WO2004046103A2 - Arylpyridine compounds - Google Patents

Abstract

The present invention relates to arylpyridine compounds and methods of using them.

Description

ARYLPYRIDINE COMPOUNDS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 60/427,606, filed November 18, 2002, the disclosure of which is incorporated herein by reference in its entirety.

FIELD

The present invention relates to arylpyridine compounds and methods of using them.

BACKGROUND

Fluorescent chemosensors, molecules that change their fluorescence in response to substrate binding, offer an extremely sensitive optical method for the real-time monitoring of molecular interactions. The majority of fluorescent chemosensors operate by one of three mechanisms: (1) suppression of photoinduced electron transfer or enhancement of heavy-atom quenching; (2) variation of the distance between two fluorophores, modulating the efficiency of interchromophore energy transfer; and (3) alteration of the microenvironment of a solvatochromic fluorophore (e.g., by displacement from a cyclodextrin cavity). Such chemosensors are finding increased use in fields as diverse as biology, medical analysis, and environmental monitoring. The majority of fluorescent chemosensors, however, are based on polycyclic aromatic fluorophores, many of which are not amenable to extensive synthetic variation (Rurac , K et al, Chem. Soc. Rev. 2002, 31, 116-127, Valeur, B et ah, Coord. Chem. Rev. 2000, 205, 3-40, deSilva et al, Chem. Rev. 1997, 97, 1515-1566). This in turn limits the tuning of emission wavelengths and variation of binding domains. A need exists for chemosensors that are easy to produce and can be easily modified so that they can be used in a variety of applications requiring variant emission wavelengths (Mello and Finney, Angew Chem. Int. Ed. 2001, 40(8), 1536-1538; Mello and Finney, Org. Lett., 2001 3(26), 4263-4265; Fang et al, Org. Lett, 2003 5(7), 967-970).

SUMMARY

The present invention provides arylpyridine compounds and methods of using them. The compounds of the present invention are fluorophores and include compounds of Formula 1:

Formula 1

or a salt or ester form thereof, wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl;

R², R³ and R⁴ are independently hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy;

R⁵ is hydrogen, aryl, or heteroaryl; R⁶ is hydrogen, aryl, heteroaryl, or C(=O)X;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; and wherein both R⁵ and R⁶ cannot both be hydrogen. The present invention also provides intermediates having formulas 9 and 10

Formula 9 Formula 10

wherein B is aryl, heteroaryl or C(=O)Z, wherein Z is -NH₂, -O-aryl, -O-heteroaryl, -NYalkyl, -NYaryl, -NYheteroaryl; and Y is hydrogen, alkyl, aryl, or heteroaryl.

The present invention provides methods of synthesizing fluorophores and in particular, methods of synthesizing fluorophores using compounds of Formula 9 or Formula 10.

The present invention also provides methods of detecting the presence, absence or concentration of analyte in a sample. The method comprises combining the sample with an arylpyridine of the present invention and measuring a signal from the arylpyrdine. In one aspect, the step of measuring the signal is performed by measuring fluorescence emission.

The present invention also provides methods of detecting a selected biological target. The method comprises contacting the biological target with an arylpyridine of the present invention and measuring a signal from the compound. In one aspect, the step of measuring the signal is performed by measuring fluorescence emission. In one aspect, the invention provides methods of imaging a selected biological target using the compounds of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Titration of compound 1, 2,6-Bis-[4-methoxy-2-(2-methoxy-ethoxymethyl)- phenylj-pyridine, (10^"5 M in CH₃CN), with metal ions.

Figure 2. Response of compound 4, 2-(4-methoxy-2-methyl-phenyl)-pyridine, (10^"5 M in CH₃CN), to metal ion.

Figure 3. Emission of representative compounds 7-11 in CH₃CN.

DETAILED DESCRIPTION

The present invention provides, mter alia, fluorescent pyridines for use in biological and chemical applications.

The fluorophores of the present invention include compounds of Formula 1 :

Formula 1

wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl; R², R³ and R⁴ are independently hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy;

R is hydrogen, aryl, or heteroaryl with the proviso that R⁵ is not a monocyclic 6- membered aromatic ring containing one or more nitrogen atoms;

R⁶ is hydrogen, aryl, heteroaryl, or C(=O)X;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; wherein both R⁵ and R⁶ cannot both be hydrogen, provided that when R², R³ and R⁴ are hydrogen or alkoxy and R⁵ is alkoxyphenyl substituted with one alkyl group on the phenyl ring, R⁶ is aryl, heteroaryl or C(=O)X; provided that when R², R³ and R⁴ are hydrogen or alkoxy and R⁵ is a phenyl ring substituted with C(=O)alkyl, R⁶ is heteroaryl, C(=O)X, or aryl, wherein aryl is not unsubstituted phenyl; and provided that when R¹, R², and R³ are hydrogen and R⁴ is hydrogen or alkyl, R⁵ is not phenyl, tolyl, or 2,6-dimethylphenyl.

The fluorophores of the present invention include compounds of Formula 1 :

Formula 1

wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl;

R², R³ and R⁴ are independently hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy;

R⁵ is hydrogen, aryl, or heteroaryl with the proviso that R⁵ is not a monocyclic 6- membered aromatic ring containing one or more nitrogen atoms;

R⁶ is hydrogen, aryl, heteroaryl, or C(=O)X; X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; wherein both R⁵ and R⁶ cannot both be hydrogen, provided that when R², R³ or R⁴ is alkoxy and R⁵ is a phenyl ring substituted with alkoxy, R⁶ is aryl, C(=O)X, or heteroaryl; provided that when R², R³ or R⁴ is alkoxy and R⁵ is a phenyl ring substituted with C(=O)alkyl, R⁶ is heteroaryl, C(=O)X, or aryl, wherein aryl is not unsubstituted phenyl; and provided that when R¹, R², and R³ are hydrogen and R⁴ is hydrogen or alkyl, R⁵ is not phenyl, tolyl, or 2,6-dimethylphenyl.

The fluorophores of the present invention include compounds of Formula 1 :

Formula 1

wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl;

R , R and R are independently hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy;

R⁵ is hydrogen, aryl, or heteroaryl with the proviso that R⁵ is not a monocyclic 6- membered aromatic ring containing one or more nitrogen atoms;

R⁶ is hydrogen, aryl, heteroaryl, or C(=O)X;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; wherein both R⁵ and R⁶ cannot both be hydrogen, and provided that when R², R³ and R⁴ are hydrogen or alkoxy, and R⁵ is alkoxyphenyl substituted with one alkyl group on the phenyl ring, R⁶ is C(=O)X, heteroaryl, or aryl wherein aryl is not unsubstituted phenyl; provided that when R², R³ and R⁴ are hydrogen or alkoxy and R⁵ is a phenyl group substituted with C(=O)alkyl, R⁶ is C(=O)X, heteroaryl, or aryl, wherein aryl is not unsubstituted phenyl; and provided that when R¹, R², and R³ are hydrogen and R⁴ is hydrogen or alkyl, R⁵ is not phenyl, tolyl, or 2,6-dimethylphenyl.

