieties are useful in a broad range of specific binding assays for the presence of analyte in a sample. "Presence" shall mean herein the qualitative and/or quantitative detection of an analyte. Such assays may be directed at any

analyte which may be detected by use of the improved chemiluminescent moiety in conjunction with specific binding reactions. These assays include, without limitation, immunoassays, protein binding assays and nucleic acid hybridization assays. In a typical immunoassay, the analyte is immunoreactive and its pr -sence in a sample may be determined by virtue of its immunoreaction w Λi an assay reagent. In a typical protein binding assay, the presence of analyte in a sample is determined by the specific binding reactivity of the analyte with an assay reagent where the reactivity is other than immunoreactivity. Examples of this include enzyme-sub strate recognition and the binding affinity of avidin for biotin. In the typical nucleic acid hybridization assay, the presence of analyte in a sample is determined by a hybridization reaction of the analyte with an assay reagent. Analyte nucleic acid (usually present as double stranded DNA or RNA) is usually first converted to a single stranded form and immobilized onto a carrier (e.g., nitrocellulose paper). The analyte nucleic acid may alternatively be eiectrophoresed into a gel matrix. The immobilized analyte may then be hybridized (i.e., specifically bound) by a complementary sequence of nucleic acid. The foregoing specific binding assays may be performed in a wide variety of assay formats. These assay formats fall within two broad categories. In the first category, the assay utilizes a chemiluminescent conjugate which comprises the improved chemiluminescent moiety attached to a specific binding material. "Specific binding material" means herein any material which will bind specifically by an immunoreaction, protein binding reaction, nucleic acid hybridization reaction, and any other reaction in which the material reacts specifically with a restricted class of biological, biochemical or chemical species. In this category of assays, the chemiluminescent conjugate participates in a specific binding reaction and the presence of analyte in the sample is proportional to the formation of one or more specific binding reaction products containing the chemiluminescent conjugate. The assay is performed by allowing the requisite specific binding reactions to occur under suitable reaction conditions. The formation of specific binding reaction products containing the chemiluminescent conjugate is determined by measuring the chemiiuminescence of such products containing the chemiluminescent conjugate or by measuring the

chemiiuminescence of unreacted or partially reacted chemiluminescent conjugate not contained in such products.

This first category of assay formats is illustrated by sandwich assays, competitive assays, surface antigen assays, sequential saturation assays, competitive displacement assays and quenching assays.

In a sandwich format, the specific binding material to which the chemiluminescent moiety is attached, is capable of specifically binding with the analyte. The assay further utilizes a reactant which is capable of specifically binding with the analyte to form a reactant-analyte- chemiluminescent conjugate complex. The reactant may be attached to a solid phase, including without limitation, dip sticks, beads, tubes, paper or polymer sheets. In such cases, the presence of analyte in a sample will be proportional to the chemiiuminescence of the solid phase after the specific binding react. 3ns are completed. Such assay formats are discussed further in U.S Patent Nos. 4,652,533, 4.383,031 , 4.380,580 and 4,226,993, which are inorporated herein by reference.

In a competitive format, the assay utilizes a reactant which is capable of specifically binding with the analyte to form an analyte- reactant complex and w th the specific binding material, to which the chemiluminescent moiety is attached, to form a chemilu minescent conjugate-reactant complex. The reactant may be attached to a solid phase, or alternatively reaction products containing the reactant may be precipitated by use of a second antibody or by other known means. In this competitive format, the presence of analyte is "proportional," i.e., inversely proportional, to the chemiiuminescence of the solid phase or precipitate. A further discussion of this assay format may be found in the immediately above mentioned U.S. patents.

. In another assay format, the analyte may occur on or be bound to a larger biological, biochemical or chemical species. This type of format is illustrated by a surface antigen assay. In this format, the specific binding material is capable of specifically binding with the analyte and the presence of analyte is proportional to the anaiyte-chemiluminescent conjugate complex formed as a reaction product. This is illustrated by attaching the chemiluminescent moiety to an antibody which is specific to a surface antigen on a cell. The presence of the cell surface antigen will be indicated by the chemiiuminescence of the cells after the completion of the reaction. The cells themselves may be used in

conjunction with a filtration system to separate the analyte- chemiluminescent conjugate complex which is formed on the surface of the cell from unreacted chemiluminescent conjugate. This is discussed further in U.S. Patent No. 4,652,533. The improved chemiluminescent moiety may be used in additional assay formats known in the art including without limitation sequential saturation and competitive displacement, both of which utilize a chemiluminescent conjugate where both ( 1 ) the specific binding material, to which the moiety is attached, and (2) the analyte specifically bind with a reactant. In the case of sequential saturation, the analyte is reacted with the reactant first, followed by a reaction of the chemiluminescent conjugate with remaining unreacted reactant. In the case of competitive displacement, the chemiluminescent conjugate competitively displaces analyte which has already bound to the reactant. In a quenching format, the assay utilizes a reactant which is capable of specifically binding with the analyte to form an analyte - reactant complex and with the specific binding material, to whic the chemiluminescent moiety is attached, to form a chemiluminescent conjugate-reactant complex. A quenching moiety is attached to the reactant. When brought into close proximity to the chemiluminescent moiety, the quenching moiety reduces or quenches the chemiiuminescence of the chemiluminescent moiety. In this quenching format, the presence of analyte is proportional to the chemiiuminescence of the chemiluminescent moiety. A further discussion of this format may be found in U.S. Patent Nos. 4,220,450 and 4,277.437, which are incorporated herein by reference.

In consideration of the above discussed assay formats, and in the formats to be discussed below, the order in which assay reagents are added and reacted may vary widely as is well known in the art. For example, in a sandwich assay, the reactant bound to a solid phase may be reacted with an analyte contained in a sample and after this reaction the solid phase containing complexed analyte may be separated from the remaining sample. After this separation step, the chemiluminescent conjugate may be reacted with .the complex on the solid phase. Alternatively, the solid phase, sample and chemiluminescent conjugate may be added together simultaneously and reacted prior to separation. As a still further but less preferred alternative, the analyte in the sample

and the chemiluminescent conjugate may be reacted prior to addition of the reactant on the solid phase. Similar variations in the mixing and reaction steps are possible for competitive assay formats as well as other formats known in the art. "Allowing under suitable conditions substantial formation" of specific binding reaction products shall herein include the many different variations on the order of addition and reaction of assay reagents.

In the second category of assay formats, the assay utilizes an unconjugated improved chemiluminescent moiety. The presence of analyte in the sample is proportional to the formation of one or more specific binding reaction products which do not themselves contain the chemiluminescent moiety. Instead, the chemiluminescent moiety chemiluminesces in proportion to the formation of such reaction products. . In one example of this second category of assays, the assay utilizes a reactant capable of binding with the analyte to form an analyte- reactant complex which causes the chemiluminescent moiety to chemiluminesce. This is illustrated by a simple enzyme-substrate assay in which the analyte is the substrate glucpse and the reactant is the enzyme glucose oxidase. Formation of the enzyme-substrate complex triggers the chemiluminescent moiety. Such enzyme-substrate assay for glucose is disclosed in U.S. Patent Nos. 3,964,870 and 4,427,770, both of which are incorporated herein by reference. This enryme-substrate assay is a specific binding assay in the sense that the substrate specifically binds to the active site of the enzyme in much the same way that an antigen binds to an antibody. In this assay, the enzyme specifically binds with the substrate which results in the production of peroxide which, in turn, causes the chemiluminescent moiety to chemiluminesce.

Also included in the second category of assays are those assays in which the formation of the reaction products promotes or inhibits chemiiuminescence by the chemiluminescent moiety in a less direct manner. In this assay, a first reactant, which is cross reactive with the analyte, is attached to an enzyme such as glucose oxidase close to its active site. A second reactant which is specific for both the analyte and the immunoreactive material is added to the sample and the altered enzyme in the presence of the substrate (i.e., glucose). When the second reactant binds to the first reactant located near the active site on the enzyme, the

second reactant blocks the active site in a way that the substrate cannot bind to the enzyme at the active site or the binding of the substrate at the active site is significantly decreased. The second reactant blocking the enzyme in this manner inhibits the enzyme from producing peroxide which, in turn, would have triggered the chemiluminescent moiety. Analyte in the sample, however, will tie up the second reactant, thus preventing the second reactant from inhibiting the production of peroxide. The presence of analyte will be proportional to the chemiiuminescence of the moiety. The assays contained in the above two categories of assay formats may be heterogeneous or homogeneous. In heterogeneous assays, the reaction products, whose formation is proportional to the presence of analyte in the sample, are separated from other products of the reaction. Separation can be achieved by any means, including without limitation, separation of a liquid phase from a solid phase by filtration, microfiltration, double antibody precipitation, centrifugation, size exclusion chromatography, removal of a solid phase (e.g., a dip stick) from a sample solution or electrophoresis. For example, in a sandwich assay the reactant-analyte-chemiluminescent conjugate complex is separated from unreacted chemiluminescent conjugate. In a surface antigen assay, the analyte-chemiluminescent conjugate complex is separated form unreacted chemiluminescent conjugate. In a competitive assay, the reactant-chemiluminescent conjugate complex is separated from unreacted chemiluminescent conjugate. In a sequential saturation assay and in a competitive displacement assay, the reactant- chemiluminescent conjugate complex is separated from unreacted chemiluminescent conjugate. Alternatively, in homogeneous assays the reaction products are not separated. After the assay reagents have been allowed to react, the chemiiuminescence may be measured from the whole assay mixture whether such miiture is in solution , on a solid phase or distributed between various membrane layers of a dip stick or other solid support. The glucose assay using glucose oxidase and a chemiluminescent moiety illustrates a simple homogeneous assay in which separation is unnecessary. The quenching assay illustrates a more complex homogeneous assay in which separation is unnecessary. It is contemplated that either category of assay formats may give rise to either heterogeneous or homogeneous formats.

Finally, "measuring the chemiiuminescence" shall include, where relevant, the act of separating those specific binding reaction products, the formation of which are proportional to the presence of analyte in the sample, from other reaction products. It shall also include, where relevant, the acts of (i) treating the chemiluminescent moiety with acid to cleave a Z (i.e., R n X) group from the moiety, and/or (ii) triggering the chemiluminescent moiety to chemiluminesce in the case of those assay formats in which the formation of the reaction products does not itself trigger the chemiluminescent moiety.

SYNTHESIS OF MOIETIES The following examples show the synthesis of certain chemiluminescent moieties of the present invention.

E∑ample 1

A preferred chemiluminescnet moiety of the present invention is (2,6-dimethyl-4-nitro)phenyl-3-(3-succinimidyl-oxycarbonyi) propyloxy-N-methyl-acridinium-9-carboxylate fluorosulfonate (or other counter ions such as chloride, trifluoroacetate, sulfate, etc.) which has the following formula:

( 2 ,6 -dimethyl-4-nitro) phenyl-3 -( 3 -succinimidyl-oxycar onyl) propyloιy-N-methyl-acridiniu m -9-carboxyiate fluorosulfonate is synthesized according to the following scheme:

CONTINUED NEXT PAGE

SUBSTITUTE SHEET

(10)

SUBSTITUTE SHEET

Condensation of isatin ( 1 ) with resorcinol (2) provides the 3-hydroxy acridine-9-carboιyiic acid (3). Reaction with benzyl-4-bromo butyrate gives the ester (4) with the 3-hydroxy group etherified. Hydrolysis using base removes both the benzyl groups resulting in the dicarboxyiic acid (5). Selective rebenzylation of the carboxylic function of the propylo∑y group gives 9-carboιylic acid (6). Esterification with 2,6-dimethyl-4- nitrophenol and methylation of the acridine nitrogen gives (8 ). Deprotection of the carboxyl group with HBr and condensation with N- hydroxysuccinimide using DCC provides (2,6-dimethyl-4-nitro) phenyl-3- (3 -succinimidyl-oxycarbonyl) propyloxy-N- ethyl- acridiniu m -9 - carboiylate fluorosulfonate. These reactions are described in further detail in the following.

