Fluorescent acridinium salts, synthesis thereof and use for detection of cardiolipin

Abstract

The present invention relates to a novel substituted acridinium salts as fluorescent dyes, as well as methods of their manufacturing and use of the disclosed compounds for the detection of cardiolipin.

Claims

1. A compound of Formula I: ##STR00008## wherein R represents C.sub.1-15 alkyl, C.sub.1-3 deuterated alkyl, or C.sub.1-6-alkylene-silyl(C.sub.1-3-alkyl).sub.3; and X.sup.− represents chloride, bromide, or iodide.

2-3. (canceled)

4. The compound of claim 1, wherein the compound is selected from the group consisting of: 3,6-di(azetidin-1-yl)-10-methylacridin-10-ium iodide; 3,6-di(azetidin-1-yl)-10-(methyl-d.sub.3)acridin-10-ium iodide; 3,6-di(azetidin-1-yl)-10-nonylacridin-10-ium iodide; 3,6-di(azetidin-1-yl)-10-dodecylacridin-10-ium iodide; and 3,6-di(azetidin-1-yl)-10-(3-(trimethylsilyl)propyl)acridin-10-ium iodide.

5. The compound of claim 4, wherein the compound has the structure: ##STR00009##

6. The compound of claim 4, wherein the compound has the structure: ##STR00010##

7. A process for the synthesis of a compound of Formula I: ##STR00011## wherein: R represents C.sub.1-15 alkyl, C.sub.1-3 deuterated alkyl, or C.sub.1-6-alkylene-silyl(C.sub.1-3-alkyl).sub.3; and X.sup.− represents chloride, bromide, or iodide; comprising reacting a compound 1: ##STR00012## with C.sub.1-12 alkyl halide; C.sub.1-3 deuterated alkyl halide, C.sub.1-6-alkylene-silyl(C.sub.1-3-alkyl).sub.3 halide in the presence of potassium phosphate.

8. The compound of claim 1, wherein R is C.sub.1-12 alkyl, deuterated methyl, or C.sub.1-3-alkylene-silyl(C.sub.1-3-alkyl).sub.3.

9. The compound of claim 8, wherein R is CH.sub.3, CD.sub.3, C.sub.9H.sub.19, C.sub.12H.sub.25, or (CH.sub.2).sub.3—Si(CH.sub.3).sub.3.

10. The compound of claim 9, wherein X.sup.− is iodide.

11. The process of claim 7, wherein R is C.sub.1-12 alkyl, deuterated methyl, or C.sub.1-3-alkylene-silyl(C.sub.1-3-alkyl).sub.3.

12. The process of claim 11, wherein R is CH.sub.3, CD.sub.3, C.sub.9H.sub.19, C.sub.12H.sub.25, or (CH.sub.2).sub.3—Si(CH.sub.3).sub.3.

13. The process of claim 12, wherein X.sup.− is iodide.

14. A method, comprising contacting the compound of claim 1 with cardiolipin.

15. The method of claim 15, further comprising detecting a loss in fluorescence intensity.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0020] Searching for fluorescent compounds to be used for the determination of cardiolipin we unexpectedly discovered that 3,6-di(azetidin-1-yl)-10-substituted-acridin-10-ium salts of Formula I exhibit superior PLQY compared to NAO. Our finding is astonishing, because it is well known, that NAO is widely used dye for the detection of cardiolipin. We discovered that high quantum yield is typical for a number of compounds in series of 3,6-di(azetidin-1-yl)-10-substituted-acridin-10-ium salts, especially if there is silyl group present in alkyl chain, CH.sub.3 or CD.sub.3 in position 10 of this scaffold.

[0021] Scheme 1 describes the preparation of compounds of Formula I of the present invention. All of the final compounds of the present invention can be prepared by procedures described in these charts or by procedures analogous thereto, which procedures would be well known to one of ordinary skill in organic chemistry. All of the variables used in the scheme are as defined below or as in the claims.

General Procedure of Compounds Preparation of Formula I (Scheme 1)

[0022] Quaternization of acridines is a challenging task. All trusted reports in literature confirm requiring of elevated temperature, excess of alkylating agent and prolonged heating. It results in the formation of difficult separable crude mixtures due to diamino acridines are sensitive to prolonged heating. In our hands quaternization of 1 proceeded even slower than the same reaction with acridine orange. Surprisingly, we have found that the treatment of 1 with alkyl halides in the presence of inorganic salts (e.g. phosphates, carbonates) led to the fast completion of the reaction. Notably, reaction time was reduced from 2-3 days to less than 1 hour. Moreover, much smaller number of by-products has been detected making isolation of I easier.

##STR00002##

EXAMPLES

[0023] Preparation of the disclosed compounds of the present invention is described in the following examples, which are intended as an illustration of and not a limitation upon the scope of the invention.

