WO1987006138A1

WO1987006138A1 - Dihydrorhodamines and halogenated derivatives thereof

Info

Publication number: WO1987006138A1
Application number: PCT/US1987/000819
Authority: WO
Inventors: Berma Mcdowell Kinsey; Amin I. Kassis; Stanley James Adelstein
Original assignee: President And Fellows Of Harvard College
Priority date: 1986-04-11
Filing date: 1987-04-07
Publication date: 1987-10-22
Also published as: JPS63503071A; AU7357887A; EP0263877A1

Abstract

Dihydrorhodamines can be made by reduction of rhodamines and can be covalently halogenated by conventional procedures. The compounds are useful as labelling, imaging, and therapeutic agents for mammalian cells.

Description

DIHYDRORHODAMINES AND HALOGENATED DERIVATIVES THEREOF

This invention relates to novel dihydrorhodamines and to covalently halogenated labelling, imaging compounds or therapeutic compounds and pertains more specifically to radiohalogenated dihydrorhodamine compounds. The compounds of the invention can be used for detecting, labelling, imaging, or therapeutically treating viable mammalian cells.

They are of particular value for use with certain types of tumor cells because of their increased retention by such cells as compared to retention by normal cells.

It has previously been proposed to use rhodamine 123, a permeant cationic dye as a mitochondrial stain of viable mamalian cells and as a probe for the study of mitochondrial function and membrane potential. This compound is retained longer in certain types of tumor cells than in normal cells and exhibits selective toxicity to certain tumor cells in vitro and in vivo .

It has previously been proposed by Padmanabhan et al., J.Nucl .Med. , Vol. 26, 124 (1985) and by Thakur et al., 5th Int.Symp. on Radiopharm.Chem., 11-p-7 (1984) tthhaait rhodamine 123 be radioiodinated with ¹²⁵I and 1³¹I and used as an imaging or radiotherapeutic agent. However, it has now been found that the positive charge on the quaternary nitrogen atom of the rhodamine in solution prevents the formation of covalently iodinated rhodamines; instead, conventional iodination procedures such as the chloramine T or iodogen procedures lead to the formation of the corresponding quaternary ammonium iodide, an ionic compound in which the iodine is subject to ionization and replacement by other non-radioactive ions in an aqueous medium such as a biological fluid. It has also been reported by Gallop et al., Biotechniques, Vol. 2, pp.32-36 (1984) that hydrogenated ethidium bromide is taken up by and reoxidized inside viable mammalian cells. It has now been found that dihydrorhodamines and halogenated dihydrorhodamines are taken up, oxidized to the fluorescent parent rhodamines, and retained by mammalian cells. Furthermore, unlike the rhodamines themselves, the dihydrorhodamines can be covalently halogenated by conventional halogenation procedures, and the halogen of such compounds is internalized by the cells, thus providing the cells with enhanced susceptibility to toxic radiation. Consequently, halogenated and radiohalogenated dihydrorhodamines are useful as detecting, labelling, imaging or therapeutic agents.

The present invention comprises a compound having the structure

in which R₁,R₂,R₃,R₄ and R₅ are hydrogen or lower alkyl (preferably having 1-5 carbon atoms), and X₁,X₂,X₃ and X₄ are hydrogen, lower alkyl (preferably having 1-5 carbon atoms), halogen, or a radiohalogen, Y is -COOR₅ or -SO₃R₅, Z is hydrogen, -COOR₅, or -SO₃R₅, and water-soluble salts of such compounds. Preferably the salts are those which are physiologically acceptable; they include, for example, the chloride perchlorate, tetrafluoroborate, and internal salts where, for example, both Y and Z are -SO₃H. The invention also comprises bringing into contact with a mammalian cell an aqueous solution comprising a compound having the structure

in which R₁-R₅, X₁-X₄ , and Y and Z are as defined above, to cause said compound to be taken up by the cell. Preferably at least one of X₁, X₂, X₃ and X₄ is halogen or radiohalogen, the remainder being hydrogen.

The dihydrorhodamine compounds of the present invention can be made by hydrogenation or reduction of the corresponding rhodamine compound by conventional procedures such as by reduction with a borohydride, e.g. sodium borohydride in aqueous medium. Among such corresponding rhodamine compounds are rhodamine B, rhodamine 3B, rhodamine 6-G, rhodamine 19, rhodamine 110, rhodamine 116, rhodamine 123, sulforhodamine B, and including various water-soluble salts. The structure of rhodamine 19 is

while that of rhodamine 110 is

and that of rhodamine 116 is

The dihydrorhodamine products have the structure (1) given above in which X₁,X₂,X₃ and X₄ are hydrogen. The dihydrorhodamines can be covalently halogenated by any conventional procedure such as the chloramine T, iodogen, or N-chlorosuccinimide procedure, or any other procedure effective to introduce one or more atoms of chlorine, bromine, iodine or astatine into an aromatic amine. The products, containing halogen covalently bonded in any one or more of the four available positions X₁-X₄ in the amino substituted benzene rings can, if desired, be separated from each other by chromatography. However, separation is not essential and mixtures containing two or more different halogenated compounds are useful in the same way as the individually pure compounds.