4-bromobutyryi chloride ( 13.8 g, 75 mmole) was placed in a 100 ml round bottom flask. The flask was cooled to -20 * C using a dry ice/ carbon tetrachloride bath. Ethyl acetate (50 ml) containing N-methylmorpholine (7.58 g, 75 mmole, 8.2 ml) was added carefully. Using an addition funnel, benzyl alcohol (6.97 g, 6.67 ml, 6.64 mmoles) was added dropwise. After the addition the bath was removed and the mixture was stirred for 2 hours. The product was transferred to a separatory funnel using ethyl acetate (50 ml), washed once with sodium bicarbonate ( 10%), then twice with water, and dried with anhydrous sodium sulfate. Evaporation of solvents gave benzyl-4-bromo-butyrate as an oil (yield - 91%).

Isatin ( 1) ( 1.88 g) was slowly added to a solution of potassium hydroxide (5.07 g. 0.09 mole) dissolved in water (3.5 ml). The reaction flask was heated to about 50 * C in an oil bath. About 10 ml more water was added dropwise. Resorcinai (2) ( 10 g, 0.089 mole) was added and the temperature was raised to 100 * C as stirring was continued, resulting in the formation of a molten mixture. More isatin ( 1 ) ( 1.88 g) was added. The reaction flask (3-necked round bottom) was attached to a nitrogen inlet and the water vapors were flushed out by the stream of nitrogen. The mixture was stirred for 4 hours at 125 * C. Water (70 ml) was added and the contents were dissolved by continued stirring. After transferring the mixture to an erlenmeyer flask the volume was brought up to 200 mi with water. The pH was adjusted to 2.0 with concentrated hydrochloric acid. Filtration and washing of the solids with water gave the crude acridine acid. It was then dissolved in 2N NaOH ( 100 ml) and the solution was filtered through celite. The celite bed was washed with 200 mi of 1 N NaOH.

The filtrate was acidified with concentrated HC1 to pH 2.0 . The precipitate of 2-hydroxy-acridine-9-carboxyiic acid (3) was filtered, washed with water and was dried in vacuum over P2O5 (yield - 42%).

3 -hydroxy-9 -acridine carboxylic acid (3) (4 g, 0.017 mole), benzyl- 4-bromobutyrate ( 14.6 g, 0.057 mole) and cesium carbonate (22.16 g, 0.068 mole) were dissolved in DMSO ( 125 ml) in a 250 ml round bottom flask. The flask was warmed to about 50 * C in an oil bath. After stirring the mixture at that temperature for 1 hour, the mixture was poured into water ( 1 liter). The precipitated product was extracted with chloroform after making the aqueous suspension basic with sodium bicarbonate. Drying and evaporation of chloroform gave 3-(3-benzyloxycarbonyl)-ρropyloxy- 9-(3-benzyloxy-carbonyl-propyi) acridine carboxylate (4) which was chromatographed on a silica gel column using chloroform as solvent. Fractions with Rf value of 0.36 on TLC with CHC^/EtOAc, 9/ 1, were pooled. The solvents were evaporated (yield - 55%).

3-(3-benzyioxycarbonyl)-propyioxy-9-(3-benzyloxycarbonyl propyi) acridine carborylate (4) (4.93 g, 8.3 mmole) was added to a mixture of 2N NaOH (300 ml) and methanol (300 ml). The mixture was stirred at room temperature for 48 hours. The methanol was removed on a rotary evaporator and the solution was acidified with concentrated hydrochloric acid to pH 6.0. The precipitated solids were filtered, washed with water and dissolved in ethyl acetate. The solution was dried and then the solvents were evaporated to give 3-(3-carboxy) ρroρyloxy-acridine-9- carboxylic acid (5) (yield - 92.8%). 3-(3-carboxy) propyloxy-acridine-9 -carboxylic acid (5) ( 1.5 g, 4.6 mmole) was dissolved in DMAP (80 ml. l ,3-dimethyl-3.4,5,6-tetrahydro- 2(IH) pyrimidone) with warming. Benzyl alcohol (0.5 ml, 0.52 g, 4.8 mmole), 1 ,3-dicycloheιylcarbodiimide ( 1.04 g, 5.0 mmole) and N.N- dimethyi aminopyridine (0.2 g, 1.6 mmole) were added to the reaction which was previously cooled in a bath of dry ice/CCi^ The mixture was stirred for 15 hours with a nitrogen inlet at room temperature. The miiture was added to saturated sodium chloride (320 ml). 3-(3- benzyloxycarbonyl) ρropyloxy-acridine-9-carboxylic acid (6) was filtered and was washed with a small amount of water. The product was chromatographed on a silica gel column using CHC^ /MeOH, 95/5 as solvent (yield - 26%).

3-(3-benzyloxycarbonyl) propyioxy-acridihe-9-carboxylic acid (6) (0.5 g, 1.2 mmole) and p-toluene sulfonyl chloride (0.46 g, 2.4 mmole) were dissolved in pyridine (20 ml). The solution was cooled in a bath of dry ice/CCl4 for 15 minutes. 2,6-dimethyl-4-nitrophenol (0.2 gm, 1.2 mmole) was added and the cooling bath was removed and the mixture was stirred for 15 hours at room temperature. It was added to water (450 ml) and the pH was adjusted to 2.0 with concentrated hydrochloric acid. The product was filtered, washed with water and was dried in vacuum. The crude product was chromatographed on a silica gel column using chloroform as solvent. Fractions with an Rf value of 0.8 on TLC with CHCI3 /EtOAc, 1:1 , were pooled. Evaporation of solvents gave (2,6- dimethyl-4- nitro) phenyl-3-(3-benzyloxycarbonyl)-propyloxy-acridine- 9-carboxyiate (7) (yield - 47%).

The acridine (7) (0.32 g. 0.56 mmole) was dissolved in anhydrous methylene chloride (4 ml) and methyl fluorosulfate (0.27 ml, 3.36 mmole, 6 molar equivalent) was added. The mixture was stirred for 15 hours at room temperature. Anhydrous ether (20 ml) was added. (2,6-dimethyl-4- nitro)phenyl-3 -( 3 -benzyioxycarbonyl) propyloxy-acridiniu m-9 - carboxylate fluorosulfonate (8) was filtered and washed with ether (50 mi). The yield was quantitative.

The benzyl- rotected acridinium ester (8) (250 ng) was treated with 30% HBr/Acetic acid (3 ml) for 2 hours at 55 * C. Anhydrous ether (20 mi) was added to precipitate the product. Filtration and washing of the solids with ether gave (2,6-dimethyl-4-nitro)phenyl-3-(3-carboxyl) propyioxy-acridinium-9-carboxylate fluorosulfonate (9). Crystallization from acetonitrile provided the pure compound (yield - 80%).

The deprotected acridinium (9) (67 mg, 0.13 mmole) in a 50 ml 2- necked round bottom flask was dissolved in anhydrous acetonitrile ( 10 ml). Dicyclohexylcarbodiimide (DCC, 33 mg, 0.16 mmole) was added and the mixture stirred for 45 minutes at room temperature. N- hydroxysuccinimide ( 17 mg, 0.15 mmole) was added and reaction continued for 2.5 hours. More DCC ( 14 mg) and N-hydroxysuccinimide (8 mg) were added and followed again by the same amounts after 1.5 hours. After 1.5 hours after the last addition, glacial acetic acid ( 1.7 1) was added to quench excess DCC. The solvent was removed In vacuo.

The crude product was purified on a semi-preparative C j g -

Dynamax HPLC column (commercially available from Rainin Instrument

Co., Inc., Woburn, Massachusetts) using CH3CN/H2O (0.1 % Trifluoroacetic acid) 60/40, as the mobile phase at a flow rate of 1.8 ml/min, using 360 nm for detection. The fraction at retention time 9.4 minutes was collected and the solvents were removed in vacuo. The (2,6-dimethyl-4-nitro)- phenyl-3 -(3 -succinimidyl-oxycarbonyl) propyloxy-acridiniu m-9 - carboxylate fluorosulfonate ( 10) was dried under vacuum in a dessicator containing phosphorus pentoxide (yield - 33 %). MS: FAB, thioglycerol matrix, 586 (M + ). HPLC: Rainin Ci s Dynamax ( 10 mm x 25 mm). CH3CN/H2O (0.1 % trifluoroacetic acid), 60:40, flow rate 1.8 mi/ min, retention time 9.4 min, detected at 360 nm.

Eiample 2 Another preferred moiety of the present invention is (2,6- dimethoxy-3-chlorosulfonyl)phenyl-N-methyl-acridinium-9-carb oιylate fluorosulfonate which has the following formula:

(2,6-dimethoxy-3-chlorosulfonyl)phenyl-N-methyl-acridiniu m-9- carboxylate fluorosulfonate is synthesized according to the following scheme:

(11)

Esterification of acridine-9-carboxylic acid ( 1 1 ) with 2 ,6 - dimethoxy phenol ( 12 ) via the acid chloride provides the ester ( 13 ). Methylation of the acridine nitrogen with methylfluorosulfate and subsequent chlorosulfonation with chloro sulfonic acid gives the label ( 15). These reactions are described in further detail in the following.

Acridine-9-carboxyiic acid ( 1 1 ) (6.10 g, 0.027 moles) in a 250 ml round bottom flask was mixed with thionyl chloride ( 130 ml) and the mixture was refluied for 2 hours with stirring. The excess thionyl chloride was removed in a rotary evaporator. The residue was treated with benzene (75 mi) and the solvent was removed in vacuo to remove traces of thionyl chloride. The residue of acridine-9-carbonyl chloride ( 1 1 ) was miied with pyridine ( 130 ml) and 2,6-dimethoxyphenol ( 12) (4.16 g. 0.027 moles) was added. The mixture was warmed using a water bath (about 60 * C) to dissolve all the solids. After 15 hours of stirring at room temperature the mixture was poured into 1 liter of water. The suspension was acidified with concentrated hydrochloric acid to pH 2.0. The solid product was filtered, washed with water and dissolved in chloroform. Drying (anhydrous sodium sulfate) and evaporation of chloroform gave (2,6- dimethoιy)ρhenyl-acridine-9 -carb oxylate ( 1 3 ). This was chromatographed on a silica gel column using CHCI3 /EtOAc, 98:2 as solvent. The fractions with Rf value of 0.1 on TLC with the same solvent were pooled and evaporation of the solvents gave the pure ester ( 13) (yield - 30%).

(2,6-dimethoxy)phenyi-acridine-9-carboxylate ( 13) (2.0 1 g, 5.6 mmole) was dissolved in dichloromethane ( 1 10 ml, anhydrous) in a 250 ml round bottom flask. Methyl fluorosulfate (4.60 ml, 6.48 g, 56 mmoles) was added and the mixture was stirred at room temperature for 15 hours. Anhydrous ether ( 100 ml) was added and the precipitated bright yellow solids were filtered after stirring the suspension for 0.5 hours. The solid was washed well with ether (about 100 ml) and then with pentane (50 ml). The acridinium was recrystallized from acetonitrile to provide pure 2,6- dim ethoxy- phenyl- acridinium -9 -carboxylate fluorosulfonate ( 14) (yield - 8 1 % ).