Example 1

3,6-di(azetidin-1-yl)-10-methylacridin-10-ium iodide (I-1)

[0024] ##STR00003##

[0025] To a preheated suspension of 1 (25 mg, 0.086 mmol) in 4 ml of toluene at 100° C. potassium phosphate (42 mg, 0.2 mmol) was added followed by the addition of iodomethane (0.5 ml). Resulting mixture was stirred under reflux for 40 min. Then reaction mixture was filtered through aluminum oxide pad and washed with 30 ml of CH.sub.2Cl.sub.2/CH.sub.3OH (50:1) mixture. Volatiles were evaporated to yield 28 mg of I-1 as red solid (75%).

[0026] .sup.1H NMR (400 MHz, Methanol-d.sub.4/CDCl.sub.3) δ 8.34 (s, 1H), 7.67 (d, 2H), 6.59 (dd, 2H), 6.17 (d, 2H), 4.20 (t, 8H), 3.98 (s, 3H), 2.55-2.44 (m, 4H). .sup.13C NMR (101 MHz, Methanol-d.sub.4/CDCl.sub.3) δ 155.3, 143.7, 143.0, 133.3, 117.1, 112.5, 90.7, 51.6, 36.3, 16.0. HRMS (ESI): calcd. for C.sub.20H.sub.22N.sub.3.sup.+ [M].sup.+ calcd. 304.1808, found 304.1823.

Example 2

3,6-di(azetidin-1-yl)-10-(methyl-d.SUB.3.)acridin-10-ium iodide (I-2)

[0027] ##STR00004##

[0028] To a preheated suspension of 1 (25 mg, 0.086 mmol) in 4 ml of toluene at 100° C. potassium phosphate (42 mg, 0.2 mmol) was added followed by the addition of iodomethane-d.sub.3 (0.3 ml). Resulting mixture was stirred under reflux for 15 min. Then reaction mixture was filtered through aluminum oxide pad and washed with 30 ml of CH.sub.2Cl.sub.2/CH.sub.3OH (50:1) mixture. Volatiles were evaporated to yield 23 mg of 1-2 as red solid (62%).

[0029] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.31 (s, 1H), 7.63 (d, 2H), 6.57 (dd, 2H), 6.11 (dd, 2H), 4.23-4.10 (m, 8H), 2.55-2.44 (m, 4H). HRMS (ESI): calcd. for C.sub.20H.sub.19D.sub.3N.sub.3.sup.+ [M].sup.+ calcd. 307.2002, found 307.2005.

Example 3

3,6-di(azetidin-1-yl)-10-nonylacridin-10-ium iodide (I-3)

[0030] ##STR00005##

[0031] To a preheated suspension of 1 (25 mg, 0.086 mmol) in 4 ml of dichlorobenzene at 170° C. potassium phosphate (42 mg, 0.2 mmol) was added followed by the addition of 1-iodononane (0.5 ml). Resulting mixture was stirred under reflux for 45 min. Then reaction mixture was evaporated, and residue was purified by flash chromatography on aluminum oxide using mixture of CH.sub.2Cl.sub.2/C.sub.2H.sub.5OH (10:1) as eluent to yield 36 mg of 1-3 (76%).

[0032] .sup.1H NMR (400 MHz, Chloroform-d) δ 8.58 (s, 1H), 7.83 (d, 2H), 6.68 (dd, 2H), 6.17 (d, 2H), 4.55 (t, 2H), 4.28 (t, 8H), 2.64-2.56 (m, 4H), 1.94-1.90 (m, 2H), 1.62-1.55 (m, 2H), 1.38-1.16 (m, 10H), 0.85 (t, 3H). HRMS (ESI): calcd. for C.sub.28H.sub.38N.sub.3.sup.+ [M].sup.+ calcd. 416.3060, found 416.3058.

Example 4

3,6-di(azetidin-1-yl)-10-dodecylacridin-10-ium iodide (I-4)

[0033] ##STR00006##

[0034] To a preheated suspension of 1 (25 mg, 0.086 mmol) in 4 ml of dichlorobenzene at 170° C. potassium phosphate (42 mg, 0.2 mmol) was added followed by the addition of 1-iodododecane (0.5 ml). Resulting mixture was stirred at 170° C. for 10 min. Then reaction mixture was filtered through aluminum oxide pad to yield 27 mg of I-4 (54%).

[0035] .sup.1H NMR (400 MHz, Acetonitrile-d.sub.3) δ 8.43 (s, 1H), 7.72 (d, 2H), 6.68 (dd, 2H), 6.10 (d, 2H), 4.44-4.30 (m, 2H), 4.21 (t, 8H), 2.58-2.42 (m, 4H), 1.84-1.78 (m, 2H), 1.60-1.52 (m, 2H), 1.48-1.39 (m, 2H), 1.37-1.24 (m, 14H), 0.92-0.86 (m, 3H). .sup.13C NMR (101 MHz, Acetonitrile-d.sub.3) δ 156.3, 144.1, 143.5, 134.2, 117.9, 113.3, 91.1, 52.4, 48.3, 32.6, 30.4, 30.4, 30.3, 30.2, 30.1, 29.9, 27.3, 26.4, 23.4, 16.7, 14.4. HRMS (ESI): calcd. for C.sub.31H.sub.44N.sub.3.sup.+ [M].sup.+ calcd. 458.3535, found 458.3535.