The dihydrorhodamine compounds after halogenation with a halogen radioisotope such as chlorine, bromine, iodine or astatine may be used as labelling, imaging or therapeutic agents in the same way as rhodamine 123 or any other such agent simply by dissolving the compound in an aqueous medium such as normal saline or any physiologically acceptable medium and bringing it into contact with the viable mammalian cells it is desired to treat, whereupon the compounds are taken up by the cells and located in the cytoplasm.

The following specific examples are intended to illustrate more fully the nature of the present invention without acting as a limitation upon its scope. Example 1 Dihydrorhodamine 123: Rhodamine 123 hydrochloride (100 mg, 0.26 mmol; Eastman Kodak) was dissolved in 50 ml of water and 50 ml of Ch₂Cl₂ was added. Excess solid NaBH₄ was added in portions with vigorous magnetic stirring until almost all the color was discharged. After 1 hr, the pale orange organic layer was separated and the water layer extracted twice with CH₂Cl₂. The combined organic layers were passed through anhydrous Na₂SO₄ and evaporated under reduced pressure. The crude oily reaction mixture was purified by flash chromatography (C) using 10% ethylacetate in CH₂Cl₂ as eluent, giving 80 mg of a slightly pinkish-orange oil (88% yield). The purified oil was dissolved in a small amount of CH₂Cl₂ and precipitated with hexane. The pinkish-white solid dihydrorhodamine 123 was washed with hexane, and a portion submitted for analysis. (m.p.= 163-165°C; calc. for C₂₁H₁₈N₂O₃: 72.96% C, 5.50% H, 71.88% N; found: 72.82% C, 5.24% H, 8.09% N) . ¹HNMR (CDCl₃) ^σ= 3.93 (s,-CO₂CH₃), 5.93 (s, C5' ) , 6.15 (d of d, J = 2, 8, 2H, C7', 3'), 6.30 (d, 2H, C9' , 1'), 6.70 (d, 2H, C6', 4'), 7.0-7.17 (m, 3H, C3 , 4 , 5), 7.65 (d of d,1H, C6) . The product possessed the structure (1) above in which R₁,R₂,R₃, and R₄ are hydrogen, R₅ is methyl, and X₁ ,X₂ ,X₃ and X₄ are hydrogen.

Example 2

Iododihydrorhodamine 123 (A) : Dihydrorhodamine 123 (43 mg, 0.12 mmol) was dissolved in 50 mL of CCl₄ and 1 mL of CH₂Cl₂, and cooled in ice under red light. A solution of 17 mg Nal in 0.5 mL MeOH and 0.5 mL 0.2 M NH₄OAc was added with stirring and a cold solution of 17 mg (0.13 mmol) of N-chlorosuccinimide in so mL CCl₄ was added in small portions. The reaction mixture was allowed to warm to room temperature and stirring was continued for 1 hr. Water and NaBH₄ were added, the organic layer separated, dried over anhydrous Na₂SO₄ and evaporated under reduced pressure. The red oily product was subjected to flash chromatography using 5% EtOAc in CH₂Cl₂, and 26 mg of A (44%) along with 7 mg of C (9%) were recovered. A sample was dissolved in a minimal amount of ether, precipitated with hexane, and submitted for analysis. (calc. for

C₂₁H₁₇IN₂O₃: 53-41% C, 3.63% H, 26.87% I; found: 53.21% C, 3.97% H, 27.08% I). ' HNMR

(CDCl₃): σ= 3.88 (s, -CO₂CH₃), 5.93 (s, C-5 ' ) , 6.15 (d of d, J=3. 8 Hz, C-7 ' ) , 6.28 (d, J=3 , C-9 ' ) ,

6.38 (s, C-1'), 6.67 (d, J=8 Hz, C-6'), 7.15 (s, C-4 ' ) ,

6.97-7.20 (m, C-3,4,5), 7.67 (m, C-6).

3 ' -Iododihydrorhodamine 123 (A) , 1'-iododihydrorhodamine 123 (B) , 3',7'-diiododihydrorhodamine 123 (C) , 1' , 7 '-diiododihydrorhodamine 123 (D) : A solution of

86.0 mg (0.25 mmol) of dihydrorhodamine in 100 mL CCl₄ was prepared with the addition of 3-4 mL of CH₂Cl₂.

