Xanthene dyes having a fused (C) benzo ring

• US 4,945,171 A
• Filed: 08/10/1987
• Issued: 07/31/1990
• Est. Priority Date: 08/10/1987
• Status: Expired due to Term

• Current Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Haugland, Richard P., Whitaker, James
• Primary Examiner(s): Pal, Asok
• Application Number:
  US07/083,459
• Time in Patent Office: 1,086 Days
• Field of Search: 549/224, 549/297, 549/344, 546/15
• US Class Current: 549/224
• CPC Class Codes: C07D 493/10 Spiro-condensed systems
• CPC Class Codes: C09B 11/28 Pyronines ; Xanthon, thioxanthon, selenoxanthan, telluroxanthon dyes
• CPC Class Codes: G01N 33/84 involving inorganic compounds or pH

Abstract

Synthesis and applications of fluorescent dyes which are derivatives of benzo[c]xanthenes is described. The dyes exhibit pH dependent absorption and fluorescence spectra with pKas near the normal physiological range. Unlike fluorescein, the dyes exhibit emission of different characteristic wavelengths dependent on the pH of the medium. This permits several methods of measuring the pH of the medium in contact with the indicator including measuring two emissions with one excitation, selectively exiting the acid and base forms independently and measuring their emission at either single or dual wavelengths, or measuring the characteristic pH dependent absorption or fluorescence excitation spectral. Methods are presented for making the indicators permeant to cell membranes for the measurement of intracellular pH.

Claims

1. A compound of the formula:

##STR6## wherein;

R¹ is hydrogen, acetyl or methyl,

R² is hydroxyl, acetoxyl, dimethylamino, diethylamino, ethylamino or methoxyl

R³ is hydrogen, hydroxyl, halo, methyl or carboxyethyl

R⁴ is hydrogen,

R⁵ is hydrogen, carboxyl or acetoxymethoxycarbonyl, and

R⁶ is hydrogen, carboxyl or acetoxymethoxycarbonyl.

2. The compound of claim 1 wherein R¹, R³, R⁴, R⁵ and R⁶ are hydrogen, and R² is hydroxy.

3. The compound of claim 1 wherein R³, R⁴, R⁵ and R⁶ are hydrogen, R¹ is acetyl and R² is acetoxy.

4. The compound of claim 1 wherein R¹, R³, R⁴ and R⁵ are hydrogen, R² is hydroxy and R⁶ is carboxy.

5. The compound of claim 1 wherein R¹, R³, R⁴ and R⁶ are hydrogen, R² is hydroxy and R⁵ is carboxy.

6. The compound of claim 1 wherein R³, R⁴ and R⁵ are hydrogen, R¹ is acetyl, R² is acetoxy and R⁶ is carboxy.

7. The compound of claim 1 wherein R³, R⁴ and R⁶ are hydrogen, R¹ is acetyl, R² is acetoxy and R⁵ is carboxy.

8. The compound of claim 1 wherein R¹, R⁴ and R⁵ are hydrogen, R² is hydroxy, R³ is chloro and R⁶ is carboxy.

9. The compound of claim 1 wherein R¹, R⁴ and R⁶ are hydrogen, R² is hydroxy, R³ is chloro and R⁵ is carboxy.

10. The compound of claim 1 wherein R⁴ and R⁵ are hydrogen, R¹ is acetyl, R² is acetoxy, R³ is chloro and R⁶ is carboxy.

11. The compound of claim 1 wherein R⁴ and R⁶ are hydrogen, R¹ is acetyl, R² is acetoxy, R³ is chloro and R⁵ is carboxy.

12. The compound of claim 1 wherein R¹, R³, R⁴ and R⁵ are hydrogen, R² is N,N-dimethylamino and R⁶ is carboxy.

13. The compound of claim 1 wherein R¹, R³, R⁴ and R⁶ are hydrogen, R² is N,N-dimethylamino and R⁵ is carboxy.

14. The compound of claim 1 wherein R³, R⁴ and R⁵ are hydrogen, R¹ is acetyl, R² is N,N-dimethylamino and R⁶ is acetoxymethoxycarbonyl.

15. The compound of claim 1 wherein R³, R⁴ and R⁶ are hydrogen, R¹ is acetyl, R² is N,N-dimethylamino and R⁵ is acetoxymethoxycarbonyl.

16. The compound of claim 1 wherein R¹, R³, R⁴ and R⁵ are hydrogen, R² is N,N-diethylamino and R⁶ is carboxy.

17. The compound of claim 1 wherein R¹, R³, R⁴ and R⁶ are hydrogen, R² is N,N-diethylamino and R⁵ is carboxy.

18. The compound of claim 1 wherein R³, R⁴ and R⁵ are hydrogen, R¹ is acetyl, R² is N,N-diethylamino and R⁶ is acetoxymethoxycarbonyl.

19. The compound of claim 1 wherein R³, R⁴ and R⁶ are hydrogen, R¹ is acetyl, R² is N,N-diethylamino and R⁵ is acetoxymethoxycarbonyl.

20. The compound of claim 1 wherein R¹, R⁴ and R⁵ are hydrogen, R² is N-ethylamino, R³ is methyl and R⁶ is carboxy.

21. The compound of claim 1 wherein R¹, R⁴ and R⁶ are hydrogen, R² is N-ethylamino, R³ is methyl and R⁵ is carboxy.

22. A compound of the formula:

##STR7## wherein;

R¹ is hydrogen, acetyl or methyl,

R² is N,N-dialkylamine, of which each alkyl group consists of 3 methylene groups which, in conjunction with R³ and R⁴, complete two six-membered non-aromatic rings chemically bonded to the aromatic ring of benzo[c]xanthene, and

R⁵ and R⁶, which may be the same or different, are hydrogen, carboxyl or acetoxymethoxycarbonyl.

23. The compound of claim 22 wherein:

R¹ and R⁵ are hydrogen; and

R⁶ is carboxyl.

24. The compound of claim 22 wherein:

R¹ and R⁶ are hydrogen; and

R⁵ is carboxyl.

25. A compound of the formula:

##STR8## wherein;

R¹ is hydrogen, a metal or non-metal salt, an alkyl or aryl ether containing from 1 to 18 carbon atoms, or an ester of an aliphatic acid containing from 1 to 18 carbon atoms;

R² is hydroxyl, amino, or an amino modified by formation of an alkylether, alkylester, alkylamide or alkylamine derivative containing 1 to 18 carbon atoms;

R⁵ and R⁶, which may be the same or different, are carboxy, sulfo, or alkylamido containing 1 to 6 carbon atoms; and

R³, R⁴, R⁷ and R⁸, which may be the same or different, are alkoxyl containing from 1 to 6 carbon atoms, alkyl containing from 1 to 6 carbon atoms, haloalkyl containing from 1 to 6 carbon atoms, or halo.