The fluorophores of the present invention include compounds of Formula 1:

Formula 1

wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl;

R², R³ and R⁴ are independently hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy;

R⁵ is hydrogen, aryl, or heteroaryl with the proviso that R⁵ is not a monocyclic 6- membered aromatic ring containing one or more nitrogen atoms;

R⁶ is hydrogen, aryl, heteroaryl, or C(=O)X;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; wherein both R⁵ and R⁶ cannot both be hydrogen, provided that when R², R³, or R⁴ is alkoxy and R⁵ is a phenyl ring substituted with alkoxy or C(=O)alkyl, R⁶ is aryl, wherein aryl is not unsubstituted phenyl, heteroaryl, or C(=O)X and provided that when R¹, R², and R³ are hydrogen and R⁴ is hydrogen or alkyl, R⁵ is not phenyl, tolyl, or 2,6-dimethylphenyl. A preferred group of compounds include compounds of Formula 1 wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl,

-NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

A preferred group of compounds include compounds of Formula 1 wherein

R² and R³ are independently hydrogen, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R^!; X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl,

-NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; and

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino.

A preferred group of compounds include compounds of Formula 1 wherein

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or

CHaR¹;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl,

-NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; and

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino.

A preferred group of compounds include compounds of Formula 1 wherein

R⁵ is hydrogen, aryl, or heteroaryl, with the proviso that R⁵ is not a monocyclic 6- membered aromatic ring containing one or more nitrogen atoms, and wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or OHfeR¹; X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; and

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino.

A preferred group of compounds include compounds of Formula 1 wherein

R⁶ is hydrogen, aryl, heteroaryl, or C(=O)X; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R^!;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl,

-NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; and

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino.

Accordingly, compounds of Formula 1 include those wherein,

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino;

R² and R³ are independently hydrogen, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH R ;

R is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH2R¹;

R is hydrogen, aryl, or heteroaryl, with the proviso that R⁵ is not a monocyclic 6- membered aromatic ring containing one or more nitrogen atoms and wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R^!;

R⁶ is hydrogen, aryl, heteroaryl or C(=O)X; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R^J;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl. Preferred compounds of Formula 1 include those wherein aryl is phenyl or polyphenyl and heteroaryl is imidazole, benzimidazole, thiophene, benzothiophene, furan, benzofuran, oxazole, benzoxazole, thiazole, benzothiazole, isoxazole, or benzisoxazole wherein said aryl and heteroaryl groups are optionally substituted. Preferred optional substituents include C(=O)X and CH2R¹, wherein

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; and

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino.

Preferred compounds of Formula 1 include those wherein R⁶ is phenyl, polyphenyl, imidazole, benzimidazole, thiophene, benzothiophene, furan, benzofuran, oxazole, benzoxazole, thiazole, benzothiazole, isoxazole, or benzisoxazole wherein said aryl and heteroaryl groups are optionally substituted. Preferred optional substituents include C(=O)X and CH₂R^!, wherein

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; and

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino.

Preferred compounds of Formula 1 include those wherein,

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino;

R² and R³ are independently hydrogen, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH_..R¹;

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R¹; R⁵ is hydrogen, aryl, or heteroaryl with the proviso that R⁵ is not a monocyclic 6- membered aromatic ring containing one or more nitrogen atoms and wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R²;

R⁶ is heteroaryl, wherein said heteroaryl is imidazole, benzimidazole, thiophene, benzothiophene, furan, benzofuran, oxazole, benzoxazole, thiazole, benzothiazole, isoxazole, or benzisoxazole and wherein said heteroaryl group is optionally substituted. Preferred optional substituents include C(=O)X and CH2R¹;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

Preferred compounds of Formula 1 include those wherein,

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino;

R² and R³ are independently hydrogen, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH^R¹;

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R¹;

R⁵ is hydrogen, aryl, or heteroaryl; with the proviso that R⁵ is not a monocyclic 6- membered aromatic ring containing one or more nitrogen atoms and wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R¹;

R⁶ is substituted phenyl or optionally substituted polyaryl. Preferred optional substituents include C(=O)X and CH2R¹;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

Preferred compounds of Formula 1 include those wherein,

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino; R and R are independently hydrogen, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH R^];

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or

R⁵ is hydrogen, aryl, or heteroaryl with the proviso that R⁵ is not a monocyclic 6- membered aromatic ring containing one or more nitrogen atoms and wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R¹; and

R⁶ is C(=O)X wherein X is alkyl, -OH, or NH₂.

The fluorophores of the present invention include compounds of Formula 2:

Formula 2 wherein

R¹ is hydrogen, -O-alkyl, -O- aryl, -O- -heteroaryl;

R², R³ and R⁴ are independently hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy;

R⁷ is hydrogen, aryl, heteroaryl, or C(=O)X;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; provided that when R², R³, and R⁴ are alkoxy or hydrogen, R⁷ is C(=O)X, aryl or heteroaryl; and

1 7 ά. provided that when R , R , R and R are hydrogen, R is not hydrogen or alkyl.

The fluorophores of the present invention include compounds of Formula 2:

Formula 2 wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl;

R², R³ and R⁴ are independently hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy;

R⁷ is hydrogen, aryl, heteroaryl, or C(=O)X;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; provided that when R , R , and R are alkoxy or hydrogen, R is C(=O)X, aryl or heteroaryl; and provided that when R¹, R², and R³ are hydrogen, R⁴ is not hydrogen or alkyl.

The fluorophores of the present invention include compounds of Formula 2:

Formula 2 wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl;

R², R³ and R⁴ are independently hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy;

R⁷ is hydrogen, aryl, heteroaryl, or C(=O)X;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; provided that when R², R³, and R⁴ are alkoxy or hydrogen, R⁷ is C(=O)X, aryl or heteroaryl, wherein aryl is not unsubstituted phenyl; and provided that when R¹, R², R³, and R⁷ are hydrogen, R⁴ is not hydrogen or alkyl.