In a dry two neck 25 ml round bottom flask were placed the (2,6- dimethoxy)ρhenyl- 10-methyi acridinium-9-carboιylate fluoro-sulfonate ( 14) ( 101.7 mg, 0.215 mmole), a magnetic stirring bar and anhydrous CH 2CI2 (5 ml). The suspension was stirred and cooled to -20 * C in a

CCl4/dry ice bath. Chlorosulfonic acid (72 1, 0.125 g, 1.07 mmole) was added and stirring continued at -20 * C for 30 minutes. The reaction mixture was then allowed to warm slowly to room temperature and stirred for an additional 2 hours. Anhydrous ether (5 ml) was added to the reaction flask causing the formation of a light yellow precipitate. It was filtered and washed thoroughly with ether. Drying under vacuum gave ( 2,6-dimethoxy-3-chlorosulfonyl)phenyl acridiniu m - -carboxylate fluoros u lfonate ( 1 5 ) (yield - 93.4 % ). MS : F AB , dithiothreitol/dithioerythrytol matrix, 472 (M + ).

Eτample 3 Another preferred moiety of the present invention is (2,6- dimethyl-4-bromo)phenyl-N-methyl-acridinium-9-carboxylate fluorosulfonate which has the following formula:

9-(2,6-dimethyl-4-bromo)phenyl-N-methyl-acridinium-9- carboxylate fluorosulfonate was synthesized by the same esterification procedure as (2,6-dimethoxy-3 -chlorosulfonyl)phenyl-N-methyi- acridinium- -carboxylate fluorosulfonate, with the substiution of 2,6- dimethyl-4-bromo-phenol for the substituted phenol employed in the (2,6-dimethoxy-3-chlorosulfonyl)phenyl-N-methyi-acridinium-9 - carboiyiate fluorosulfonate synthesis.

Fτamole 4

Another preferred moiety of the present invention is (2,6 dimethyl-3-chlorosulfonyi)phenyl-N-methyl-acridinium-9-carbo xylate fluorosulfonate which has the following formula:

(2,6-dimethyl-3-chlorosulfonyl)phenyl-N-methyl-acridinium -9- carboxylate fluorosulfonate was synthesized by the same method as (2,6- dimethoxy-3-chlorosulfonyl)ρhenyl-N-methyi-acridinium-9 -carboxylate fluorosulfonate with the substitution of 2,6-dimethylphenol for 2,6- dimethoxy phenol in the esterification step.

Exampl 5. Another moiety of the present invention is (2,6-dimethyl-4- nitro)phenyl-3-(3-succinimidyloxycarbonyl)propyioxy-9, 10-dihydro-N- methyl-acridan-9-carboxylate which has the following formula:

(2,6-dimethyl-4-nitro)phenyi-3-(3-succinimidyloxycarbonyl)pr opyloxy- 9,10-dihydro-N-methyl-acridan-9-carboxylate was synthesized from the acridinium acid (9). Reduction of the acid (9 ) with sodium cyanoborohydride gives the acridan which is then converted to the NHS

ester by the mixed anhydride method. These reactions are described in further detail in the following.

The acridinium acid (9) (210 mg, 0.37 mmole) was dissolved in a 1: 1 mixture of acetonitrile and 0.1M phosphate buffer, pH 5.2 (60 ml). A solution of sodium cyanoborohydride ( 190 mg) in acetonitrile (5 mi) was added dropwise to the acridinium solution. This results in the bleaching of the yellow color of the solution. Stirring was continued for 15 minutes. Acetonitrile ( 100 ml) was added and the solvents were removed in a rotary evaporator. The residue as a suspension in water is extracted with ethylacetate. The organic layer was washed with water and dried. Removal of solvents gave ( 2,6-dimethyl-4-nitro ) phenyl-3 -(3 - carboxyl)propyloxy-9,10-dihydro-acridan-9-carboiylate (yield « 90%).

The acridan acid ( 125 mg, 0.255 mmole) and N-methylmorpholine

(28 1) were dissolved in anhydrous acetonitrile ( 15 ml). The mixture was cooled in a CC j /dry ice bath under nitrogen. Isobutylchloroformate (35

1) was added, the mixture was stirred for 3 minutes and N- hydroxysuccinimide (35 mg) dissolved in acetonitrile (2 ml) was added. After stirring at -20" C for 15 minutes the CCl-j/dry ice bath was removed and the reaction allowed to warm up to room temperature. After 2 hours the solvents were evaporated and the residue extracted into ethyl acetate. The insoluble N-methyimorpholine hydrochloride salt was removed by filtration. The filtrate was concentrated and hexane (20 ml) was added. Cooling results in crystallization of (2,6-dimethyl-4-nitro)phenyi-3-(3- succinimidyloιy-carbonyl)ρropyloxy-9,10-dihydro-N-methyl-a cridan-9- carboxylate. The crystals were finally filtered and washed with hexane (yield - 70%). MS: FAB, dithiothreitol/dithioerythrytol matrix. 588 (M + + 1). HPLC: Waters Ci8 Novapak (3.9 mm x 15 mm) (commercially available from Millipore Corporation, Waters Chromatography Division, Milford, Massachusetts), CH3CN/H2O (0.1 % trifluoracetic acid) 60:40, flow rate 1.0 ml/min, retention time 6.34 min, detected at 280 nm.

- Example 6 Another moiety of the present invention is (2,6 -dimethyl-4- nitro)phenyl-N-methyi-acridinium-9-carboxylate-3-oxo-butyrim idate chloride, hydrochloride which has the following formula:

(2,6-dimethyl-4-nitro)phenyi-N-methyl-acridinium-9-carbox ylate-3 -oxo- butyrimidate chloride, hydrochloride is synthesized according to the following scheme:

SUBSTITUTE SHEET

Reaction of 3 -hydroxy-9-acridine carboxylic acid ( 1 6 ) with 4- bromobutyronitrile gives an ester ( 17). Hydrolysis of the ester and reesterification with 2,6-dimethyl-4-nitrophenol provides ( 1 9 ). Methyiation with methyl fluorosulfate and conversion of the cyano group to the imidate ester using hydrogen chloride gas and methanol provides (2,6 -dimethyl- 4-nitro)phenyl-N- methyl-acridiniu m - 9 - carboxylate-3 -oxo-butyrimidate chloride, hydrochloride (2 1 ). These reaction are described in further detail in the following.

3-Hydroxy-9-acridine carboxylic acid ( 16 ) (2 g, 8.4 mmole), 4- bromobutyronitrile (5.87 ml, 8.74 g, 34 mmole) and cesium carbonate ( 1 1.08 g, 34 mmole) were dissolved in anhydrous DMSO (50 ml) in a 100 ml round bottom flask. The mixture was warmed to about 50 * C in a water bath with stirring. After 3 hours the mixture was poured into water (600 ml). The solids were filtered and were dissolved in chloroform. Drying and evaporation of the solvent gave 3-(3-cyaήo)propoxyl-acridine-9- carboiyiic acid-(3-cyano)propyl ester ( 17). It was then dissolved in toluene (50 ml) and cyclohexane ( 150 mi) was added. The pure product ( 17) separated, and was then filtered and dried. The dried product was purified by thin layer chromatography with ethylacetate as the mobile phase (R f - 0.58) (yield - 78.6%).

The cyanopfopyl ester ( 17) (3.73 g. 10 mmole) was dissolved in a mixture of 0.5N NaOH (90 ml) and methanol (90 mi) and stirred in a water bath at 60" C using a reflex condenser for 2.5 hours. The methanol was removed in a rotary evaporator and the product was extracted with ethyl acetate after acidifying the aqueous phase with concentrated hydrochloric acid. Drying and evaporation of the solvent provides 3-(3 - cyano) propoxyl-acridine-9-carboxyiic acid ( 18) (yield - 80%).

The carboxylic acid ( 18) (4.62 g, 15 mmole) was dissolved in pyridine ( 130 ml) and the solution was cooled in a CCl-j/dry ice bath, p- toluenesulfonyl chloride (5.8 g. 30 mmole) was added and the bath was removed. After 15 minutes of stirring at room temperature, 2,6-dimethyl- 4-nitrophenol (2.8 g, 16.8 mmole) was added. After 18 hours at room temperature, water ( 10 ml) was added and the solvents were removed in vacuo. The residue was dissolved in chloroform (200 ml) and the organic layer was washed with saturated sodium bicarbonate (2 x 100 ml), water (2 i 100 ml), 1 N HC1 ( 1 x 100 ml) and finally with water (2 x 100 ml). Drying and evaporation of the solvent gave (2,6-dimethyl-4-nitro)phenyi-3- ( 3 -

cyano) propoxyl-acridine-9 -carb oxylate ( 1 9 ) which was chromatographed in a silica gel column using ethylacetate/hexane (7:3) as solvent (yield = 74.5%).

The ester (19) ( 1.6 g, 3.52 mmole) was dissolved in dry methylene chloride (50 ml) and under nitrogen methyl fluorosulfate ( 1.6 ml, 17.6 mmole) was added. The solution was stirred at room temperature for 20 hours. Anhydrous ether ( 100 ml) was added and the precipitated (2,6- dimethyl- 4-nitro)phenyl-3 -( 3 -cyano) prop o∑yi- acridiniu m -9 - carboxylate fluorosulfonate (20) was filtered, washed with ether and dried in vacuo (yield - 84.7%).

The acridinium ester (20) (4 mg. 7.4 x 10"3 mmoie) was dissolved in methanol (0.5 ml) in a 5 ml 2-necked flask. Anhydrous hydrogen chloride gas was bubbled carefully for 10 minutes. Anhydrous ether (3 ml) was added. The precipitated (2,6-dimethyl-4-nitro)phenyl-N-methyl- acridinium-9-carboxylate-3-oxo-butyrimidate chloride, hydrochloride (21) was collected and washed with ether. The solid was dried in vacuum and was stored in a dessicator containing phosphorus pentoxide.

Example 7 Another moiety of the present invention is (2,6-dimethyl-4- nitro)phenyl-N- methyl- phenanthridinium-6 -carboxylate fluorosulfonate which has. the following formula:

(2,6-dimethyl-4-nitro)phenyl-N-methyl-phenanthridinium-6- carboxylate fluorosulfonate is synthesized according to the following scheme:

(27)

SUBSTITUTE SHEET

2-aminobiphenyl ( 16.9 g, 0.1 mol) was dissolved in anhydrous pyridine (30 ml) and acetic anhydride (10,5 ml, 0.1 1 mol) was added. The solution was shaken briefly and cooled to room temperature and let stand for 15 hours. After the addition of water (50 ml) N-acetyl-2- aminobiphenyl (22) was filtered off and recrystallized from, aqueous ethanol to give 19.6 g of white needles (yield - 93%).

N-acetyl-2 -aminobiphenyl (22) ( 19 g, 0.09 mol) was gently refluxed with freshly distilled phosphoryl chloride (45 ml, 0.49 mol) for 80 minutes. The solution was then cooled in ice and the precipitate (6- methyiphenanthridine-hydrochloride) was filtered off, dissolved in water and made alkaline with aqueous ammonia. The solution was then extracted with ether (4 x 75 ml). The extract was dried over sodium sulfate and the ether was removed in vacuo. The resulting yellow oil was dissolved in boiling cyclohexane (400 ml) and on cooling formed white needles of 6-methylphenanthridine (23) (yield - 63%).

6-(2-hydroxy- l -hydroxymethylethyl)-phenanthridine (24) was prepared by treating 6-methylphenanthridine (23) with formaldehyde according to the method of Morgan and Walls, J. Chem. Soc. 34:2447 ( 1931 ). which is incorporated herein by reference. 6-(2-hydroxy- l - hydroxymethyiethyD-phenanthridine (24) formed as white needles (yield = 57%).