A solution of 150 mg (1.0 mmol) Nal in 5 mL of MeOH and 5 mL of 0.2 M NH₄OAc was added and the mixture cooled in an ice bath with stirring. A cold solution of 133.5 mg (1.0 mmol) of N-chlorosuccinimide in 100 mL of CCl₄ was added all at once and the mixture stirred in an ice bath for 0.5 hr. Stirring was continued at room temperature for 0.5 hr., after which water and a small amount of solid NaBH₄ were added. The organic layer was separated after vigorous shaking, and the water layer washed several times with CH₂Cl₂. The combined organic layers were filtered through anhydrous Na₂SO₄ and the solvent removed under reduced pressure. Flash chromatography on a

1.5 cm column was done on the residue, using 85 mL of 1% EtOH in CH₂Cl₂, 50 mL of 5% EtOH in CH₂Cl₂, and 40 mL of 10% EtOH in CH₂Cl₂, collecting 5 mL fractions. Four products were isolated and characterized by NMR.

A: 23 mg, 21% yield, R_f (in 5% EtOH/CH₂Cl₂)

= 0.35. Identical with previously prepared A,

B: 13 mg, 11% yield, R_f (in 5% EtOH/CH₂Cl₂)

= 0.60. (calc. for C₂₀H₁₅IN₂O₃ : 53.41% C, 3.63% H, 26.87% I; found: 53.31% C, 3.92% H, 26.08% I). ΗNMR (CDCl₃): σ= 3.98 (s, -CO₂CH₃), 6.08 (s, C-5), 6.30 (d of d,

J = 3, 8 Hz, C-7') 6.35 (d, J = 8 Hz, C-3' ) , 6.56 (d, J = 3 Hz, C-9'), 6.78 (m, C-4 ' 6 ' ) , 6.88-7.42 (m, C-3, 4, 5), 7.22 (m, C-6). C: 26 mg, 17% yield, R_f (in 5% EtOH/CH₂Cl₂)

= 0.72. (calc. for C₂₀H₁₄I₂N₂O₃ : 42.17% C, 2.70% H, 42.43% I; found 42.98% C, 3.04% H,

41.70% I). 'HNMR (CDCI₃) : σ = 4.00 (s, -CO₂CH₃), 6.05 (s, C-5'), 6.43 (s, C-l1,9'), 7.22 (s, C-4',6'), 6.98-7.23 (m, C-3, 4, 5), 7.75 (d of d, J = 2, 7 Hz, C-6). D: 18 mg, 12% yield, R_f (in 5% EtOH/CH₂Cl₂)

= 0.87. (calc. for C₂₀H₁₄I₂N₂O₃ : 42.17% C, 2.70% H, 42.43% I; found: 41.20% C, 2.90% H, 42.72% I). 'HNMR (CDCI₃): σ= 3.97 (s, -CO₂CH₃), 6.08 (s, C-5'), 6.32 (d, J = 8 Hz, C-3'), 6.62 (s, C9"), 6.73 (d, J = 8 Hz, C-4 ' ) ,

7.22 (s, C-6'), 6.87-7.33 (m, C-3, 4, 5), 7.73 (d of d, J = 3, 8 Hz, C-6) .

1',3',7'-Triiododihydrorhodamine (E) : To a solution of 86 mg (0.25 mmol) of dihydrorhodamine in 20 mL of methanol and 6 mL of 0.2 M NH₄OH was added a solution of 187.5 mg (1.25 mmol) Nal in 10 mL of methanol and 2.5 mL of 0.2 M NH₄OH. Nitrogen was bubbled into the stirred solution and 150 mg (0.35 mmol, 1.4 meq) of Iodogen was added all at once. Stirring was continued under N₂ for 1 hr. The reaction mixture was filtered, and methanol removed from the filtrate under reduced pressure. The filter cake was washed several times with CH₂Cl₂, and the water layer extracted several times with CH₂Cl₂. The CH₂Cl₂ fractions were combined and the solvent removed under reduced pressure. The residue was dissolved in 2-3 mL of CH₂Cl₂, absorbed onto a small amount of silica gel, and the CH₂Cl₂ removed under reduced pressure. The dry silica gel reaction mixture was applied to the top of a 1.5 cm column for flash chromatography using 25% EtOH in hexane. Three main products were recovered: 26 mg (17%) of C (R_f in 25% EtOH in hexane = 0.27; 11 mg (7%) of D (R_f = 0.43), and 14 mg (8%) of E (R_f = 0.58). (calc. for C₂₀H₁₃I₃N₂O₃ : 34.84% C, 2.09% H, 52.58% I; found: 34.61% C, 2.30% H, 53.73% I). 'HNMR (CDCl₃): σ= 3.98 (s, -CO₂CH₃), 6.10 (s, C-5'), 6.58 (s, C-1'), 7.22 (s, C-4 ' , 6 ' ) , 6.80-7.30 (m, C-3, 4, 5), 7.75 (m, C-6).