The fluorophores of the present invention include compounds of Formula 2:

Formula 2

wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl;

R , R and R are independently hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy;

R⁷ is hydrogen, aryl, heteroaryl, or C(=O)X;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; provided that when R², R³, or R⁴ is alkoxy, R⁷ is C(=O)X, aryl or heteroaryl; and provided that when R¹, R², and R³ are hydrogen, R⁴ is not hydrogen or alkyl.

The fluorophores of the present invention include compounds of Formula 2:

Formula 2 wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl;

R², R³ and R⁴ are independently hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy;

R⁷ is hydrogen, aryl, heteroaryl, or C(=O)X;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; provided that when R², R³, and R are alkoxy or hydrogen, R⁷ is C(=O)X, aryl or heteroaryl, wherein aryl is not unsubstituted phenyl; and provided that when R¹, R², and R³ are hydrogen, R⁴ is not hydrogen or alkyl.

The fluorophores of the present invention include compounds of Formula 2:

Formula 2

wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl;

R², R³ and R⁴ are independently hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy;

R⁷ is hydrogen, aryl, heteroaryl, or C(=O)X;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; provided that when R², R³, or R⁴ is alkoxy, R⁷ is C(=O)X, aryl or heteroaryl, wherein aryl is not unsubstituted phenyl; and provided that when R¹, R², and R³ are hydrogen, R⁴ is not hydrogen or alkyl. A preferred group of compounds include compounds of Formula 2 wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl,

-NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

A preferred group of compounds include compounds of Formula 2 wherein

R² and R³ are independently hydrogen, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R^!; X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl,

-NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; and

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino.

A preferred group of compounds include compounds of Formula 2 wherein

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CHzR¹;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; and

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino.

A preferred group of compounds include compounds of Formula 2 wherein

R⁷ is hydrogen, C(=O)X, aryl, or heteroaryl; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R^J;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl,

-NYheteroaryl; Y is hydrogen, alkyl, aryl, or heteroaryl; and

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino.

Accordingly the present invention provides compounds of Formula 1 wherein,

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino;

R² and R³ are independently hydrogen, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R ;

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CHzR¹.

R is C(=O)X, hydrogen, aryl, or heteroaryl; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R¹;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

Preferred compounds of Formula 2 include those wherein aryl is phenyl or polyphenyl and heteroaryl is imidazole, benzimidazole, thiophene, benzothiophene, furan, benzofuran, oxazole, benzoxazole, thiazole, benzothiazole, isoxazole, or benzisoxazole wherein said aryl and heteroaryl groups are optionally substituted. Preferred optional substituents include C(=O)X and CEfeR¹, wherein

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl; and

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino.

Preferred compounds of Formula 2 include those wherein R⁷ is phenyl, polyphenyl, imidazole, benzimidazole, thiophene, benzothiophene, furan, benzofuran, oxazole, benzoxazole, thiazole, benzothiazole, isoxazole, or benzisoxazole wherein said aryl and heteroaryl groups are optionally substituted. Preferred optional substituents include C(=O)X and

wherein

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl; and

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino.

Preferred compounds of Formula 2 include those wherein,

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino;

R² and R³ are independently hydrogen, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R¹;

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CHzR¹;

R⁷ is heteroaryl, wherein said heteroaryl is imidazole, benzimidazole, thiophene, benzothiophene, furan, benzofuran, oxazole, benzoxazole, thiazole, benzothiazole, isoxazole, or benzisoxazole and wherein said heteroaryl group is optionally substituted. Preferred optional substituents include C(=O)X and CH₂R¹;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

Preferred compounds of Formula 2 include those wherein,

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino;

R² and R³ are independently hydrogen, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R*; R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CHsR¹;

R⁷ is substituted phenyl or optionally substituted polyaryl. Preferred optional substituents include C(=O)X and CH₂R^!;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

Preferred compounds of Formula 2 include those wherein,

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino;

R and R are independently hydrogen, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R^J;

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CHzR¹;

R⁷ is C(=O)X wherein X is -OH, -O-alkyl, or -NH₂.

The present invention provides compounds of Formula 3

wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy;

R⁵ is hydrogen, aryl, or heteroaryl; and

R⁶ is hydrogen, aryl, heteroaryl, or C(=O)X; X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; with the proviso that both R⁵ and R⁶ cannot both be hydrogen.

A preferred group of compounds include compounds of Formula 3 wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl,

-NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

A preferred group of compounds include compounds of Formula 3 wherein

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or

CH₂R¹;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl,

-NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; and

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino.

A preferred group of compounds include compounds of Formula 3 wherein

R⁵ is hydrogen, aryl, or heteroaryl; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CHjR¹;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl,

-NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; and

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino. A preferred group of compounds include compounds of Formula 3 wherein

R⁶ is hydrogen, C(=O)X, aryl, or heteroaryl; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R^!;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl,

-NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; and

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino.

Accordingly the present invention provides compounds of Formula 3 wherein,

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino;

R is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R¹;

R⁵ is hydrogen, aryl, or heteroaryl; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R^!;

R⁶ is C(=O)X, hydrogen, aryl, or heteroaryl; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R^];

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

Preferred compounds of Formula 3 include those wherein aryl is phenyl or polyphenyl and heteroaryl is imidazole, benzimidazole, thiophene, benzothiophene, furan, benzofuran, oxazole, benzoxazole, thiazole, benzothiazole, isoxazole, or benzisoxazole wherein said aryl and heteroaryl groups are optionally substituted. Preferred optional substituents include C(=O)X and wherein

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; and R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino.

Preferred compounds of Formula 3 include those wherein R⁶ is phenyl, polyphenyl, imidazole, benzimidazole, thiophene, benzothiophene, furan, benzofuran, oxazole, benzoxazole, thiazole, benzothiazole, isoxazole, or benzisoxazole wherein said aryl and heteroaryl groups are optionally substituted. Preferred optional substituents include C(=O)X and wherein

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; and

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino.

Preferred compounds of Formula 3 include those wherein,

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino;

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CHsR¹;

R⁵ is hydrogen, aryl, or heteroaryl; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R^!;

R⁶ is heteroaryl, wherein said heteroaryl is imidazole, benzimidazole, thiophene, benzothiophene, furan, benzofuran, oxazole, benzoxazole, thiazole, benzothiazole, isoxazole, or benzisoxazole and wherein said heteroaryl group is optionally substituted. Preferred optional substituents include C(=O)X and CH₂R^!;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

Preferred compounds of Formula 1 include those wherein, R is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino;

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R ;

R is hydrogen, aryl, or heteroaryl; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH_..R¹;

R⁶ is substituted phenyl or optionally substituted polyaryl. Preferred optional substituents include C(=O)X and CH₂R¹;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

Preferred compounds of Formula 3 include those wherein,

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino;

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH.R¹;

R⁵ is hydrogen, aryl, or heteroaryl; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R ;

R⁶ is C(=O)X wherein X is -OH, -O-alkyl, or -NH₂.