A mixture of 6 -(2-hydroxy-l -hydroxy methyl)- phenanthridine (24) (6 g, 31 mmoles) and finely powdered selenium dioiide (3.8 g, 34 mmoles) was refluxed in ethyl acetate ( 125 mi) for 10 hours. The deep red solution was then filtered while hot through celite, before evaporating to dryness. The resulting solid was digested in warm 1M hydrochloric acid ( 125 ml), filtered and partially neutralized with sodium bicarbonate. The initial red precipitate was filtered off before completely neutralizing the solution. The resulting pale yellow solid was filtered off and recrystallized from acetone/pet. ether to give 2.7 g of 6-formylphenanthridine (yield = 42%). 6 -carboxy phenanthridine (25) was prepared by chromic acid oxidation of 6-formylphenanthridine according to the method of Morgan and Walls, J. Chem. Soc. 34:2447 ( 193 1 ), which is incorporated herein by reference The product (25) formed as a white powder (yield - 60%). 6-carboxyρhenanthridine (25) (662 mg, 3 mmoles) was dissolved in anhydrous pyridine ( 14 ml) and cooled to O 'C. p-toluene-sulfonyl chloride ( 1.15 g, 6 mmoles) was added followed by 2,6-dimethyl-4-nitro-

phenol (50 1 mg, 3 mmoles) and the mixture was allowed to stand overnight at 4 * C. The resulting brown solution was stirred into iced water. The precipitate was filtered off and was chromatographed on a silica gel using chloroform/hexane ( 1 : 1 ) to obtain (2,6 -dimethyl-4- nitro)phenyl-phenanthridine-6-carboxylate (26 ) (yield = 60 %).

In a dry two neck 25 ml round bottom flask the ester (26) (369 mg, 1 mmole) was suspended in anhydrous methylene chloride (5 ml). The suspension was cooled in a dry ice/CCl4 bath under nitrogen. Chlorosulfonic acid (342 1, 6 mmole) was added and stirring continued at -20 * C for 30 minutes. The mixture was then allowed to warm slowly to room temperature and stirred for an additional 2 hours. Anhydrous ether (20 ml) was added and the precipitated solids were filtered and washed with ether. Drying gave (2,6 -dimethyi-4-nitro)phenyl-N- methyl- phenanthridinium-6-carboxylate fluorosulfonate (27) (yield - 90%).

Eiamole g Another moiety of the present invention is (2,6-dimethyi-4- nitro)phenyl-5,6-dihydro-N-methyl-phenanthridinium-6-carboxy late fluorosulfonate which has the following formula:

(2,6-dimethyl-4-nitro)phenyl-5,6-dihydro-N-methyl- phenanthridinium-6-carboxylate fluorosulfonate is synthesized from the unreduced phenanthridinium analog (27). The phenanthridinium (27) (398 mg, 0.8 mmole) was dissolved in a 1:1 miiture of acetonitrile and 0.1 M phosphate b uffer, pH 5.2 (80 ml). A solution of sodiu m cyanoborohydride (410 mg) in acetonitrile ( 10 ml) was added dropwise to the phenanthridinium solution. This resulted in the bleaching of the yellow color of the solution. Stirring was continued for 15 minutes.

Acetonitrile ( 100 ml) was added and the solvents were removed in a rotary evaporator. The residue was suspended in water and eitracted with ethylacetate. The organic layer was washed with water and dried. Removal of solvents gave (2,6-dimethyl-4-nitro)phenyl-5,6-dihydro-N- methyi-phenanthridinium-6-carboxyiate fluorosulfonate (yield = 90%).

Eτamole 9 Another moiety of the present invention is (2,6-dimethoxy-3 - chlorosulfonyl-phenyl)-2-phenyl-N-methyl-quinoiinium-4-carbo xyiate fluorosulfonate which has the following formula:

(2,6-dimethoxy-3-chlorosulfonyl-ρhenyl)-2-phenyl-N-methy i- quinolinium-4-carboxylate fluorosulfonate is synthesized according to the following scheme:

SUBSTITUTE SHEET

Acetophenone (29) ( 120 g, 1 mol) and isatin (30) ( 147 g, 1 mol) were refluxed for 10 hours in water and ethanol, with potassium hydroxide (17 g). 2-phenyl-quinoiine-4-carboxylic acid (3 1) was recovered from ethanol as white needles (yield 84%). 2-phenyl-quinoline carboxylic acid (3 D (735 mg, 3 mmoles) was dissolved in anhydrous pyridine ( 14 ml) and cooled in an icewater bath. p-Toiuene sulfonyl chloride ( 1.15 g. 6 mmoles) was added and the mixture was stirred for 15 mins. 2,6-dimethoxy phenol (462 mg, 3 mmoles) was added and the mixture was stirred at room temperature for 15 hours. The solution -was poured into ice water (300 ml) and the (2 ,6 - dimethoxy)phenyl-2-phenyl-quinoline-4-carboιylate (32) was filtered. The solids were dried and purified on a silica gel column using chloroform /hexane ( 1:1 ) (yield - 50%).

The ester (32) (38 1 g, 1 mmole) was dissolved in anhydrous methylene chloride (3 ml) and methyl fluorosulfate (492 1, 0.69 g, 6 mmoles) was added. After stirring for 15 hours at room temperature under nitrogen anhydrous ether (20 ml) was added. The (2,6-dimethoxy)phenyl- 2-phenyl-quinoline-4-carboxylate-N-methyl fluorosulfonate (33) was filtered and washed with ether and dried (yield - 95%). In a dry two neck 25 ml round bottom flask the ester (33) (200 mg,

0.4 mmole) was suspended in anhydrous methylene chloride (5 ml). The suspension is cooled in a dry ice/CCl-j b ath under nitrogen.

Chiorosulfonic acid ( 144 1, 2 mmole) was added and stirring continued at -20 * C for 0.5 hours. The mixture was then allowed to warm slowly to room temperature and stirred for an additional 2 hours. Anhydrous ether (20 ml) was added and the precipitated ( 2 ,6 -dimethoxy-3 - chlorosulfonyl)phenyl-2-pheήyl-N-methyl-quinolinium-4-carbo ιylate fluorosulfonate (34) was filtered and washed with ether and dried (yield - 90 % ).

-Tarrmlβ 1 0 Another moiety of the present invention is (2,6-dimethyl-4- bromo)phenyi-2 -phenyl- 1.4-dihydro-N-methyi-quinoline-4-carboxylate which has the following formula:

(2,6-dimethyl-4-bromo)phenyl-2-phenyl- l ,4-dihydro-N-methyl- quinoline-4-carboxyiate was synthesized from the (2,6-dimethyl-4- bromo)phenyl-2 -phenyl-N- methyl-quinoliniu m-4-carb oxylate by reduction with sodium cyanoborohydride. The unreduced quinolinium was obtained by the same procedure as the (2,6-dimethyl-4-bromo)ρhenyl acridinium moiety described above, with the substitution of quinoline for acridine.

The quinolinium ester (500 mg, 0.9 mmole) was dissolved in a 1 :1 mixture of acetonitrile and 0.1 M phosphate buffer pH 5.2 (80 ml). A solution of sodium cyanoborohydride (56 mg, 0.9 m mole) in acetonitrile/buffer mixture ( 10 ml) was added. After stirring for 2 minutes, the mixture was acidified to pH 2.0 and acetonitrile ( 100 ml) was added. The solvents were removed in a rotary evaporator. The residue was suspended in water and was extracted with ethylacetate. Drying and evaporation of the ethyl acetate gave (2,6-dimethyl-4-bromo)phenyl-2- phenyl- l ,4-dihydro-N-methyl-quinoline-4-carboιylate (yield = 70%).

fiτamr?le 1 1 Another moiety of the present invention is (2,6-dimethyl-4- bromo)phenyi-2-phenyl- 1 ,2,3, 4-tetrahydro-N- methyl-quinoline-4- carboxylate which has the following formula:

(2,6-dimethyi-4-bromo ) phenyl-2-phenyl- l,2,3,4-tetrahydro-N- methyl-quinoline-4-carboxylate was synthesized from the (2,6-dimethyl- 4-bromo)phenyl-2-phenyl-N-methyl-quinolinium-4-carboxylate by reduction with sodium cyanoborohydride. The unreduced quinolinium was obtained by the same procedure as the (2,6-dimethyl-4-bromo)phenyl acridinium compound described above, with the substitution of quinoline for acridine.

The quinolinium ester (500 mg, 0.9 mmole) was dissolved in a 1 :1 mixture of acetnitrile and 0.1 M phosphate buffer pH 5.2 (80 ml). A solution of sodium cyanoborohydride (560 mg, 9.0 m mole) in acetonitrile/buffer mixture (20 ml) was added. After stirring for 30 minutes, the mixture was acidified to pH 2.0 with 0.1 N hydrochloric acid and stirred for an additional 5 minutes. Acetonitrile ( 100 ml) was added and the solvents were removed in a rotary evaporator. The residue was suspended in water and was extracted with ethylacetate. Drying and evaporation of the ethyl acetate gave (2,6-dimethyl-4-bromo)phenyl-2- phenyl- l,2.3,4-tetrahydro-N-methyl-quinoline-4-carboxyiate (yield = 80 % ).

Fτamnlβ 1 2

Another preferred moiety of the present invention is (2,6 - dimethyl-4-trimethyiammonio)phenyl-N-methyi-acridinium-9- carboxylate difiuorosulfonate which has the following formula:

(2,6-dimethyi-4-trimethylammonio)phenyl-N-methyl-acridini um-9- carboxylate difluorosulfonate is synthesized according to the following scheme:

SUBSTITUTE

(2,6-dimethyl-4-trimethylammonio)phenyl-N-methyl-acridiniu m-9- carboxylate (35) was obtained by esterification of acridine-9 -carboxylic acid with 2,6-dimethyl-4-nitroρhenol (36 ). The product (37) was reduced to the (2,6-dimethyl-4-amino)phenyl-acridine-9-carboxylate (38 ) with zinc. Two methyl groups were introduced on the amino group by treatment with methyl iodide. Quaternization and acridinium formation was then accomplished using methyl fluorosulfate. These reactions are described in further detail in the following.

Acridine-9-carboxylic acid (35 ) (3.05 g, 0.0 14 moles) in a 250 ml round bottom flask was mixed with thionyl chloride (65 ml) and the mixture was refluxed for 2 hours with stirring. The excess thionyl chloride was removed in a rotary evaporator. The residue was treated with benzene (75 ml) and the solvent was removed in vacuo to remove traces of thionyl chloride. The residue of acridine-9-carbonyl chloride was mixed with pyridine (65 mi) and 2,6-dimethyl-4-nitrophenol (36) (2.25 g, 0.0 1 4 moles) was added. The mixture was warmed using a water bath (about 60 *

C) to dissolve all the solids. After 15 hours of stirring at room temperature the mixture was poured into 1 liter of water. The suspension was acidified with concentrated hydrochloric acid to pH 2.0. The solid product was filtered, washed with water and dissolved in chloroform. Drying

(anhydrous sodium sulfate) and evaporation of chloroform gave the crude ester.

The crude ester was chromatographed on a silica gel column using CHCi3/EtOAc 98:2 as solvent. The fractions with Rf value of 0.6 on TLC with the same solvent were pooled and evaporation of the solvents gave pure (2,6-dimethyl-4-nitro)phenyl-acridine-9-carboxylate (37) (yield - 30%).

The (2,6-dimethyl-4-nitro)phenyl ester (37) ( 1.16 g, 3.1 mmole) was dissolved in acetic acid (50 ml) by warming in an oil bath at about 65 * C. Stannous chloride ( 1.5 g) was dissolved in concentrated hydrochloric acid ( 10 ml) and was added to the ester solution. The mixture was stirred for 45 minutes and was then poured into water (750 mi). Extraction with chloroform (3 x 200 ml) removed unreacted (2,6-dimethyl-4-nitro)phenyl ester. The aqueous layer was made basic with sodium bicarbonate and was extracted with chloroform (4 x 200 ml). Drying and evaporation of the chloroform gave (2,6-dimethyl-4-amino)phenyi-acridine-9-carboxylate (38) (yield = 25%).