Example 5

3,6-di(azetidin-1-yl)-10-(3-(trimethylsilyl)propyl)acridin-10-ium iodide (I-5)

[0036] ##STR00007##

[0037] To a preheated suspension of 1 (25 mg, 0.086 mmol) in 4 ml of dichlorobenzene at 170° C. potassium phosphate (42 mg, 0.2 mmol) was added followed by the addition of 3-iodopropyl trimethylsilane (0.2 ml). Resulting mixture was stirred under reflux for 30 min. Then reaction mixture was evaporated, and residue was purified by flash chromatography on aluminum oxide using mixture of CH.sub.2Cl.sub.2/C.sub.2H.sub.5OH (10:1) as eluent to yield 31 mg of 1-5 (67%).

[0038] .sup.1H NMR (400 MHz, CDCl.sub.3) δ 8.71 (s, 1H), 7.91 (d, 2H), 6.67 (dd, 2H), 6.22 (d, 2H), 4.70-4.56 (m, 2H), 4.29 (t, 8H), 2.62-2.55 (m, 4H), 1.99-1.74 (m, 2H), 0.91-0.73 (m, 2H), 0.03 (s, 9H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 155.2, 143.8, 142.6, 133.9, 117.3, 112.3, 90.4, 51.5, 50.8, 20.8, 16.1, 13.9, −1.7. HRMS (ESI): calcd. for C.sub.25H.sub.34N.sub.3.sup.+ [M].sup.+ calcd. 404.2517, found 404.2523.

[0039] Photo-physical properties of I-1-I-5 was measured in aqueous HEPES [4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid] buffer solution (20 mM, pH 7.4); NAO bromide was used as reference compound. Similar to NAO derivatives I-1-I-5 has absorption maxima at 494-498 nm, and emission maxima at 528-529 nm (Table 1). However, surprisingly, the introduction of azetidinyl moieties instead of dimethylamino groups in NAO led to increase of D from 15.5% to 47.9% (I-3). Moreover, the trimethylsilylpropyl substituent in position 10 improved PLQY up to 60.7% (I-5). Notably, 10-methyl (I-1) and 10-methyl-d.sub.3 (I-2) exhibit similar value of PLQY, 59.9% and 61.5%, correspondingly. The introduction of longer alkyl chains such as nonyl and dodecyl led to PLQY decrease.

TABLE-US-00001 TABLE 1 Photoluminescence properties of I-1-I-5 Compound λ.sub.abs, nm λ.sub.em, nm Φ, % NAO 498 528 15.5 I-1 497 529 59.9 I-2 497 529 61.5 I-3 498 529 47.9 I-4 494 528 16.1 I-5 497 529 60.7

[0040] Mitochondrial membrane consists of 4 general phospholipids: phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI), and unique negatively charged phospholipid-cardiolipin (CL). I-5 interacts with CL/DOPC liposomes (3:1), this interaction can be observed as a fluorescence intensity drop from 65800 a.u. to 6600 a.u., similarly to NAO (from 13300 a.u. to 1260 a.u.).

[0041] I-5 is selective towards CL, since fluorescence intensity does not significantly decrease in the presence of DOPC liposomes without cardiolipin (7.7% drop), and I-5/CL optimal molar ratio was determined to be 2:1. Besides, NAO fluorescence intensity loss during interaction of DOPC was detected at 5.6% level. Notably, NAO fluorescence is not stable during experiments. It dropped by 15.5% in 30 minutes, however, fluorescence intensity of I-5 remaining the same.

[0042] I-5 was titrated with CL in 0.05-8 μM range and trustful linear regression curve (R.sup.2=0.9944) was obtained (FIG. 1 represents linear regression curves for NAO and I-5 titration with cardiolipin). Consequently, we state that I-5 can be successfully used for qualitative and quantitative cardiolipin assay with superior fluorescence intensity and greater linear slope of the titration curve (−6259±250) compared to commercially available NAO (−1222±49).

[0043] Therefore, we claim water-soluble acridinium derivatives with improved fluorescence characteristics for selective CL detection.

[0044] Liposomes preparation. Vesicles were prepared by classic thin film method. Desired volume of stock solutions of DOPC (25 mg/ml, CHCl.sub.3) and CL (5 mg/ml, EtOH) was completely evaporated on a vacuum line, and the lipid films were re-suspended in HEPES buffer (20 mM, pH 7.4) to acquire 100:300 μM CL/DOPC or 400 μM DOPC liposome 1.sup.st stock solutions. Obtained large multilamellar liposomes were sonicated in a bath-type sonicator at room temperature for 30 min following by extrusion through a 100 nm polycarbonate filter for 21 times. The quality of the resulting small unilamellar vesicles was monitored by dynamic light scattering (DLS) technique. These stock solutions were diluted 5-fold to acquire 2.sup.nd stock solutions that were used in the fluorometric experiments.

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