The present invention provides compounds of Formula 4:

wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; and

R⁶ is C(=O)X. hydrogen, aryl, or heteroaryl.

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

A preferred group of compounds include compounds of Formula 4 wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl,

-NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

A preferred group of compounds include compounds of Formula 4 wherein

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or

CH.R¹;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl,

-NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; and

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino.

A preferred group of compounds include compounds of Formula 4 wherein

R⁶ is C(=O)X, hydrogen, aryl, or heteroaryl; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R^J;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl,

-NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; and R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino.

Accordingly the present invention provides compounds of Formula 4 wherein,

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino;

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CHsR¹;

R⁶ is C(=O)X, hydrogen, aryl, or heteroaryl; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R¹;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

Preferred compounds of Formula 4 include those wherein aryl is phenyl or polyphenyl and heteroaryl is imidazole, benzimidazole, thiophene, benzothiophene, furan, benzofuran, oxazole, benzoxazole, thiazole, benzothiazole, isoxazole, or benzisoxazole wherein said aryl and heteroaryl groups are optionally substituted. Preferred optional substituents include C(=O)X and CH₂R¹, wherein

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl; and

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino.

Preferred compounds of Formula 4 include those wherein R is phenyl, polyphenyl, imidazole, benzimidazole, thiophene, benzothiophene, furan, benzofuran, oxazole, benzoxazole, thiazole, benzothiazole, isoxazole, or benzisoxazole wherein said aryl and heteroaryl groups are optionally substituted. Preferred optional substituents include C(=O)X and wherein X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl; and

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino.

Preferred compounds of Formula 4 include those wherein,

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino;

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH.R¹;

R is heteroaryl, wherein said heteroaryl is imidazole, benzimidazole, thiophene, benzothiophene, furan, benzofuran, oxazole, benzoxazole, thiazole, benzothiazole, isoxazole, or benzisoxazole and wherein said heteroaryl group is optionally substituted. Preferred optional substituents include C(=O)X and CH R^!;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

Preferred compounds of Formula 4 include those wherein,

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino;

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CHsR¹;

R⁶ is substituted phenyl or optionally substituted polyaryl. Preferred optional substituents include C(=O)X and CH₂R^X;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl. Preferred compounds of Formula 4 include those wherein,

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino;

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R^!;

R⁶ is C(=O)X wherein X is -OH, -O-alkyl, -or -NH₂..

Representative fluorophores of the present invention include compounds having Formula

5:

Formula 5 wherein A is C(=O)X, aryl or heteroaryl,

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

In a preferred embodiment of Formula 5, A is C(=O)X wherein X is -OH, -NH₂, or -Oalkyl.

In a preferred embodiment of Formula 5, A is phenyl or polyphenyl wherein said phenyl or polyphenyl group is optionally substituted.

In a preferred embodiment of Formula 5, A is imidazole, benzimidazole, thiophene, benzothiophene, furan, benzofuran, oxazole, benzoxazole, thiazole, benzothiazole, isoxazole, or benzisoxazole wherein said heteroaryl group is optionally substituted.

Preferred optional substituents on the aryl and heteroaryl groups include C(=O)X and CH₂R¹ wherein

X is -O-alkyl, -OH, -NH₂, -O-aryl, -O-heteroaryl, -NYalkyl, -NYaryl, - NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl; and R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino.

In a preferred embodiment, X is phenyl, benzofuranyl, CO₂CH₃, or benzoxazolyl.

Representative fluorophores of the present invention include compounds having

Formulas 6

Formula 6 wherein A is C(=O)X, aryl or heteroaryl,

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

In a preferred embodiment of Formula 6, A is C(=O)X wherein X is -OH, -NH₂, or -Oalkyl.

In a preferred embodiment of Formula 6, A is phenyl or polyphenyl wherein said phenyl or polyphenyl groups are optionally substituted.

In a preferred embodiment of Formula 6, A is imidazole, benzimidazole, thiophene, benzothiophene, furan, benzofuran, oxazole, benzoxazole, thiazole, benzothiazole, isoxazole, or benzisoxazole wherein said heteroaryl group is optionally substituted.

In a preferred embodiment of Formula 6, A is phenyl or polyphenyl wherein said phenyl or polyphenyl group is optionally substituted

In a preferred embodiment, A is phenyl, benzofuranyl, CO₂CH₃, or benzoxazolyl.

Preferred optional substituents on the aryl and heteroaryl groups include C(=O)X and CH₂R^! wherein

X is -O-alkyl, -OH, -NH₂, -O-aryl, -O-heteroaryl, -NYalkyl, -NYaryl, - NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl; and R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino.

Representative fluorophores of the present invention include compounds having Formula 7:

Formula 7 wherein A is hydrogen, C(=O)X, aryl or heteroaryl,

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

In a preferred embodiment of Formula 7, A is C(=O)X wherein X is -OH, -NH , or -Oalkyl.

In a preferred embodiment of Formula 7, A is phenyl or polyphenyl wherein said phenyl or polyphenyl group is optionally substituted.

In a preferred embodiment of Formula 7, A is imidazole, benzimidazole, thiophene, benzothiophene, furan, benzofuran, oxazole, benzoxazole, thiazole, benzothiazole, isoxazole, or benzisoxazole wherein said heteroaryl group is optionally substituted.

In a preferred embodiment, A is phenyl, benzofuranyl, CO₂CH₃, or benzoxazolyl.

Preferred optional substituents on the aryl and heteroaryl groups include C(=O)X and CH₂R* wherein

X is -O-alkyl, -OH, -NH₂, -O-aryl, -O-heteroaryl, -NYalkyl, -NYaryl, - NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl; and

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino. Representative fluorophores of the present invention also include compounds having Formula 8:

wherein A is hydrogen, C(=O)X, aryl or heteroaryl,

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

In a preferred embodiment of Formula 8, A is C(=O)X wherein X is -OH, -NH₂, or -Oalkyl.

In a preferred embodiment of Formula 8, A is phenyl or polyphenyl wherein said phenyl or polyphenyl group is optionally substituted.

In a preferred embodiment of Formula 8, A is imidazole, benzimidazole, thiophene, benzothiophene, furan, benzofuran, oxazole, benzoxazole, thiazole, benzothiazole, isoxazole, or benzisoxazole wherein said heteroaryl group is optionally substituted.

