Studies directed towards nonyl acridine orange analogues having the potential to act as FRET donors with the PDT drug Pc 4

Abstract

A group of nonyl acridine orange analogues (NAO) was prepared which were designed to have the potential of possessing visible bands allowing them to act in cells as fluorescence resonance energy transfer (FRET) donors with the photodynamic therapy drug Pc 4. The existence of Pc 4-FRET with the analogues of NAO in MCF-7c3 cells was probed. The results suggest that NAO analogues giving strong FRET with Pc 4 in cells can be found.

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Introduction

Substantial effort has been devoted to gaining an understanding of the mechanism of photodynamic therapy (PDT) in which Pc 4, Fig. 1a, is used as the photosensitizer.^1–12 Some of this effort has been focused on the use of a derivative of acridine orange commonly called nonyl acridine orange (NAO), Fig. 1b and c.¹³ In cells NAO binds with cardiolipin (CL), Fig. 1d, an important component of the inner membrane of mitochondria.

Fig. 1 Structures of (a) (hydroxy)(dimethylaminopropyldimethyl)siloxysilicon phthalocyanine, Pc 4, (b) acridine orange, 1, (c) nonyl acridine orange, NAO, (d) cardiolipin, CL (R₁–R₄ are fatty acid chains having two double bonds).

In previous work, we used fluorescence resonance energy transfer (FRET) between Pc 4 and NAO to study the localization of Pc 4 in cells since the localization of NAO in cells is well studied and since FRET requires close association between its participants.^14,15 No fluorescence was observed when cells containing Pc 4 were excited at 488 nm, approximately at an absorption band of NAO, nor when cells containing NAO were excited at 633 nm, approximately at an absorption band of Pc 4. When cells containing Pc 4 were excited at 633 nm a band was observed at 675 nm and when cells containing NAO were excited at 488 nm a band at 525 nm was observed. With cells containing both Pc 4 and NAO, excitation at 488 nm caused a decrease in the 525 nm NAO band and the appearance of the 675 nm band of Pc 4. This indicated that FRET had occurred between Pc 4 and NAO, and that the Pc 4 had co-localized with the NAO in the mitochondrial membrane. It further suggested that cells containing both Pc 4 and an analogue of NAO designed to give stronger FRET with Pc 4 could provide an improved method of probing the location of Pc 4 in cells. Access to such a method is important because Pc 4-PDT in cells is thought to oxidize mitochondrial CL and the oxidation of CL is responsible for the release of cytochrome c from the mitochondria, a release that is important because it is an early step in cell apotosis.¹⁶

One way to obtain NAO-Pc 4 efficient analogues would be to take advantage of the dependence of FRET efficiency on the overlap integral of the emission band of the energy donor and the absorption band of the acceptor.¹⁷ Since the donor band of NAO, ∼495 nm is too short to overlap well with the acceptor band of Pc 4, ∼670 nm, this requires finding NAO analogues having bands with wavelengths longer than 495 nm.

Work based on this approach is described here. We have made a set of acridine orange analogues and then quaternized them to obtain a set of nonyl acridine orange analogues having the potential to have useful donor bands. In addition, we have prepared some NAO analogues and carried out studies of the occurrence of FRET between them and Pc 4 in MCF-7c3 cells.

Experimental procedures

Instrumental

The ¹H NMR spectra were recorded with an INOVA 400 MHz spectrometer (Varian, Palo Alto, CA), and the UV-vis spectra were recorded with a PerkinElmer Lambda 25 spectrometer (PerkinElmer, Shelton, CT) equipped with Fisher Scientific quartz cells (SCC 283, 1.00 cm, Pittsburgh, PA). The fluorescence spectra were collected with a Cary Eclipse spectrophotometer (Varian, Palo Alto, CA), and the mass data were determined by electron impact (EI), high-resolution fast-atom bombardment (FAB), and high-resolution electrospray (ESI) techniques with a KRATOS MS25RFA spectrometer (Ion Tech, Manchester, UK).

Synthesis

Acridine orange, 1. Acridine orange was purchased from Sigma-Aldrich (Milwaukee, WI).