The amino ester (38) (64 mg, 0.18 mmole) was dissolved in nitromethane (5 ml). Methyl iodide ( 1 ml) and pyridine (0.1 ml) were added. The mixture was stirred at room temperature for 15 hours. Methanol (2 ml) was added and the mixture was then stirred for an additional 2 hours. The solvents were evaporated and the residue was treated with water ( 10 ml) and was then extracted with chiroform (4 x 20 ml) after the solution was made basic. Drying and evaporation of the chloroform gave ( 2,6-dimethyl-4-dimethylamino)phenyl-acridine-9 - carboxyiate (39) (yield = 50%). The dimethyiamino ester (39) ( 154 mg. 0.41 mmole) was dissolved i methylene chloride (2 ml). Methyl fluorosupfate (265 1, 3.28 mmole) was added and the mixture was stirred at room temperature for 15 hours. Amhydrous ether ( 15 ml) was added and the precipitated solids were filtered and washed with ether. Drying gave (2,6-dimethyl-4- trimethylammonio)phenyl-N-methyl-acridinium-9-carboxylate (40 ) (yield - 50%). MS: FAB, thioglycerol matrix, m/e 400 (M + ).

LABELLING PR TETN AND OTHER MATERIAL WTTff CHEMTLtlMTNESCENT MOTETTES

Example 1 A conjugate of the present invention comprises progesterone b ound to a β-D-thioglucose adduct of (2 ,6 -dimethoxy-3 - chiorosulfonyl)phenyl-N-methyl-acridinium-9-carboιylate fluorosulfonate. The progesterone conjugate of the β-D-thioglucose adduct of (2,6-dimethoιy-3-chlorosulfonyl)phenyl-N-methyl-acridinium- 9-carboιyiate fluorosulfonate has the following formula:

The progesterone conjugate is synthesized according to the following scheme:

(49)

SUBSTITUTE S

3,5-dimethoxy-4-hydroxy benzamide (35) (3.0 g. 15.2 mmole) was dissolved in anhydrous pyridine ( 15 ml) and the solution was cooled in a dry ice/CG.4 bath. Acetyl chloride ( 1.4 ml, 1.54 g, 19.7 mmole) was added and the mixture was kept stirred at room temperature for 2 hours. Methanol ( 1 mi) and water (5 ml) were added and the solvents were removed under reduced pressure. The residue was treated with water ( 0 mi) acidified with dilute hydrochloric acid and was extracted with ethyl acetate. Washing with water, drying and evaporating of the ethyl acetate gave 2,6-dimethoιy-4-carbozamido-phenyl acetate (36) (2.2 g) which was recrystallized from ethyl acetate (yield - 60%).

The phenyl acetate (36) ( 1.27 g, 5.33 mmole) was dissolved in anhydrous tetrahydrofuran ( 125 ml). Diborane solution in THF ( 1.0 M, 10.9 ml, 10.9 mmoles) was added and the mixture was refluxed for 4 hours. After cooling to room temperature water (2 ml) and hydrochloric acid ( 1.0 N, 5 ml) were added. After stirring for 30 minutes the solvents were removed in vacuum. The residue was extracted with chloroform. The chloroform was then dried and removed in vacuo . 2,6-dimethoxy-4- aminomethyi phenol (37) was used in the next step without further purification. To a solution of the crude amine (37) in anhydrous pyridine ( 10 ml) benzylchloroformate ( 1.050 mi, 1.25 g, 7.3 mmole) was added and the mixture was stirred for 3 hours at room temperature. Water (5 mi) was added and the solvents were removed in vacuo . To the residue water (30 ml) was added and the mixture was acidified with dilute HCl. Extraction with ethyl acetate, washing with water, drying and evaporation of the solvent gave 2,6-dimethoxy-4-(benzyloxycarbonyi amino)methyl phenol (38) as an oil (yield * 70% overall).

Acridine-9-carboxylic acid (754 mg, 3.38 mmole) was dissolved in anhydrous pyridine ( 14 ml). p-Toluene sulfonyl chloride ( 1.28 g, 6.76 mmole) was added and the mixture was stirred at room temperature for 30 minutes. 2,6-dimethoxy-4-(benzyloxy carbonyl amino)methyl phenol (38) ( 1.18 g, 3.76 mmole) was added and the mixture was stirred at room temperature for 15 hours. Water ( 10 ml) was added and solvents were removed in vacuo . The residue was dissolved in chloroform and the chloroform layer was washed successively with water, 0.1 N HCl and sodium bicarbonate solution. Drying and evaporating of chloroform gave the crude ester which was chromatographed on a silica gel column using

CHCI3 /Ethyl acetate, 1 : 1. as the solvent. Evaporation of the solvents from the pooled fractions gave [2,6 -dimethoxy-4-(benzyloxycarbonyi amino)methyl]phenyl-acridine-9-carboιylate (39 ) (yield = 22%).

The acridine (39) (296 mg, 0.57 mmole) was dissolved in anhydrous methylene chloride (5 ml). Methyl fluorosulfate (277 1. 3.4 mmole) was added and the mixture was stirred at room temperature for 5 hours. Anhydrous ether (25 ml) was added and the precipitated [2,6-dimethoxy-4- (benzyioxycarbonyl amino) methyllphenyl-acridiniu m - -carb oxylate fluorosulfonate (40) was filtered and washed with ether and dried (yield = 99 % ).

The acridinium (40) ( 107 mg, 0.169 mmole) was suspended in anhydrous methylene chloride (2 ml). Chlorosulfonic acid (53 1, 92 mg, 0.797 mmole) was added after the flask was cooled in a dry ice/CCl4 bath. It was stirred for 30 minutes and the bath was removed. After further stirring at room temperature for 1.5 hours anhydrous ether (20 ml) was added. The precipitated product was filtered and dried in vacuo . The (2,6- dimethoxy-4-aminomethyl-3-chlorosulfonyl)phenyl-acridinium-9 - carboxylate fluorosulfonate (41 ) was directly used in the next reaction.

The sulfonyl chloride (41 ) ( 129 mg) was stirred at room temperature in a mixture of methanol ( 12.5 ml) and water ( 12.5 ml) for 3 hours. Acetonitrile (35 ml) was added and the solvents were evaporated. The residue was dried in vacuum over phosphorous pentoxide. The (2,6- dimethoιy-4-aminomethyl-3-oιosulfonyl)ρhenyl-acridinium-9 - carboxylate fluorosulfonate (42) was used directly for the next reaction. Progesterone hemisuccinate (90 mg, 0.209 mmole) and N- methylmorpholine (22 1, 209 mmole) were dissolved in anhydrous DMF (2 ml). The solution was chilled in dry ice/CCl4 bath and isobutylchloroformate (30 1, 0.229 mmole) was added. After 2 minutes a solution of the acridinium (42) ( 10 1 mg, 0.143 m mole) in dimethylsulfoiide (2 ml) containing N-methylmorpholine (3.14 1, 0.28 mmole) was added. Stirring was continued at -20 * C for 10 minutes and the cooling bath was removed. After stirring at room temperature for 7 hours, 3 drops of water were added. The solvents were removed in vacuo and ethyl acetate was added to the residue. The oily precipitate was washed repeatedly with ethyl acetate. Upon trituration with acetonitrile (2 ml) the oil separated as solids. The product was purified on HPLC using C j g

Dynamax semi-prep column ( 10 mm x 250 mm) (commercial! available

from Rainin Instrument Co., Inc., Wob urn, Massachusetts) using CH3CN/H2O (0.1 % trifluoroacetic acid), 55/45 as mobile phase at a flow rate of 2.75 ml/min. The peak appearing at retention time of 6.00 minutes was collected. Evaporation of solvents gave the conjugate (43) (yield - 30 %). MS: FAB, thioglycerol matrix, 895 (M + , without any counterions).

The progesterone conjugate (43) ( 1.1 mg) in a mixture of CH3CN ( 1 ml) and H2O (200 1) was treated with β-D-thioglucose (0.29 mg) as a solution in water (72 1). After 10 minutes the solvents were removed completely under vacuum to provide the β-D-thiogiucose adduct depicted above.

Example 14

The following procedure for attaching to protein is generally applicable to moieties of the present invention. Mouse IgG (Sigma, 1 mg) was dissolved in 0.9 ml phosphate buffer

( 100 mM, pH 8.0, 150 mM). The solution was then divided into three equal portions of 0.33 mg/0.3 ml (0.0022 moles). About 0.3 mg of a moiety of the present invention was dissolved in about 0.4 mi DMF so as to obtain 0.022 moles of moiety in 15 1 DMF. 0.022 moles of the compound of the present invention was mixed with 0.0022 moles of IgG in a plastic microcentrifuge tube. After 15 minutes, an additional 0.022 moles of compound was added to the microcentrifuge tube (compound to protein molar ratio was 20:1 ). After an additional 15 minutes, excess amounts of the compound of the present invention were quenched with lysine HCl solution ( 10 1 in lOOmM p/ buffer, pH 8.0) for 15 minutes.

Alternatively, aliquots of 0.0055 moles of the compound of the present invention was used instead of 0.022 moles (compound to protein molar ratio was 5:1 ). Biorad glass columns ( 1 cm x 50 cm) (commercially available from

Biorad, Chemical Division, Richmond, California) were packed with previously swelled and de-aerated Sephadex G-50-80 in phosphate buffer ( 100 mM, pH 6.3, 150 mM NaCl, 0.001 % TMS) to a bed volume of 45 ml. The reaction solution was run through the columns at a flow rate of 0.3-0.4 ml/min. 0.5 ml fractions were collected. Labelled protein fractions were detected by diluting 20 1 from each fraction to 1 ml and determining the

chemiiuminescence produced with 30 1 of the diluted solution. Labelled fractions were then pooled.

The pooled conjugate fractions were dialyzed to improve the purity of immunore active conjugate. The pooled fractions were dialyzed against 500 mi pH 6.3 phosphate buffer ( 100 mM, pH 6.3. 150 mM NaCl. 0.001 % TMS) over a period of 24 hours with three buffer changes.

Example π Moieties containing an unreduced heterocyclic ring or ring system can be converted to their equivalent reduced forms while such unreduced moieties are attached to protein or other material. This can be accomplished by using a reducing agent such as sodiu m cyanoborohydride. The procedure for reduction of an acridinium/IgG conjugate is described below. The same procedure is applicable to the reduction of other conjugates.

The IgG labelled with a representative acridinium ( 100 g) in phosphate buffer (400 1) (pH 6.0, 100 mM, 150 mM. NaCl, 0.00 1 % Thimerosai) was treated with a freshly prepared solution ( 10 1) containing sodium cyanoborohydride ( 10"? moles). After two hours of incubation at room temperature the conversion of the acridinium label on the antibody to the acridan is complete as seen from the UV-Vis spectra indicating appearance of a band at 280 nm and disappearance of the band at 360 nm. This reduced forms retained all their immunological properties.

ASS Y PROTOCOLS

Example 1$ I. Components A) Labelled Antibody (conjugate): Affinity purified rabbit anti - p r oiactin conj u g ate d to ( 2 , 6 - d i m e thoxy - 3 - chlorosulfonyl)phenyl-N-methyl-acridinium-9-carboιylate fluorosulfonate. Storage buffer: 10 mM phosphate buffer, 100 mM NaCl pH 6.0, 0.001 % Thimerosai, 0.4% BSA. B) Capture antibody: Rabbit anti-proiactin (6 g/ml) as a solid phase on Nunc tubes (commercially available from Midland Scientific, Roseville, Minnesota).