In a preferred embodiment, A is phenyl, benzofuranyl, CO₂CH₃, or benzoxazolyl.

Preferred optional substituents on the aryl and heteroaryl groups include C(=O)X and CH₂R^! wherein

X is -O-alkyl, -OH, -NH₂, -O-aryl, -O-heteroaryl, -NYalkyl, -NYaryl, - NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl; and

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino.

The present invention also provides salts and esters of Formulas 1 to 8 that have fluorescent properties. Such salts include, for example, salts that can be formed where acidic protons present in the compounds are capable of reacting with inorganic or organic bases. Suitable inorganic salts include, for example, those formed with the alkali metals or alkaline earth metals, e.g. sodium and potassium, magnesium, calcium, and aluminum. Suitable organic salts include, for example, those formed with organic bases such as the amine bases, e.g. ethanolamine, diethanolamine, triethanolamine, tromethamine, N methylglucamine, and the like. Pharmaceutically acceptable salts can also include acid addition salts formed from the reaction of basic moieties, such as amines, in the parent compound with inorganic acids (e.g. hydrochloric and hydrobromic acids) and organic acids (e.g. acetic acid, citric acid, maleic acid, and the alkane- and arene-sulfonic acids such as methanesulfonic acid and benzenesulfonic acid). Pharmaceutically acceptable esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the compounds, e.g. C^ alkyl esters. When there are two acidic groups present, an acceptable salt or ester can be a mono-acid-mono-salt or ester or a di-salt or ester; and similarly where there are more than two acidic groups present, some or all of such groups can be salified or esterified. Compounds named in this invention can be present in unsalified or unesterified form, or in salified and/or esterified form, and the naming of such compounds is intended to include both the original (unsalified and unesterified) compound and its salts and esters.

The terms "alkyl" and "alkylene," as used herein, whether used alone or as part of another group, refer to substituted or unsubstituted aliphatic hydrocarbon chains, the difference being that alkyl groups are monovalent (i.e., terminal) in nature whereas alkylene groups are divalent and typically serve as linkers. Both include, but are not limited to, straight and branched chains containing from 1 to about 12 carbon atoms, preferably 1 to 6 carbon atoms, unless explicitly specified otherwise. For example, methyl, ethyl, propyl, isopropyl, butyl, /-butyl and t- butyl are encompassed by the term "alkyl." Specifically included within the definition of "alkyl" are those aliphatic hydrocarbon chains that are optionally substituted.

The carbon number as used in the definitions herein refers to carbon backbone and carbon branching, but does not include carbon atoms of the substituents, such as alkoxy substitutions and the like.

The term "aryl", as used herein, whether used alone or as part of another group, is defined as a substituted or unsubstituted aromatic hydrocarbon ring group having 5 to about 50 carbon atoms with from about 6 to about 14 carbon atoms or about 6 to about 10 carbon atoms being preferred. The "aryl" group can have a single ring or multiple condensed rings. The term "aryl" includes, but is not limited to phenyl, -naphthyl, β-naphthyl, biphenyl, anthryl, tetrahydronaphthyl, phenanthryl, fluorenyl, indanyl, biphenylenyl, and acenaphthenyl. Specifically included within the definition of "aryl", unless otherwise specified, are those aromatic groups that are optionally substituted. As used herein, the term "heteroaryl", whether used alone or as part of another group, is defined as a substituted or unsubstituted aromatic heterocyclic ring system (monocyclic or bicyclic). Heteroaryl groups can have, for example, from about 3 to about 50 carbon atoms, with from about 4 to about 10 being preferred. Representative heteroaryl groups include furan, thiophene, indole, azaindole, oxazole, thiazole, isoxazole, isothiazole, imidazole, N- methylimidazole, pyridine, pyrimidine, pyrazine, pyrrole, N-methylpyrrole, pyrazole, N- methylpyrazole, 1,3,4-oxadiazole, 1,2,4-triazole, 1 -methyl- 1,2,4-triazole, lH-tetrazole, 1- methyltetrazole, benzoxazole, benzothiazole, benzofuran, benzisoxazole, benzimidazole, N- methylbenzimidazole, azabenzimidazole, indazole, quinazoline, quinoline, and isoquinoline. Bicyclic aromatic heteroaryl groups include phenyl, pyridine, pyrimidine or pyridizine rings that are (a) fused to a 6-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom; (b) fused to a 5- or 6-membered aromatic (unsaturated) heterocyclic ring having two nitrogen atoms; (c) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one nitrogen atom together with either one oxygen or one sulfur atom; or (d) fused to a 5-membered aromatic (unsaturated) heterocyclic ring having one heteroatom selected from O, N or S. Specifically included within the definition of "heteroaryl" are those aromatic heterocyclic rings that are optionally substituted.

The term "alkoxy" as used herein, refers to the group R_a-O- wherein R_a is an alkyl group as defined above. Specifically included within the definition of "alkoxy" are those alkoxy groups that are optionally substituted.

The term "polyalkoxy" refers to polyethylene glycol, e.g., (poly-(OCH₂CH₂)_n-R) where R is alkyl and n is from 1 to about 10.

The term "polyphenyl" refers to a pheny ring with one or more phenyl rings attached to it, wherein said phenyl ring can have, for example, from one to about four additional phenyl rings attached to it.

The term "halogen" or "halo" refers to chlorine, bromine, fluorine, and iodine.

The term "analyte" refers to the substance to be detected that can be present in a sample. The term analyte is used to refer to any biological or chemical molecule. The term analyte, for example, refers to ions, including cations such as Li⁺, Ca²⁺, Na⁺, Mg²⁺, Hg(II), Pb(II), Cd(-Q), As(HI), Cu(II), Ni(II) and other metals of biological and environmental interest. The term analyte also includes, for example, DNA and protein.

The term "sample" as used herein includes both biological and environmental samples. The sample can comprise any number of things, including but not limited to, bodily fluids (including, but not limited to blood, urine, semen, lymph, saliva, anal and vaginal secretions, perspiration and semen), environmental samples (including, but not limited to air, agricultural, water, and soil samples); biological warfare agent samples, research samples, purified samples, such as purified genomic DNA, RNA, proteins, raw samples (such as bacteria, virus, genomic DNA).

The present invention also provides intermediates used to synthesize fluorophores of the present invention. Intermediates of the present invention include Formula 9 and Formula 10

Formula 9 Formula 10

wherein B is aryl, heteroaryl or C(=O)Z, wherein Z is -NH₂, -O-aryl, -O-heteroaryl, -NYalkyl, -NYaryl, -NYheteroaryl; and Y is hydrogen, alkyl, aryl, or heteroaryl.