3,6-Bis(dimethylamino)-10H-acridine-9-thione, 2. 2 was synthesized according to the method of Elslager.¹⁸

9-Methylmercapto-3,6-bis(dimethylamino)acridine, 3. 3 was prepared from 2 according to the method of Elslager.¹⁸

9-Methoxy-3,6-bis(dimethylamino)acridine, 4. A solution of 3 (66 mg, 0.21 mmol), phenol (9 mg, 0.1 mmol) and CH₃OH (6 mL, 0.15 mol) was heated (reflux) for 24 h and evaporated nearly to dryness by rotary evaporation (40 °C). The damp solid was chromatographed (basic Al₂O₃ V, CH₂Cl₂–CH₃OH solution, 90 : 1), washed (ether), vacuum dried (40 °C) and weighed (21 mg, 34%). UV-vis (CH₃OH) λ_max nm (log ε): 476 (4.1). NMR (CDCl₃): δ 8.02 (d, J = 9.4 Hz, 2H), 7.10 (dd, J = 9.4, 2.5 Hz, 2H), 7.04 (d, J = 2.5 Hz, 2H), 4.17 (s, 3H), 3.12 (s, 12H). HRMS-FAB (m/z): (M + H)⁺ (calcd with M as C₁₈H₂₁N₃O): 296.1763: found 296.1758. The product was a yellow solid. It was soluble in dimethylformamide and CH₂Cl₂, and insoluble in H₂O and hexanes.

9-Chloro-3,6-bis(dimethylamino)acridine, 5. Under Ar, a mixture of 5 (680 mg), POCl₃ (12.3 g) and PCl₃ (1.31 g) was heated (reflux) for 3.5 h, diluted with xylenes (10 mL), and filtered. The solid was chromatographed (basic Al₂O₃ V, CH₂Cl₂–N(C₂H₅)₃ solution, 15 : 1), washed (CH₃CN), vacuum dried (40 °C) and weighed (237 mg, 35%). UV-vis (CH₃OH) λ_max, nm (log ε): 505 (4.5). NMR (CDCl₃): δ 8.12 (d, J = 9.5 Hz, 2H), 7.18 (d, J = 9.5, 2.5 Hz, 2H), 7.02 (d, J = 2.1 Hz, 2H), 3.13 (s, 12H). HRMS-EI (M)⁺ (calcd with M as C₁₇H₁₈N₃Cl): 299.1189; found 299.1189. The product was brown solid. It was soluble in dimethylformamide and CH₂Cl₂, and insoluble in H₂O and hexanes.

9-Amino-3,6-bis(dimethylamino)acridine, 6. A mixture of 5 (50 mg, 0.17) and phenol (300 mg) was treated with NH₃ gas (∼20 bubbles per min) while being heated (120 °C) for 4 h, diluted with acetone (20 mL), and filtered. The filtrate was chromatographed (basic Al₂O₃ V, CHCl₃, CH₂Cl₂–N(C₂H₅)₃ solution, 10 : 1), washed (ether), vacuum dried (40 °C) and weighed (23 mg, 48%). UV-vis (CH₃OH) λ_max, nm (log ε): 418 (4.4). NMR (CD₃OD): δ 7.99 (d, J = 9.5 Hz, 2H), 6.90 (dd, J = 9.5, 2.4 Hz, 2H), 6.47 (s, 2H), 3.12 (s, 12H). HRMS-FAB (M + H)⁺(calcd with M as C₁₇H₂₀N₄) 281.1768: found 281.1758. The product was a yellow solid. It was soluble in dimethylformamide, slightly soluble in CH₂Cl₂ and H₂O, and insoluble in hexanes.

9-Dimethylamino-3,6-bis(dimethylamino)acridine, 7. A mixture of 5 (31 mg) and NH(CH₃)₂ (3.3 mL) was stirred at room temperature in a pressure tube for 14 h, and evaporated to dryness by rotary evaporation (40 °C). The solid was chromatographed (basic Al₂O₃ V, tetrahydrofuran), washed (CH₃CN), vacuum dried (40 °C) and weighed (13 mg, 42%). UV-vis (CH₃OH) λ_max, nm (log ε): 450 (4.4). NMR (CDCl₃): δ 8.00 (d, J = 9.8 Hz, 2H), 7.04 (s, 2H), 7.02 (d, J = 2.5 Hz, 2H), 3.32 (s, 6H), 3.12 (s, 12H). HRMS-EI (M)⁺(calcd with M as C₁₉H₂₄N₄): 308.2001: found 308.1999. The product was a yellow solid. It was soluble in dimethylformamide and CH₂Cl₂, slightly soluble in H₂O, and insoluble in hexanes.