C) Solid-phase coated tubes: Dried Nunc tubes were prepared as follows:

1) 0.3 ml of the capture antibody per tube at 6 g/mi in PBS buffer (phosphate buffer saline, pH 7.2-7.4, 10 mM phosphate, 100 mM NaCl, 10 mM NaN3 ) was pipetted into

Nunc tubes.

2) Tubes were incubated for 18-24 hours.

3) Tubes were washed 2 times with the PBS buffer.

4) Tubes were blocked with 2.0% BSA in PBS buffer. Incubate for <, 4 hours at room temperature.

5) Tubes were washed 3 times with PBS buffer.

6) Tubes were dried at room temperature.

7) Tubes were stored in plastic freezer bags at 4 * C.

D) Standards: Prepared in horse serum 0, 5, 30, 100 and 200 ng/ml/ml

2. Assay Protocol 1) ' 25 1 of sample or standard was pipetted into the antibody- coated tubes. 2) 100 1 of labelled antibody was added.

3) Tubes were vortexed gently.

4) Tubes were incubated for 1 hour at room temperature on a rotator.

5) Tubes were washed 3 times with deionized water. 6) Chemiiuminescence was counted for 2 seconds [pump 1 : 0.1

N HN0 3 + 0.25% H 2 0 2 ; pump 2: 0.25 N NaOH + 0.125% CTACl in a

Lu maTag" Analyzer (commercially available from London Diagnostics, Eden Prairie, Minnesota).

Example 17

1 . Components A) Progesterone Conjugate of the β-D-thiogiucose adduct of (2,6-dimethoxy-3-chlorosulfonyi)phenyl-N-methyl-acridinium-9 - carboxylate fluorosulfonate: 20 pg/ml progesterone conjugate in phosphate buffer (pH 6.0, 100 mM phosphate, 150 mM NaCl, 0.1 % human serum albumin, 0.00 1 % Thimerosai).

B) Primary antibody: Rabbit anti-progesterone (Cambridge Medical Diagnostics) in phosphate buffer (pH 6.0, 200 mM phosphate, 150 mM NaCl, 0.1 % human serum albumin, 0.0 1 CHAPS, 5 g Danazol). C) Solid-phase coated tubes: Dried Nunc tubes coated with 2.5 g of Goat anti-Rabbit fc and. blocked with 0.5% BSA. Tubes were prepared as follows:

1 ) Tubes were ncubated for 1 hour with 2.5 g/ml Goat anti-Rabbit fc (500 1) at room temperature. 2) Tubes were washed 3 times with distilled water.

3) Tubes were immediately incubated for 3 hours with 0.5% BSA (500 1) at room temperature.

4) Tubes were washed 3 times with distilled water.

5) Tubes were dried overnight at 40% relative humidity at room temperature.

6) Tubes were stored in plastic freezer bags at 4 * C.

D) Serum matrii: Antech steer serum.

E) Standards: 0, 0.13. 0.38, 1.31. 7.31 16.6 and 37.0 ng/ml.

2. Assay Protocol

1 ) 50 1 of sample or standard was pipetted into the antibody- coated tubes.

2) 100 1 of conjugate buffer was added.

3) 100 1 of primary antibody buffer was added. 4) Tubes were vortexed gently.

5) Tubes were incubated for 2 hours at 37 * C.

6) Tubes were decanted and washed with 150 mM NaCl in 0.1 % Tween ( 1 ml) and then 3 times with distilled water.

7) Tubes were inverted and allowed to drain. 8) Chemiiuminescence was counted for 2 seconds [pump 1 : 0.1

N HNO3 + 0.25% H 2 0 2 ; pump 2: 0.25 N NaOH + 0.125% CTACl in a

LumaTag" Analyzer (commercially available from London Diagnostics, Eden Prairie, Minnesota).

Example 18 1 . Components

A) Labelled Ab: Affinity purified goat anti-TSH conjugated to (2.6-dimethoxy-3-chlorosulfonyi)ρhenyi-N-methyl-acridinium- 9- - carboxylate fluorosulfonate.

B) Storage buffer: 100 mM phosphate. 0.145 M NaCl, 0.00 1 % Thimerosai, 0.4% BSA, 0.1 mg/ml mouse-globulins, and 0.1 mg/ml goat-globulins, pH 6.0 .

C) Capture antibody: Monoclonal-anti-TSH (2 g/mi) as a solid phase on Nunc tubes. Procedure for preparation of solid- phase Nunc tubes:

1 ) 0.4 ml of the capture antibody at 2 g/ml in PBS buffer (phosphate buffer saline, pH 7.2-7.4, 10 mM phosphate, 100 mM NaCl, 10 mM Na^ ) was added to each tube.

2) Tubes were incubated for 18-24 hours.

3) Tubes were washed 3 times with the PBS buffer.

4) Tubes were blocked with 2.0% BSA in PBS buffer and incubated for <. 4 hours at room temperature. 5) Tubes were washed 3 times with PBS buffer.

6) Tubes were dried at room temperature.

7) Tubes were stored in plastic freezer bags at 4 * C.

D) Standards: Prepared in horse serum.O, 0.5, 2.5, 10, 25 and 100 IU/ml

Assay Protocol

1) 200 1 of sample was pipettd into the coated tubes.

2) 100 1 of labelled antibody was added.

3) Tubes were vortexed gently. 4) Tubes were incubated for 2 hours at room temperature on a shaker.

5) Tubes were washed using a Biotomic washer (commercially available from Ocean Scientific, Inc., Garden Grove, Claifornia).

6) Chemiiuminescence was counted for 2 seconds [pump 1: 0.1 N HNO3 + 0.25% H 2 0 2 ; pump 2: 0.25 N NaOH + 0.125% CTACl in a

LumaTag" Analyzer (commercially available from London

Diagnostics, Eden Prairie, Minnesota).

Example 19

1 , Components

A) Labelled Ab: Affinity purified rabbit anti-proiactin conj ug ated to ( 2 ,6 - dimethyl-4 -nitro ) phenyl-N- methyl - acridinium-9 -carboxylate fluorosulfonate. Storage buffer: 10 mM phosphate buffer, 100 mM NaCl pH 6.0, 0.00 1 % Thimerosai, 0.4% BSA.

B) Capture antibody: Rabbit anti-proiactin (6 g/ml) as a solid phase on Nunc tubes.

C) Solid-phase coated tubes: Dried Nunc tubes were prepared as follows:

1 ) 0.3 ml of the capture antibody per tube at 6 g/ml in PBS buffer (phosphate buffer saline, pH 7.2-7.4, 10 mM phosphate, 100 mM NaCl, 10 mM NaN3 ) was pipetted into

Nunc tubes.

2) Tubes were incubated for 18-24 hours.

3) Tubes were washed 2 times with the PBS buffer.

4) Tubes were blocked with 2.0% BSA in PBS buffer. Incubate for <, 4 hours at room temperature.

5) Tubes were washed 3 times with PBS buffer.

6) Tubes were dried at room temperature.

7) Tubes were stored in plastic freezer bags at 4 * C.

D) Standards: Prepared in horse serum 0, 5, 30, 100 and 200 ng/ml/ml

2. Assay Protocol 1 ) 25 1 of sample or standard was pipetted into the antibody- coated tubes. 2) 100 1 of labelled antibody was added.

3) Tubes were vortexed gently.

4) Tubes were incubated for 1 hour at room temperature on a rotator.

5) Tubes were washed 3 times with deionized water. 6) Chemiiuminescence was counted for 2 seconds [pump 1 : 0.1

N HNO3 + 0.25% H2O2 ; pump 2: 0.25 N NaOH + 0.125% CTACl in a

LumaTag" Analyzer (commercially available from London Diagnostics, Eden Prairie, Minnesota).

STABILITY STUDIES

In accordance with the following stability studies, the moieties and conjugates of the present invention are preferably stored and used in assays at a pH between about 6.5 and about 7.5. The moieties and conjugates exhibit increased stability at other pHs under certain conditions; however, the increase is not condition-dependant within the preferred pH range.

Example 20

Comparative stability was determined by comparing the number of days (tι /2)in which a moiety lost 50% of its chemiiuminescence. Stability of certain moieties not linked to protein or other material was monitored at room temperature and at 4 * C. Moieties were stored in buffer (pH 6.0, 50 mM p/, 0.1 % BSA). (2,6-dimethyl-4-nitro)phenyi-3-(3-succinimidyl- oxycarb onyl) propyloxy-N-methyi-acridiniu m- 9 -carb oxylate fluorosulfonate showed no appreciable loss of counts after 98 days at both room temperature and at 4 * C. (2,6-isopropyl-4-nitro)phenyl-3-(3- succinimidyl-oxycarbonyl) pro pyloxy-N- methyl- acridiniu m -9 - carboxyiate fluorosulfonate showed no appreciable loss of counts after 312 days at room temperature and 4 * C. For (2,6-dimethyl-4- trimethylammσnio)phenyi-N-methyl-acridinium-9-carboxyiate difluorosulfonate tι 2 was 105 days at room temperature and tι 2 was not reached after 105 days at 4 * C. For (2,6-dimethoxy-3 - chlorosulfonyl)phenyl-N-methyl-acridinium-9 -carboxylate fluorosulfonate, ι/2 was 50 days at room temperature, tχ/2 at 4 * C was not reached after 130 days. For (2,6-dimethoxy-3 -chiorosulfonyl-4- propanoic)phenyl-N-methyl-acridinium-9 -carboxylate fluorosulfonate, tl /2 ' w ' as 24 days at room temperature and > 95 days at 4 * C. For (2.6- dimethyi)phenyl-N-methyl-acridinium -9 -carboxylate fluorosulfonate, ι /2 at room temperature was 50 days. In comparison, moieties not having the substitutions of moieties of the present invention had an average [/2 less than 8 days at room temperature and approximately 32 days at 4 * C.

Example 21 The comparative stability of the phenyl-, (2,6-dimethyl)phenyl- and ( 2 ,6 -dimethoxy-3 -chlorosulfonyl)phenyl-, ( 2 ,6 -dimethyi- 3 - chlorosulfonyDphenyl-, (2,6-dimethyl-4-nitro)phenyl-, (2,6-dimethyl-4- trimethylammonio)ρhenyl-, and (2,6-dimethyl-4-bromo)phenyl- N- methyl- acridinium esters was observed in phosphate buffer ( 100 mM py, 150 mM NaCl, 0.001 % Thimerosai, 0.005% human serum albumin) at pH 6.3 and pH 8.0 while incubated at 35 * C and 45 * C. Stability data for these compounds at pH 6.3 at 35 * C and 45 * C is shown in Figs. 1 and 2, respectively. Stability data for these compounds at pH 8.0 at 35 * C and 45 "C is shown in Figs. 3 and 4, respectively. All of the substituted phenyl compounds, with the exception that the (2,6-dimethyl)phenyl- compound, showed increased stability in comparison with the naked compound. The (2, 6 -dimethyDphenyl- compound, was less stable than the naked phenyl compound at pH 6.3 at 35 * C, but showed increased stability at pH 8.0 at 35 * C and at both pHs at 45 * C.