In a preferred embodiment, B is phenyl or polyphenyl and heteroaryl is imidazole, benzimidazole, thiophene, benzothiophene, furan, benzofuran, oxazole, benzoxazole, thiazole, benzothiazole, isoxazole, or benzisoxazole wherein said aryl and heteroaryl groups are optionally substituted. Preferred optional substituents include C(=O)X and

wherein

X is -O-alkyl, -OH, -NH₂, -O-aryl, -O-heteroaryl, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl; and

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl, and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino.

In another preferred embodiment B is iodine, phenyl, or CO₂CH₃.

In all of the embodiments listed herein, the alkyl carbon chains are preferably from 1 to about 12 carbon atoms, more preferably from 1 to about 6 carbon atoms. In all of the embodiments listed herein, the aryl rings are preferably from 6 to about 14 carbon atoms, more preferably from 6 to about 10 carbon atoms. The following synthetic schemes can be used to prepare representative fluorophores of the present invention. The skilled practitioner will know how to make use of variants of these process steps. Scheme 1 demonstrates how to prepare a representative intermediate of the present invention.

Scheme 1

Synthesis of 4 begins with pyridine derivative citrazinic acid. Treatment with POCl₃ at elevated temperature afforded the corresponding 2-6, dichloroisonicotinoyl chloride. To facilitate purification, the reaction was quenched with methanol; methyl ester 1 was isolated in 76% yield after passage through a plug of silica gel to remove colored impurities. Saponification then provided acid 2 in quantitative yield without purification. Transformation to 3 was effected by conversion to the acyl azide, thermal Curtius rearrangement, and hydrolysis of the resulting trifluoroacetamide to provide 2,6-dichloro-4-aminopyridine (3), (Pfister, J. R., Wymann, W. E. Synthesis 1983, 38). While this method for converting 2 to 3 is nominally 3 steps, it requires only a single extractive workup and the steps can thus be carried out in rapid succession. 3 was converted directly to 4 by diazotization and reaction with potassium iodide (it is necessary to stir 3 in cold hydrochloric acid for several hours prior to diazotization in order to obtain an acceptable yield) providing 4 in reasonable yield and excellent purity after trituration with acetone. This short sequence of reactions allows preparation of 4 in -35% overall yield, requires no chromatography beyond a single filtration through a plug of silica gel, and has allowed the routine preparation of 5-10 g quantities of this intermediate. Unlike many 4-halopyridines, 4 is stable for several months at room temperature if protected from light. 4 can be ready transformation to fluorophores of the present invention.

Representative fluorophores can be prepared using one or more of the following synthetic schemes. The skilled practitioner will know how to make use of variants of these process steps. In the following synthetic schemes, THF is tetrahydrofuran.

Taking advantage of the higher reactivity of iodine relative to chlorine in Pd(II) catalyzed reactions, it is possible to selectively replace iodine with an aryl or heteroaryl substituent without displacing the chlorine substituents.

The present invention provides methods comprising reacting an intermediate of Formula 9 or Formula 10 with ArB(OH)₂ or ArZnCI and an organic solvent. Analyte binding domains can be attached to ArB(OH)₂ or ArZnCI prior to coupling to dichloropyridine.

The fluorophores of the present invention can act as sensors, e.g., chemosensors or biosensors, that are triggered by analyte molecules to generate a measurable signal, e.g., fluorescence. The sensors of the present invention comprise a core pyridine molecule attached to one or more substituents. The C(=O)X substituents of the present invention are particularly desirable because they permit the fluorophore to be attached to other molecules, biomolecules (e.g., DNA, proteins, oligonucleotides, and the like), polymers and surfaces (e.g., glass, metal, and the like). Such attachment is desirable for application of such fluorophores as, for examples, labels or in sensing devices.

In certain embodiments of the present invention, the substituents therefore can comprise one or more analyte binding groups. For example, an analyte binding group such as a polyether metal binding domain can be attached to a methyl substituent on a phenyl ring as shown in Formula 11. R₆ is as described above.

The fluorophores of the present invention include fluorophores attached to or comprising one or more analyte-binding groups. Analyte-binding groups useful in the present invention can include any chemical group that can be incorporated into a fluorophore, e.g., by covalent bonding, and react with an analyte to be quantified.

Several analyte-binding moieties that can be incorporated into the sensors of the invention are known in the art. One such analyte-binding moiety is the ethylene diamine group. This particular group is useful for chelating divalent cations such as Zn²⁺, Ca²⁺, or Mg²⁺. Another exemplary analyte-binding moiety is guanidinium which is useful for binding acids such as carboxylic acid. Yet another exemplary analyte-binding moiety is trimethyl ethylenediamine which is useful for binding various metal ions including, for example, Ag(I), Zn(II), Hg(I), Hg(II), Fe(I), Fe(II), Co(II), Cu(I), Cu(H), Ni(II), and Cd(II). Many other analyte- binding moieties or groups useful in the present invention are known.

The fluorophores of the present invention can be used to determine the presence and/or concentration of an analyte in a sample. Typically, a small amount of the fluorophore (e.g., in solution or organic solvent) is added to the sample and the sample is analyzed for a signal emitted from the fluorophore. The concentration of fluorophore in the sample can be, for example, from about 10^"6 M to about 10^"4 M. The signal can be measured by measuring fluorescence, i.e., a change in fluorescence emission or an emission wavelength shift. Methods of detecting fluorescence are known in the art and as such are not described in detail. For example, methods of performing assays on fluorescent materials are described in, Brand, L. et al. (eds.), "Fluorescence Spectroscopy" in Methods in Enzymology, Academic Press, 1997, Vol. 278; Dewey, T.G. (ed.), Biophysical and Biochemical Aspects of Fluorescence Spectroscopy, Plenum Publishing, 1991; Guilbault, G.G. (ed.), Practical Fluorescence, Second Edition, Marcel Dekker, 1990; Lakowicz, J.R. (ed.), Topics in Fluorescence Spectroscopy: Techniques, Vol. 1, 1991; Principles, Vol. 2, 1991; Biochemical Applications, Vol. 3, 1992; Probe Design and Chemical Sensing, Vol. 4, 1994; Nonlinear and Two-Photon Induced Fluorescence, Vol. 5, 1997; Protein Fluorescence, Plenum Publishing, Vol. 6, 2000; Lakowicz, J.R., Principles of Fluorescence Spectroscopy, Second Edition, Plenum Publishing, 1999; Naleur, B., Molecular Fluorescence: Principles and Applications, John Wiley and Sons, 2002, each of which is incorporated by reference herein in its entirety and for all purposes.