9-Phenylethynyl-3,6-bis(dimethylamino)acridine, 8. With the procedure of Wan as a guide,¹⁹ a mixture of 5 (33 mg), phenylacetylene (28 mg), Pd(PPh₃)₄ (15 mg), N(C₂H₅)₃ (0.1 mL) and tetrahydrofuran (2 mL) was heated (reflux) under N₂ for 8 h, and extracted with ethyl acetate. The extract was evaporated to dryness by rotary evaporation (40 °C), and the solid was chromatographed (basic Al₂O₃ III, CHCl₃; neutral Al₂O₃ III, CHCl₃–hexanes solution, 3 : 1), vacuum dried (room temperature) and weighed (8 mg, 20%). UV-vis (CH₃OH) λ_max, nm (log ε): 542 (3.8). NMR (CDCl₃): δ 8.28 (d, J = 9.4 Hz, 2H), 7.75 (m, 2H), 7.46 (m, 3H), 7.21 (dd, J = 9.4, 2.5 Hz, 2H), 7.09 (s, 2H), 3.16 (s, 12H). HRMS-FAB (M + H)⁺ (calcd with M as C₂₅H₂₂N₃): 366.1970: found 366.1971. The product was a brown solid. It was soluble in dimethylformamide and CH₂Cl₂, and insoluble in H₂O and hexanes.

9-Cyano-3,6-bis(dimethylamino)acridine, 9. With the synthesis of cyanoacridine as a guide,²⁰ a mixture of 1 (438 mg), C₂H₅OH (2 mL) and glacial acetic acid (148 mg) was treated dropwise with a solution of KCN (230 mg) and H₂O (1 mL), heated (reflux) for 140 min, and evaporated to dryness by rotary evaporation (40 °C). The solid was washed (aqueous NaOH, 2 N) and extracted (CHCl₃), and the extract was evaporated to dryness by rotary evaporation (room temperature). The solid was chromatographed (basic Al₂O₃ III, CHCl₃–ethanol solution, 100 : 1), washed (H₂O), vacuum dried (40 °C) and weighed (275 mg, 57%). UV-vis (CH₃OH) λ_max, nm (log ε): 488 (4.6). NMR (CDCl₃): δ 8.05 (d, J = 9.4 Hz, 2H), 7.29 (dd, J = 9.4, 2.6 Hz, 2H), 7.04 (d, J = 2.5 Hz, 2H), 3.18 (s, 12H). HRMS-FAB (M + H)⁺ (calcd with M as C₁₈H₁₈N₄): 291.1610: found 291.1604. The product was an orange solid. It was soluble in dimethylformamide and CH₂Cl₂, and insoluble in H₂O and hexanes.

3,6-Bis(dimethylamino)-9-acridinecarboxamide, 10. A mixture of 9 (25 mg) and H₂SO₄ (conc, 1 mL) was heated at 90–100 °C for 2 h, treated with aqueous NaOH (2 N) until basic and filtered. The solid was vacuum dried (40 °C) and weighed (23 mg, 87%). UV-vis (CH₃OH) λ_max, nm (log ε): 503 (4.7). NMR (CDCl₃): δ 7.83 (d, J = 9.5 Hz, 2H), 7.11 (dd, J = 9.4, 2.4 Hz, 2H), 6.99 (d, J = 2.2 Hz, 2H), 3.14 (s, 12H). HRMS-FAB (M + H)⁺ (calcd with M as C₁₈H₂₀N₄O): 309.1715: found 309.1714. The product was an orange solid. It was soluble in dimethylformamide, slightly soluble in CH₂Cl₂, and insoluble in H₂O and hexanes.

2-Amino-4-nitroanisole, 11. 11 was purchased from Fisher Scientific.

2-Dimethylamino-4-nitroanisole, 12. 12 was prepared according to the procedure of Giumanini et al.²¹

3-Dimethylamino-4-methoxyaniline, 13. Under H₂, a suspension of 12 (958 mg), Pd/C (10%) and CH₃OH (50 mL) was stirred at room temperature for 2 days, filtered, and evaporated to an oil by rotary evaporation (room temperature). The oil was subjected to vacuum (40 °C) and weighed (759 mg, 94%). NMR (CDCl₃): δ 6.66 (d, J = 8.4 Hz, 1H), 6.34 (d, J = 2.6 Hz, 1H), 6.28 (dd, J = 8.4, 2.6 Hz, 1H), 3.79 (s, 3H), 2.75 (s, 6H). The product was brown oil. It was soluble in dimethylformamide, slightly soluble in CH₂Cl₂ and H₂O, and insoluble in hexanes.