Example 22 The stability of (2,6-dimethyl-4-nitro)phenyl-3-(3-succinimidyl- oxycarb onyl) pro pyloxy-N- methyl- acridiniu m - 9 - carb oxyl ate fluorosulfonate and (2,6-dimethoxy-3-chlorosulfonyi)phenyl-N-methyl- acridinium-9-carboxylate fluorosulfonate mouse IgG conjugates at various temperatures and pHs was studied and compared to the stability of 4-( 2-succinimidylcarboxyethyl)-ρhenyl-N-methyl acridiniu m -9 - carb oxylate flu oros ulfonate IgG conj ug ate s . 4 - ( 2 - succinimidylcarboxyethyl)-phenyl-N-methyl acridinium -9 -carboxylate fluorosulfonate has the following formula:

4-(2 -succinimidylcarboxyethyl)-phenyl-N- methyl acridiniu m -9 - carboxylate fluorosulfonate is different from the compounds of the present invention in that it does not have an electron withdrawing group or 2,6 -substitutions on the phenyl ring. 4-(2-succinimidyicarboxyethyD- phenyl-N-methyi acridinium-9 -carboxylate fluorosulfonate has been published in the literature and is widely used in chemiluminescent applications. As presented below, loss of counts in a certain conjugate results from the hydrolysis of the ester linkage in the chemiluminescent label. Therefore, as the stability of the labelling compound decreases, the rate of loss" of counts in the conjugate decreases.

Mouse IgG conjugates of (2,6-dimethyl-4-nitro)phenyi-3 -(3- s uccinimidyl-oxycarbonyi) propyloxy-N- methyi-acridiniu m -9 - carboxylate fluorosulfonate, (2,6-dimethoιy-3-chlorosulfonyi)phenyl-N- methyl-acridiniu m-9 -carboιylate fluorosulfonate and 4 -( 2 - succinimidylcarboxyethyl)-phenyl-N-methyl acridinium-9 -carboxylate fluorosulfonate were stored at 4 * C. The conjugates were stored in three different buffer solutions at pH 6.3, 7.3 and 8.0.

The first buffer contained 100 mM pj t 150 mM NaCl, 0.00 1 % Thimerosai, 0.1 % human serum albumin, 20 mg/L sheep IgG ("Standard Phosphate Buffer"). Stability data in Standard Phosphate Buffer at pH 6.3, 7.3 and 8.0 are shown in Figs. 5, 6 and 7 respectively. The second buffer was Standard Phosphate Buffer without sheep

IgG. Stability data in the second buffer at pH 6.3, 7.3 and 8.0 are shown in Figs. 8, 9 and 10 respectively.

The third buffer was Standard Phosphate Buffer without sheep IgG and with 0.1% bovine serum albumin instead of human serum albumin. Stability data in the third buffer at pH 6.3, 7.3 and 8.0 are shown in Figs. 11,12 and 13 respectively. In each buffer at each pH (2,6-dimethyl-4-nitro)phenyl-3-(3- succinimidyl-oiycarbonyl) pro pyioxy-N-m ethyl -acridinium -9- carboxylate fluorosulfonate and (2,6 -dimethoιy-3 - chlorosulfonyl)ρhenyl-N-methyl-acridinium- -carboxylate fluorosulfonate IgG conjugates exhibited increased stability when compared with 4-(2-succinimidylcarboxyethyl ) -ρhenyl-N-methyl acridinium-9-carboxylate fluorosulfonate IgG conjugate.

Mouse IgG conjugates of (2,6-dimethyl-4-nitro)phenyl-3-(3- succinim idyl -oxycarbonyl) propyloxy-N-methyl-acridinium-9- carboxylate fluorosulfonate, (2,6-dimethoxy-3-chlorosulfonyl)ρhenyl-N- methyl-acridinium-9-carboxylate fluorosulfonate and 4-(2- succinimidyicarboxyethyl)-phenyl-N-methyl acridinium-9 -carboxylate fluorosulfonate were stored at room temperature (about 23 * C) ("RT") and 37 * C. The conjugates were stored in Standard Phosphate Buffer at pH 6.3, 7.3 and 8.0. Stability data at room temperature at all three pHs for 4-(2- succinimidylcarboxyethyl)-phenyl-N-methyl acridinium-9 -carboxylate fluorosulfonate, ( 2,6 -dimethyl-4-nitro)phenyl-3-( 3 -succinimidyl- oxycarbonyi) ρroρyloxy-N-methyl-acridinium-9-carboxylate fluorosulfonate and (2.6-dimethoxy-3-chlorosulfonyl)phenyl-N-methyl- acridinium-9-carboxylate fluorosulfonate IgG conjugates are shown in Figs. 14, 15 and 16, respectively. Stability data for 4-(2- succinimidylcarboxyethyi)-phenyl-N-methyl acridinium-9 -carboxylate fluorosulfonate and (2,6-dimethoxy-3-chlorosulfonyl)phenyl-N-methyl- acridinium-9-carboxylate fluorosulfonate IgG conjugates at 37 * C at all three pHs are shown in Figs. 17 and 18, respectively. Figs. 19 and 20 summarize the stability data for (2,6-dimethyl-4-nitro)phenyl-3-(3- succinimidyl-oxycarbonyl) propyloxy-N-methyl-acridinium-9- carboxylate fluorosulfonate IgG conjugate at 37 * C at all three pHs. Figs. 19 and 20 show data for (2,6-dimethyl-4-nitro)phenyl-3-(3-succinimidyl- oiycarbonyl) propyloιy-N-methyl-acridinium-9-carboxyiate fluorosulfonate conjugates made using 20x mole excess (2,6-dimethyl-4- nitro)ρhenyl-3-( 3 -succinimidyl-oxycarbonyi) propyloxy-N-methyl-

acridinium-9-carboxylate fluorosulfonate and 5x mole excess (2,6- dimethyl-4-nitro)phenyi-3-(3-succinimidyl-oxycarbonyi) propyloxy-N- methyl-acridinium-9-carboxylate fluorosulfonate, respectively, in the conjugation protocol. At each pH and at room temperature and 37 * C (2,6-dimethyl-4- nitro) phenyl- 3 -(3 -succinimidyl-oxycarbonyi) propyioxy-N-methyl- acridinium-9-carboxylate fluorosulfonate and (2,6-dimethσxy-3 - chlorosulfonyl)phenyl-N-methyl-acridinium-9 -carboxylate fluorosulfonate IgG conjugates exhibited increased stability when compared with 4-(2-succinimidylcarbσxyethyl)-phenyi-N-methyl acridinium-9-carboxylate fluorosulfonate IgG conjugate.

(2,6-dimethyl-4-nitro)phenyl-3-(3-succinimidyl-oxycarbony i) ρropyloxy-N-methyi-acridinium-9 -carboxylate fluorosulfonate, (2,6- dimethoιy-3-chlorosulfonyl)phenyl-N-methyl-acridinium-9 -carboxylate fluorosulfonate and 4-(2-succinimidylcarboxyethyl)-phenyl-N-methyl acridinium -9 -carboxylate fluorosulfonate IgG conjugates were stored at 37 * C in azide buffer (50 mM P , 100 mM NaCl. 0.1 % bovine serum albumin, 10 mM sodium azide) at pH 6.9, 7.0, 7.3 and 8.0. Stability data in the azide buffer at pH 6.9, 7.0, 7.3 and 8.0 are shown in Figs. 21 , 22, 23 and 24, respectively.

At pHs above 7.3 both (2,6-dimethyi-4-nitro)phenyl-3-(3 - s uccinimidyi-oiycarbonyl) proρyloxy-N- methyl-acridiniu m-9 - car b oxylate fl u oro s u lfonate and ( 2 , 6 - d i m e thoxy - 3 - chiorosulfonyl)phenyI-N-methyl-acridinium-9-carboxylate fluorosulfonate IgG conjugates exhibited increased stability when compared with 4-(2-succinimidylcarboxyethyl)-phenyl-N- methyl acridinium -9 -carboxylate fluorosulfonate IgG conjugate. At pH 6.9 and 7.0 only (2,6 -dimethyl-4-nitro)phenyl-3 -(3 -succinimidyl-oxycarbonyi) propyioxy-N-methyl-acridinium-9-carboxylate fluorosulfonate IgG conjugate exhibited increased stability. (2,6 -dimethoxy-3 - chlorosulfonyl)phenyi-N-methyl-acridinium-9 -carboxylate fluorosulfonate IgG conjugate was not significantly more stable than the 4-( 2 -succinimidylcarb oxyethyl)-phenyl-N-methyl acridiniu m -9 - carboxylate fluorosulfonate IgG conjugate at pH 6.9 and 7.0. (2.6-dimethyl-4-nitro)phenyl-3-(3-succinimidyl-oxycarbonyi) propyloxy-N-methyl-acridiniu m-9-carboxylate fluorosulfonate, (2,6 - dimethoxy-3-chlorosulfonyl)ρhenyl-N-methyi-acridinium-9-car boxylate

fluorosulfonate and 4-(2-succinimidylcarboxyethyi)-phenyl-N- methyl acridinium-9-carboxylate fluorosulfonate IgG conjugates were stored at 37 * C in azide buffer at pH 6.3. Stability data for (2,6-dimethyi-4- nitro)phenyl-3-(3 -succinimidyl-oιycarbonyl) propyloxy-N-methyl- acridiniu m -9 -carboxylate fluorosulfonate, ( 2 ,6 - di methoxy-3 - chlorosulfonyi)phenyl-N-methyl-acridinium-9 -carboxylate fluorosulfonate and 4-( 2 -succinimidylcarboxyethyl)-phenyl-N- methyl acridinium-9-carboxylate fluorosulfonate IgG conjugates in the azide buffer at pH 6.3 at 37 * C is shown in Fig. 25. (2,6-dimethyl-4-nitro)phenyl-3-(3-succinimidyl-oxycarbonyl) propyloxy-N-methyi-acridinium-9-carboxylate fluorosulfonate and 4-(2- succinimidylcarboxyethyl)-phenyl-N-methyl acridinium-9 -carboxylate fluorosulfonate IgG conjugates were stored at room temperature and 4 * C in azide buffer at pH 5.9. Stability data for (2,6-dimethyl-4-nitro)ρhenyl- 3 -(3 -succinimidyl-oxycarbonyl) propyloxy-N-methyl-acridiniu m-9 - carboxylate fluorosulfonate and 4-(2-succinimidylcarboxyethyl)-ρhenyl- N-methyl acridinium-9-carboιylate fluorosulfonate IgG conjugates in the azide buffer at pH 5.9 is shown in Fig. 26.

At pHs less than 6.3 at 37 * C both (2,6-dimethyl-4-nitro)ρhenyl-3 : ( 3 -succinimidyl-oιycarbonyl) pr opyloxy-N- m ethyl -acridiniu m - 9 - car b oxylate fl u oro s u lfonate and ( 2 ,6 - di m ethoxy - 3 - chiorosulfonyl)phenyl-N-methyl-acridinium-9-carboxylate fluorosulfonate IgG conjugates exhibited decreased stability when compared with 4-(2-succinimidylcarboxyethyl)-phenyi-N- methyl acridinium-9-carboxylate fluorosulfonate IgG conjugate. At pH 5.9 at room tem perature and 4 * C ( 2,6-dimethyi-4-nitro)ρhenyl-3 -( 3 - s uccinimidyl-oxycarbonyl) pro pyloxy-N- m ethyl- acrid iniu m - 9 - carbσxyiate fluorosulfonate IgG conjugate exhibited decreased stability when compared with 4-(2-succinimidylcarboxyethyl)-phenyl-N-methyl acridinium-9-carboxylate fluorosulfonate IgG conjugate.

Example 23

The comparative stability of the phenyl-, (2,6-dimethyi)phenyi- and (2,6 -dimethyl-4-nitro)phenyl- N-methyl-acridan esters was observed in phosphate buffer ( 100 mM py, 150 mM NaCl, 0.00 1 %

Thimerosai, 0.005% human serum albumin) at pH 6.3 and pH 8.0 while incubated at 35 * C and 45 * C Stability data for these compounds at pH 6.3 at

35 *C and 45 "C is shown in Figs. 27 and 28, respectively. Stability data for these compounds at pH 8.0 at 35 * C and 45 * C is shown in Figs. 29 and 30, respectively. The (2,6-dimethyi)phe.nyl- and (2,6-dimethyi-4- nitro)phenyl- compounds showed increased stability in comparison with the naked phenyl compound.

