Diaminorhodamine derivative

A compound represented by the following general formula:

wherein R

1 and R

2 represent amino groups present at adjacent positions each other on the phenyl ring; R

3, R

4, R

5 and R

6 independently represent a C

1-6 alkyl group; R

7, R

8, R

9 and R

10 independently represent a hydrogen atom, a C

1-6 alkyl group, an allyl group, or a halogen atom; R

11 represents a hydrogen atom or a C

1-18 alkyl group; and X

− represents an anion, and an agent for nitric oxide measurement comprising said compound.

The compound efficiently reacts with nitric oxide to give a fluorescent compound that emits strong fluorescence by irradiation with excitation light of a long wavelength. This fluorescent compound is characterized in that said compound is detectable in a fluorescence wavelength range hardly influenced by autofluorescence of cells, and its fluorescence intensity is not attenuated under an acidic condition.

BRIEF EXPLANATION OF THE DRAWINGS

FIG. 1 depicts changes in fluorescence spectra of a compound of the formula (I) after the addition of nitric oxide. In the figure, (a) represents an excitation spectrum (Em. 580 nm), and (b) represents a fluorescence spectrum (Ex. 565 nm). The concentrations of nitric oxide were 1: 0.11 M, 2: 0.21 M, 3: 0.32 M, 4: 0.43 M, 5: 0.53 M, and 6: 0.64 M.

FIG. 2 depicts changes in fluorescence intensity of the compound of the formula (I) depending on the amount of generated nitric oxide. In the figure, (a) and (b) represent the results obtained by using NOC12 and NOC13, respectively. The curves

1,

2 and

3, and

1′,

2′ and

3′ represent the results obtained by using the aforementioned NOCs at concentrations of 10 M, 50 M and 100 M, respectively.

FIG. 3 shows correlation between fluorescence intensity and concentration of a compound (DAR-1T) of the formula (II) (calibration curve).

FIG. 4 shows results of changes in sensitivity of the compound of the formula (I) as a function of pH.

FIG. 5 shows results of measurement of nitric oxide present in individual cells. In the figure, (a) shows changes after replacement of a culture medium of stimulated cells with a culture medium containing 1 mM L-Arg (incubation: 35 minutes, nitric oxide-producing cells); (b) shows changes after replacement of a culture medium of non-stimulated cells with a culture medium containing 1 mM L-Arg (incubation: 75 minutes, nitric oxide non-producing cells); and (c) shows changes after replacement of the culture medium of the step (a) with a culture medium containing 1 mM L-Arg+10 mM NMMA (incubation: 108 minutes, in the presence of NOS inhibitor). The normal lines represent fluorescence intensity of each cell and bold lines represent the averages thereof.

1. A compound represented by the following general formula:

wherein:

2. The compound according to claim 1, wherein R

3, R

4, R

5 and R

6 are ethyl groups; and R

7, R

8, R

9 and R

10 are hydrogen atoms.

3. A composition for nitric oxide measurement comprising the compound according to claim 1.

4. A composition for nitric oxide measurement comprising the compound according to claim 2.

5. A compound represented by the following general formula:

wherein

6. A method for measuring nitrogen monoxide which comprises:

wherein R

1 and R

2 represent amino groups present at positions adjacent each other on the phenyl ring;

R

3, R

4, R

5 and R

6 each independently represent a C

1-6 alkyl group; and

R

7, R

8, R

9 and R

10 each independently represent a hydrogen atom, a C

1-6 alkyl group, an allyl group, or a halogen atom; and

wherein R

21 and R

22 are present at adjacent positions on the phenyl ring and bind to each other to form a group represented by —N=N-NR

41— that forms a ring;

R

41 represents a hydrogen atom, a C

1-18 alkyl group, or an aralkyl group which may be substituted, or R

21 and R

22 represent a combination of an amino group and a nitro group which are present at adjacent positions on the phenyl ring;

R

23, R

24, R

25, and R

26 independently represent a C

1-6 alkyl group; and

R

27, R

28, R

29, and R

30 each independently represent a hydrogen atom, a C

1-6 alkyl group, an allyl group, or a halogen atom.

7. The method according to claim 6, wherein the reacting is carried out under substantially a neutral condition.

8. The method according to claim 6, wherein the irradiating is carried out by using an excitation light having a wavelength of about 495 nm.

9. The method according to claim 6, further comprising measuring intracellular nitrogen monoxide concentration of an individual cell.

10. The method according to claim 6, wherein the reacting is carried out in the presence of an oxygen source.

11. The method according to claim 6, wherein the oxygen source is oxygen, ozone or an oxide compound.

12. The method according to claim 11, wherein the oxygen source is 2-phenyl-4,4,5,5-tetramethylimidazoline- 1-oxyl-3-oxide or its carboxy derivative.

13. A method for measuring nitrogen monoxide which comprises:

wherein R

1 and R

2 represent amino groups present at positions adjacent each other on the phenyl ring;

R

3, R

4, R

5 and R

6 are ethyl groups; and

R

7, R

8, R

9 and R

10 are hydrogen atoms; and

wherein R

21 and R

22 are present at adjacent positions on the phenyl ring and bind to each other to form a group represented by —N=N-NR

41— that forms a ring;

R

41 represents a hydrogen atom, a C

1-18 alkyl group, or an aralkyl group, or an aralkyl group which may be substituted, or R

21 and R

22 represent a combination of an amino group and a nitro group which are present at adjacent positions on the phenyl ring;

R

23, R

24, R

25, and R

26 are ethyl groups; and

R

27, R

28, R

29, and R

30 are hydrogen atoms.

14. The method according to claim 13, wherein the reacting is carried out under substantially a neutral condition.

15. The method according to claim 13, wherein the irradiating is carried out by using an excitation light having a wavelength of about 495 nm.

16. The method according to claim 13, further comprising measuring intracellular nitrogen monoxide concentration of an individual cell.

17. The method according to claim 13, wherein the reacting is carried out in the presence of an oxygen source.

18. The method according to claim 13, wherein the oxygen source is oxygen, ozone or an oxide compound.

19. The method according to claim 18, wherein the oxygen source is 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide or its carboxy derivative.

20. A composition for nitric oxide measurement consisting essentially of the compound according to claim 1.

21. A composition for nitric oxide measurement comprising the compound according to claim 1 and a diluent.

22. A composition for nitric oxide measurement consisting essentially of the compound according to claim 2.

23. A composition for nitric oxide measurement comprising the compound according to claim 2 and a diluent.