The compounds of the present invention can be used in any applications that use fluorescence. For example, the compounds of the present invention can be used in cellular imaging techniques (de Silva et a ,. Proc. Nat. Acad. Sci. 1999, 96, 8336-8337; Naleur, B., Leray, I. Coord. Chem. Rev. 2000, 205, 3-40; each of which is incorporated by reference in its entirety and for all purposes). In some embodiments, therefore, the fluorophores can be used to image a selected biological target and/or region of a subject. Region of a subject refers to a particular area or portion of the subject and in some instances to regions throughout the entire subject.

The following Examples of specific embodiments for carrying out the present invention are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

The disclosures of all publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety and for all purposes

EXAMPLES Example 1: Synthesis of fluorescent chemosensor substructures 1-4.

1 x= -Q O— 3 X= — Q O-

2 X= CH₃ 4 X= CH₃

Reagents and conditions: a) NaH, BnOH, then 2,6-dibromopyridine (55%); b) n-BuLi, ArBr; ZnCI₂; then 2-benzyloxy-6-bromopyridine and cat. Pd(PPh₃)₄ (70%); c) H₂, cat. Pd/C; d) Tf₂0, Py (79%, two steps); e) ArB(OH)₂, cat. Pd(OAc)₂, cat. BINAP, Cs₂C0₃ (64%); f) n-BuLi, ArBr; ZnCI₂; then 2-bromopyridine and cat. Pd(PPh₃)₄ (46%); g) as (f) (57%).

Example 2: K_a values (M^"1) for metal-ion binding by compounds 1-4. ion 1 2 3 4

Li⁺ 6700 9 39 6

Na⁺ — 29 20 3

Mg ⁺ 830 63 64 32

Ca ⁺ 670 9 240 9

Example 3: Synthesis of representative compounds 7-11

Reagents and conditions: a) n-BuLi, ArBr; ZnCI₂; then 2,6- dibromopyridine and cat. Pd(PPh₃)₄ (95%); b) LiOH, THF/H₂0 (100%); c) (COCI)₂; NaN₃; TFA; d) K₂C0₃, CH₃OH (78%); e) HCI, CH₃CN, NaN0₂; Kl (52%); f) n-BuLi, ArBr; ZnCI₂; then 13 and cat. Pd(PPh₃)₄ (98%); g) ArB(OH)₂, cat. Pd₂(dba)₃, cat. P(t-Bu)₃, Cs₂C0₃, (82%); h) as (f) (71 %); i) as (g) (31%); j) n-BuLi, ArBr; ZnCI₂; then 12 (80%); k) 2- aminophenol, EDCI (97%); I) 230°C (87%); m) ArB(OH)₂, cat. Pd₂(dba)₃, cat. [HP(t-Bu)₃]BF₄, Cs₂C0₃ (78%).

Example 4: Select spectral parameters for representative compounds 7-11 in CH₃CN. Excitation and emission maxima are rounded to the nearest 5 nm. Quantum yields were determined by standard methods (J.R. Lakowicz, Principles of Fluorescence Spectroscopy, 2^nd Ed, Kluwer Academic, New York, 1999, herein incorporated by reference in its entirety and for all purposes). Increasing conjugation or polarization of the core biarylpyridine by substitution at the 4-position of the pyridine can lead to emission red-shifts of almost 100 nm as illustrated by compounds 7-11. 7 8 9 10 11 ε (xlO^" 14. 9.3 33.8 4.4 14.6

³) 8 _exc 290 305 310 315 300

(nm)

^-em 350 380 400 420 440

(nm)

Δλ 60 75 90 105 140

(nm)

Φ 0.0 0.20 0.21 0.23 0.14

Claims

What is Claimed:

A compound having formula 1:

or a salt or ester form thereof, wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl;

R², R³ and R⁴ are independently hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy;

R⁵ is hydrogen, aryl, or heteroaryl with the proviso that R⁵ is not a monocyclic 6- membered aromatic ring containing one or more nitrogen atoms;

R⁶ is hydrogen, aryl, heteroaryl, or C(=O)X;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; wherein both R⁵ and R⁶ cannot both be hydrogen provided that when R², R³ and R⁴ are hydrogen or alkoxy and R⁵ is alkoxyphenyl substituted with one alkyl group on the phenyl ring, R⁶ is aryl, heteroaryl or C(=O)X; provided that when R², R³ and R⁴ are hydrogen or alkoxy and R⁵ is a phenyl ring substituted with C(=O)alkyl, R⁶ is heteroaryl, C(=O)X, or aryl, wherein aryl is not unsubstituted phenyl; and provided that when R¹, R², and R³ are hydrogen and R⁴ is hydrogen or alkyl, R⁵ is not phenyl, tolyl, or 2,6-dimethylphenyl.

A compound of claim 1 provided that when R , R and R are hydrogen or alkoxy and R⁵ is alkox yypphheennyyll ssuubbssttiittuutteedd wwiitthh oonnee aallkkyyll ggrroouupp oonn tthhee ; phenyl ring, R⁶ is C(=O)X, heteroaryl or aryl wherein aryl is not unsubstituted phenyl; and when R², R³ and R⁴ are hydrogen or alkoxy and R⁵ is a phenyl ring substituted with C(=O)alkyl, R⁶ is C(=O)X, heteroaryl, or aryl, wherein aryl is not unsubstituted phenyl

3. A compound having formula 1 :

Formula 1 or a salt or ester form thereof, wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl;

R², R³ and R⁴ are independently hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy;

R is hydrogen, aryl, or heteroaryl, with the proviso that R is not a monocyclic 6- membered aromatic ring containing one or more nitrogen atoms;

R⁶ is hydrogen, aryl, heteroaryl, or C(=O)X;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; wherein both R⁵ and R⁶ cannot both be hydrogen, provided that when R², R³ or R⁴ is alkoxy and R⁵ is a phenyl ring substituted with alkoxy, R is aryl, C(=O)X, or heteroaryl; when R², R³ or R⁴ is alkoxy and R⁵ is a phenyl ring substituted with C(=O)alkyl, R⁶ is heteroaryl, C(=O)X, or aryl, wherein aryl is not unsubstituted phenyl; and provided that when R¹, R², and R³ are hydrogen and R⁴ is hydrogen or alkyl, R⁵ is not phenyl, tolyl, or 2,6-dimethylphenyl

A compound of claim 3 provided that when R , R or R is alkoxy and R is a phenyl ring sub bssttiittuutteedd v w. ith alkoxy or C(=O)alkyl, R⁶ is C(=O)X, heteroaryl, or aryl, wherein aryl is not unsubstituted.