Example 3

A 2.5 mM solution of the dihydrorhodamine of Example 1 in CCl₄ with the addition of a few drops of CH₂Cl₂ was prepared, and to one mL of this solution was added the contents of two vials of Na ¹²⁵I (14.2 mCi). The vials were rinsed twice with 10 Wl of 0.2 M

NH₄OAc and 10 μL of methanol, and the resulting mixture stoppered and stirred in an ice bath. A 2.5 mM solution of N-chlorosuccinimide in CCl₄ was added and the vial stoppered, covered with aluminum foil, and stirred vigorously at room temperature for 1 hr . One mL of water and several 10 μl portions of 1M NaBH₄ were added and stirring continued until most of the red color had disappeared. The CCl₄ layer was removed with a pipet and the remaining water layer was washed twice with small amounts of CH₂Cl₂. Solvent was evaporated from the combined organic layers under a stream of nitrogen, and one mL of methanol and a tiny amount of solid NaBH₄ were added to colored residue. The methanol was evaporated, and the now colorless residue was taken up in 5% EtOAc/CH₂Cl₂ and flash chromatography performed using the same solvent on a 0.8 x 12 cm column, 85 drops per fraction. Fractions which showed a single spot on TLC radioautography were combined to give 4.22 mCi of A (Table 1), 1.36 mCi of B, 1.49 mCi of C, and 0.40 mCi of D. Fractions showing more than one spot on TLC were combined and rechromatographed using 25% EtOAc/hexane. From this chromatography was isolated 0.18 mCi of B, 0.53 mCi of C, 0.16 mCi of D, 0.09 mCi of F, and 0.02 mCi of unidentified material with a greater R_f on TLC than F. The total radioiodinated yield was 8.45 mCi (60%) of which 50% was A, 18% was B, 24% was C, 7% was D, and 1% was E.

TABLE 1

Compound X1 X2 X3 X4 R1 R2 R3 R4 R5

A I H H H H H H H CH₃

B H H I H H H H H CH₃

C I I H H H H H H CH₃

D I H H I H H H H CH₃

E I I I H H H H H CH₃ In order to characterize the individual compounds present in each fraction, the same reaction had been carried out using non-radioactive sodium iodide and individual fractions obtained by chromatography were characterized by NMR. The fractions containing ¹²⁵I were thus identified by comparison on TLC with NMR-authenticated material.

Other dihydrorhodamines, and halogenated and radiohalogenated derivatives thereof can be made by similar procedures.

While dihydrorhodamine 123 is itself a nonfluorescent compound, when an aqueous solution of the compound is applied to viable mammalian cells, its presence in the mitochondria of the cells is observed by fluorescent microscopy, showing that' it is oxidized within the cell to the fluorescent rhodamine 123. Halogenation of the compound does not fnterfere with its being taken up by the cells and oxidized.

What is claimed is:

Claims

1. A compound having the structure

in which R₁,R₂,R₃,R₄ and R₅ are hydrogen or lower alkyl, at least one of X₁,X₂,X₃ and X₄ is halogen or radiohalogen, the remainder being hydrogen or lower alkyl, Y is -COOR₅ or -SO₃R₅ and Z is hydrogen, -COOR₅, or -SO₃R₅, and water-soluble salts of such compounds.

2. A compound as claimed in claim 1 in which at least one of X₁,X₂,X₃ and X₄ is a radioisotope of chlorine, bromine, iodine or astatine, the remainder being hydrogen.

3. A compound as claimed in claim 1 in which R₁,R₂,R₃ and R₄ are hydrogen and R₅ is methyl, Y is -COOR₅, and Z is hydrogen.

4. A compound as claimed in claim 3 in which at least one of X₁,X₂,X₃ and X₄ is a halogen radioisotope of chlorine, bromine, iodine or astatine.

5. The method which comprises bringing into contact with a mammalian cell an aqueous solution comprising a compound having the structure

6. The method as claimed in claim 5 in which R₁,R₂R₃ and R₄ are hydrogen, R₅ is methyl, Z is hydrogen and at least one of X₁,X₂,X₃ and X₄ is a radioisotope of chlorine, bromine, iodine or astatine, the remainder being hydrogen.

7. The method as claimed in claim 6 in which at least one of X₁,X₂,X₃ and X. is a radioisotope of iodine