ADDITIONAL CHEMILUMINESCENT M TETT .<.

Example 24 A preferred chemiluminescent moiety of the present invention having an RnX group on the carbon to which the ester linkage is attached is (2,6-dimethoxy-3-chlorosulfonyl)phenyl-N-methyl-acridan- 9 -ethoxy-9 -carboxylate, which has the following formula:

(2,{5-dimethoxy-3-chlorosulfonyl)phenyl-N-methyl-acridan- 9-ethoxy-9- carb oxylate is synthesize d fro m ( 2 , 6 -di m ethoxy- 3 - chlorosulfonyl)phenyl-N-methyl-acridinium-9-carboxyiate by two alternative methods described below.

1 TTigh Perform ance T.iαuid Chrom atngranhv PT.C . A Ci s column (Rainin, Dynamax 60A, 250 mm x 10mm) was equilibrated with a mixture of ethanol and acetonitrile (up to 10 % ) containing about 0.05% of a tertiary amine (e.g., triethylamine). (2,6- dimethoxy-3-chlorosulfonyl)phenyl-N-methyl-acridinium-9-carb oxylate was dissolved in the mobile phase and injected onto the column. The major fraction, eluting with a maximum absorbance at 280nm, was collected at a flow rate of 2.0-2.5 ml/min. The solvent was immediately

completely removed under vacuum. The residue was then treated with a small amount of benzene to remove traces of alcohol and to remove m oistu re f ro m the final pro d u ct, ( 2 , 6 - di m ethoxy- 3 - chlorosulfonyl)phenyl-N-methyl-acridan-9-ethoxy-9-carboxylat e. This HPLC method is the preferred method of synthesis, since it yields a much purer product. However, the HPLC method may not be appropriate for certain nucleophiles. Where the HPLC method does not work, the nucleophilic anion method described below can be used.

2. Treatment of Starting Compound with Nucleophilic Anion

(2,6-dimethoxy-3-chlorosulfonyl)phenyi-N-methyl-acridiniu m-9- carboxylate (0.013 mmole, 7.5 mg) was dissolved in absolute ethanol (3 ml) under nitrogen. A solution of potassium-t-butoxide in absolute ethanol (2 mg/ml) was added dropwise (using a gas tight syringe) to the vigorously stirred (2,6-dimethoιy-3-chiorosulfonyl)phenyl-N-methyi-acridinium- 9- carboxylate solution until the yellow color of the solution was completely discharged. The ethanol was then completely removed under vacuum. The residue was treated with anhydrous ether (2ml) and approximately lg anhydrous sodium sulfate. Separation of the ether solution and evaporation of the solvent yielde d ( 2 ,6 - dimethoxy- 3 - chlorosulfonyi)phenyl-N-methyl-acridan-9-ethoxy-9-carboxylat e as a white solid (2.5 mg).

(2,6-dimethoxy-3-chlorosulfonyl)phenyl-N-methyl-acridan-9 - methoxy-9 -carboxylate has been produced using both the HPLC and nucleophilic anion synthetic procedures from (2,6 -dimethoxy-3 - chlorosulfonyi)phenyl-N- methyl- acridiniu m -9 -carboxylate. ( 2 ,6 - dimethyl-3-chlorosulfonyl)phenyl-N-methyl-acridan-9 -methoxy-9 - carboxylate has been produced using the nucleophilic anion synthetic method from ( 2 ,6 -dimethyl-3 -chlorosulfonyl)phenyl-N-methyl- acridinium-9 -carboxylate.

V Other Syntheses

Both synthetic procedures described above can be used to produce any of the compounds of the present invention having an R n X group. If a nucleophile other than ethanol is to donate the R n X group, the desired nucleophile is substituted for ethanol in the synthetic procedures. If a

different acridinium. phenanthridinium, etc. is desired, the compound to which the R n X group is to be added can be substituted for (2,6-dimethoxy- 3 -chlorosulfonyl)phenyl-N-methyl-acridinium -9 -carboxylate in the synthetic procedures described above. In many instances, a change in the nucleophile will also require a change in solvent (e.g., if methanol is the desired nucleophile, ethanol cannot be used as a solvent because of competition for addition). In such instances, the new nucleophile can be used as a replacement solvent, if appropriate, or a non-nucleophilic, non- protic solvent (e.g., THF) can be substituted. Other synthetic methods, including without limitation treatment with a nucleophilic solvent, can also be used to produce moieties having an RnX group.

Example 2 * >

To confirm the operability of the synthetic procedures described in Example 24, the products were analyzed by UV-Vis spectroscopy, fast atom bombardment mass spectroscopy and 300 MHz proton NMR.

Fig. 3 1 is a UV-Vis spectru m of (2,6 -dimethoxy-3 - chlorosulfonyl)phenyl-N-methyl-acridan-9-ethoxy-9-carboxyiat e

(solvent: chloroform). Fig. 32 is a fast atom bombardment mass spectrum of (2, 6-dimethoxy-3-chiorosulfonyl)phenyl-N-methyl-acridan-9 -ethoxy - 9-carboxylate (m/e- 472.1 ; M+ - OCH2 CH 3 ). The integrity of the sulfonylchloride moiety is confirmed by the isotope abundance peak at 472+2- 474.

Fig. 33 is a 300 MHz proton NMR spectrum of (2,6-dimethoxy-3- chlorosulfonyl)phenyl-N-methyl-acridan-9 -methoxy-9 -carboxylate (solvent: CDCI3 ).

Fig. 34 is a 300 MHz proton NMR spectrum of (2,6-dimethyl-3- chlorosulfonyl)phenyi-N-methyl-acridan-9 -methoxy-9 -carboxylate (solvent: CDCI3).

Example 26 (2,6-dimethoιy-3-chlorosuifonyl)phenyl-N-methyl-acridan-9- ethoxy- 9 -carboxylate was used as a label in an assay for TSH as follows:

1. Com ponents

A) Labelled Ab: Affinity purified goat anti-TSH was conjugated to (2,6-dimethoιy-3-chlorosulfonyl)phenyl-N-methyl- acridan-9-ethoxy-9-carboxylate as follows: a solution of the anti- TSH antibody (approximately l OOug) in bicarbonate buffer (0.1 M, pH 9.6) was treated with 25 moles excess of (2,6-dimethoxy-3- chlorosulfonyi)phenyl-N-methyl-acridan-9-ethoιy-9 -carboxylate as a solution in DMF. The reaction mixture was purified on a fast flow Sephadei G25 (superfine) column (Pharmacia) using an HPLC system. The protein peak was collected at a flow rate of approximately 0.75 ml/min with a mobile phase of phosphate buffer (pH 6.0 ) containing approximately 20% ethanol. After buffer exchange the labelled antibody preparation was diluted with storage buffer to provide approximately 100,000 counts/ 100 ul in a LumaTag" Analyzer (commercially available from London

Diagnostics, Eden Prairie, Minnesota) after 1: 10 dilution.

B) Storage buffer: 100 mM phosphate, 0.145 M NaCl, 0.00 1 % Thimerosai, 0.4% BSA, 0.1 mg/ml mouse-globulins, and 0.1 mg/ml goat-globulins, pH 6.0 . C) Capture antibody: Monoclonal-anti-TSH (2 ug/ml) as a solid phase on Nunc tubes. Procedure for preparation of solid-phase Nunc tubes:

1 ) 0.4 ml of the capture antibody at 2 ug/ml in PBS buffer (phosphate buffer saline, pH 7.2 -7.4, 1 0 mM phosphate, 100 mM NaCl, 10 mM Na^ ) was added to each tube.

2) Tubes were incubated for 18-24 hours.

3) Tubes were washed 3 times with the PBS buffer.

4) Tubes were blocked with 2.0% BSA in PBS b uffer and incubated for < 4 hours at room temperature.

5) Tubes were washed 3 times with PBS buffer.

6) Tubes were dried at room temperature.

7) Tubes were stored in plastic freezer bags at 4 * C.

D) Standards: Prepared in horse serum.0, 0.05, 0.1 , 0.5, 2.5, 10, 25 and 50 ulU/ml

E) Wash Solution: saline buffer containing BSA

2. Assay Protocol

1 ) 200 ul of sample was pipetted into the coated tubes.

2) 100 ui of labelled antibody was added.

3) Tubes were vortexed gently. 4) Tubes were incubated for 2 hours at room temperature on a shaker.

5) 1 ml Wash Solution was added to each tube.

6) Tubes were washed using a Biotomic washer (commercially available from Ocean Scientific, Inc., Garden Grove, California). 7) Chemiiuminescence was counted for 2 seconds {pump 1: 0.1

N HNO3 + 0.25% H 2 0 2 ; pump 2: 0.25 N NaOH + 0.125% CTACl in a

LumaTag" Analyzer (commercially available from London Diagnostics, Eden Prairie, Minnesota).

Addition of HNO3 to the assay mixture containing the labelled antibody causes the C9 ethoxy group to cleave from the acridinium molecule before the chemiluminescent reaction is triggered by the addition of NaOH. A standard curve for the assay is shown in Fig. 35.

Example 27 The co mp arative stab ility of ( 2 , 6 -dim ethoxy- 3 - chlorQsulfonyl)phenyl-N-methyl-acridinium-9-carboιylate ("DMC") and ( 2,6-dimethoxy-3-chlorosulfonyl)phenyl-N-methyl-acridan -9-ethoxy-9- carboxylate ("DME") was compared at pH 9.6 at 25 * C. The compounds were dissolved in DMF (Approximately 0.5mg/ml). lOul of the solution was then added to 1ml bicarbonate buffer (0.1M bicarbonate, 0.00025% Thimerosai, 0.1 % bovine serum albumin). The solution was diluted further to provide approximately 300,000 counts in l Oul. The data from the stability study is shown in Fig. 36. DME was shown to be more stable than DMC over time.

The comparative stability of DMC-labelled TSH conjugate and DME- labelled TSH conjugate was compared at pH 6.0 at 25, 30 and 35 * C. The data from the stability study is shown in Figs. 37-39. The DME-iabelled TSH conjugate was shown to be more stable over time.

Example 28 A preferred chemiluminescent moiety of the present invention having an R n X group on the carbon to which the ester linkage is attached and having a peri substituent is phenyl-N-methyl- 1 ,3 -dimethyl - acridan-9-methoxy-9-carboxylate, which has the following formula:

Phenyl-N-methyl- l ,3-dimethyl-acridan-9-methoxy-9-carboxylate was synthesized from phenyl-N- methyl- l ,3 -dimethyl-acridiniu m- 9 - carboxylate according to the HPLC described above. To produce phenyl- N-methyl- l ,3-dimethyi-acridinium-9-carboxylate, 3 ,5-dimethylaniline and bromobenzene were reacted under extention Ulmann reaction conditions to obtain the N.-ρhenyl-N-( 3,5 -dimethyl)phenylamine. Reaction of N-phenyl-N-(3,5-dimethyl)phenylamine with oxalyi chloride provided the intermediate N-phenyl-dimethyl isatin. Upon cyclization under basic conditions, the l,3-dimethyl-acridine-9-carboxylic acid was formed. The acridine phenyl ester was formed by esterification with phenol as previously described. Phenyi-N-methyl- 1 ,3 -dimethyl- acridinium-9-carboιylate was produced from the acridine ester as described above.

To confirm the operability of the synthetic procedure described in this example, the product was analyzed by 300 MHz proton NMR. The spectrum is shown in Fig. 40.

From the foregoing, it will be obvious to those skilled in the art that various modifications in the above-described compositions and methods can be made without departing from the spirit and scope of the invention. Accordingly, the invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Present embodiments, therefore, are to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.