5. A compound of claims 1 to 4 wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino;

R and R are independently hydrogen, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R¹;

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R¹;

R⁵ is hydrogen, aryl, or heteroaryl, with the proviso that R⁵ is not a monocyclic 6-membered aromatic ring containing one or more nitrogen atoms and wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R¹;

R⁶ is hydrogen, aryl, heteroaryl or C(=O)X; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R¹;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

6. A compound of claims 1 to 5 wherein R⁶ is phenyl, polyphenyl, imidazole, benzimidazole, thiophene, benzothiophene, furan, benzofuran, oxazole, benzoxazole, thiazole, benzothiazole, isoxazole, or benzisoxazole.

7. A compound having Formula 2:

Formula 2 or a salt or ester form thereof, wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl; R², R³ and R⁴ are independently hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy;

R⁷ is hydrogen, aryl, heteroaryl, or C(=O)X;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; provided that when R², R³, and R⁴ are alkoxy or hydrogen, R⁷ is C(=O)X, aryl or heteroaryl; and provided that when R¹, R², and R³ are hydrogen, R⁴ is not hydrogen or alkyl

8. A compound of claim 7, wherein when R², R³, and R⁴ are alkoxy or hydrogen, R⁷ is C(=O)X, aryl or heteroaryl, wherein aryl is not unsubstituted phenyl.

9. A compound having Formula 2:

Formula 2 or a salt oi ^■ ester form thereof, wherein

R¹ is hydrogen, -O-alkyl, -O- •aryl, -O-heteroaryl;

R², R³ and R⁴ are independently hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy;

R⁷ is hydrogen, aryl, heteroaryl, or C(=O)X;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; provided that when R², R³, or R⁴ is alkoxy, R⁷ is C(=O)X, aryl or heteroaryl; and provided that when R¹, R², and R³ are hydrogen, R⁴ is not hydrogen or alkyl

10. A compound of claim 9, wherein when R², R³, or R⁴ is alkoxy, R⁷ is C(=O)X, aryl or heteroaryl, wherein aryl is not unsubstituted phenyl.

11. A compound of claims 7 to 10 wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino;

R² and R³ are independently hydrogen, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CHsR¹;

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R¹;

R⁷ is hydrogen, aryl, heteroaryl or C(=O)X; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R^J;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

12. A compound of claims 7 tol 1 wherein R⁷ is phenyl, polyphenyl, imidazole, benzimidazole, thiophene, benzothiophene, furan, benzofuran, oxazole, benzoxazole, thiazole, benzothiazole, isoxazole, or benzisoxazole.

13. A compound having Formula 3

or a salt or ester form thereof, wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy;

R⁵ is hydrogen, aryl, or heteroaryl; and R⁶ is hydrogen, aryl, heteroaryl, or C(=O)X;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl;

Y is hydrogen, alkyl, aryl, or heteroaryl; with the proviso that both R⁵ and R⁶ cannot both be hydrogen.

14. A compound of claim 13 wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino;

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R¹;

R⁵ is hydrogen, aryl, or heteroaryl; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R^!;

R⁶ is hydrogen, aryl, heteroaryl or C(=O)X; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CBfcR¹;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

15. A compound of claims 13 or 14 wherein R⁶ is phenyl, polyphenyl, imidazole, benzimidazole, thiophene, benzothiophene, furan, benzofuran, oxazole, benzoxazole, thiazole, benzothiazole, isoxazole, or benzisoxazole.

16. A compound of claim 13 having Formula 4:

or a salt or ester form thereof, wherein R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; and

R⁶ is C(=O)X. hydrogen, aryl, or heteroaryl.

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

17 A compound of claim 16 wherein

R¹ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X and said alkyl group is optionally substituted with C(=O)X, alkoxy, polyalkoxy, or amino;

R⁴ is hydrogen, -O-alkyl, -O-aryl, -O-heteroaryl, alkyl, aryl, heteroaryl, amino, or alkoxy; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CH₂R¹;

R⁶ is hydrogen, aryl, heteroaryl or C(=O)X; wherein said aryl and heteroaryl groups are optionally substituted with C(=O)X or CE^R¹;

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

18. A compound of claims 16 or 17 wherein R⁶ is phenyl, polyphenyl, imidazole, benzimidazole, thiophene, benzothiophene, furan, benzofuran, oxazole, benzoxazole, thiazole, benzothiazole, isoxazole, or benzisoxazole.

19. A compound of claims 1 to 4 having formula 5

Formula 5 wherein A is C(=O)X, aryl or heteroaryl,

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl,

-NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

20. A compound of claims 1 to 4 having formula 6

Formula 6 wherein A is C(=O)X, aryl or heteroaryl,

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

21. A compound of claims 1 to 4 having formula 7

Formula 7 wherein A is hydrogen, C(=O)X, aryl or heteroaryl,

X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

22. A compound of claims 1 to 4 having formula 8

Formula 8 wherein A is hydrogen, C(=O)X, aryl or heteroaryl, X is -OH, -O-alkyl, -O-aryl, -O-heteroaryl, -NH₂, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

23. A compound having Formula 9 or Formula 10

Formula 9 Formula 10

wherein B is aryl, heteroaryl or C(=O)Z,

Z is -NH₂, -O-aryl, -O-heteroaryl, -NYalkyl, -NYaryl, -NYheteroaryl; and

Y is hydrogen, alkyl, aryl, or heteroaryl.

24. A compound having Formula 9 or Formula 10

Formula 9 Formula 10

wherein B is aryl, heteroaryl or C(=O)Z, wherein said aryl ring is not unsubstituted phenyl,

Z is -NH₂, -O-aryl, -O-heteroaryl, -NYalkyl, -NYaryl, -NYheteroaryl; and Y is hydrogen, alkyl, aryl, or heteroaryl.

25. A method comprising reacting a compound of claim 23 or 24 with ArB(OH)₂ or ArZnCI and an organic solvent.

26. A method of detecting the presence or absence or concentration of an analyte in a sample comprising adding a compound of claims 1 to 22 to a sample and measuring a signal from the compound.

27. The method of claim 26 wherein the step of measuring the signal is performed by measuring fluorescence emission from the sample.

28. A method of detecting a selected biological target comprising contacting the target with a compound of claims 1 to 22 and measuring a signal from the compound.

29. The method of claim 28 wherein the step of measuring the signal is performed by measuring fluorescence emission.

30. A method of imaging a selected biological target in a subject comprising administering to the subject a compound of claims 1 to 22.