2-[(3-Dimethylamino-4-methoxyphenyl)amino]-4-nitrobenzoic acid, 14. With the work of Goldberg et al. as a guide,^22–24 a suspension of aniline 13 (1.32 g), 2-chloro-4-nitrobenzoic acid (829 mg), potassium carbonate (770 mg), copper powder (66 mg) and 1-pentanol (5 mL) was heated (reflux) for 4.5 h, subjected to steam distillation and filtered. At 80–100 °C, the filtrate was treated with aqueous HCl (2 N) until the pH was 4, filtered, allowed to stand overnight, and refiltered. The solid was vacuum dried (40 °C) and weighed (280 mg, 11%). The product was a brown solid. It was soluble in dimethylformamide, slightly soluble in CH₂Cl₂ and H₂O, and insoluble in hexanes.

9-Chloro-2-methoxy-3-dimethylamino-6-nitroacridine, 15. With the work of Csuk et al. as a guide,²² a suspension of 14 (263 mg) and POCl₃ (3.0 mL) was heated (reflux) for 3.5 h, cooled, and added to a slurry of NH₄OH (conc, 50 mL), CHCl₃ (100 mL) and ice (50 g) while the reaction mixture was being maintained at a pH of >8. The organic phase was separated and evaporated to dryness by rotary evaporation (room temperature). The solid was washed with conc NH₄OH, vacuum dried (40 °C), and weighed (116 mg, 44%). NMR (CDCl₃): δ 9.00 (dd, J = 2.3, 0.5 Hz, 1H), 8.41 (dd, J = 9.4, 0.5 Hz, 1H), 8.22 (dd, J = 9.4, 2.3 Hz, 1H), 7.44 (s, 1H), 7.37 (s, 1H), 4.14 (s, 3H), 3.10 (s, 6H). The product was a red-brown solid. It was soluble in dimethylformamide and CH₂Cl₂, and insoluble in H₂O and hexanes.

2-Methoxy-3-dimethylamino-9-methylmercapto-6-nitroacridine, 16. With the work of El-Sherief et al. as a guide,²⁵ a solution of 16 (65 mg), thiourea (184 mg) and ethanol (3 mL) was heated (reflux) for 4.5 h and filtered. The solid was vacuum dried (40 °C) and dissolved in acetone (3 mL). The solution was treated with a suspension of CH₃I (400 mg) and K₂CO₃ (80 mg), heated (reflux) for 1 h, concentrated by rotary evaporation (room temperature), diluted with CHCl₃ (20 mL), and filtered. The filtrate was evaporated to dryness by rotary evaporation (room temperature), and the solid was vacuum dried (40 °C) and weighed (46 mg, 65%). NMR (CDCl₃): δ 9.02 (d, J = 2.3 Hz, 1H), 8.77 (d, J = 9.5 Hz, 1H), 8.23 (dd, J = 9.5, 2.3 Hz, 1H), 7.90 (s, 1H), 7.40 (s, 1H), 4.15 (s, 3H), 3.09 (s, 6H), 2.47 (s, 3H). The product was an orange-red solid. It was soluble in dimethylformamide and CH₂Cl₂, and insoluble in H₂O and hexanes.

2-Methoxy-3-dimethylamino-6-aminoacridine, 17. Under H₂, a suspension of 16 (26 mg), Pd/C (10%, 63 mg) and tetrahydrofuran (20 mL) was stirred for 24 h, filtered, and evaporated to dryness by rotary evaporation (40 °C). The solid was chromatographed (basic Al₂O₃ III, CHCl₃–C₂H₅OH solution, 50 : 1), vacuum dried (40 °C) and weighed (7 mg, 39%). NMR (CDCl₃): δ 8.32 (s, 1H), 7.72 (d, J = 8.9 Hz, 1H), 7.41 (s, 1H), 7.20 (d, J = 2.2 Hz, 1H), 7.05 (s, 1H), 6.94 (dd, J = 8.8, 2.3 Hz, 1H), 4.03 (s, 3H), 2.99 (s, 6H). The product was a yellow solid. It was soluble in dimethylformamide and CH₂Cl₂, and insoluble in H₂O and hexanes.

2-Methoxy-3,6-bis(dimethylamino)acridine, 18. A solution of aqueous formaldehyde (37%, 38 mg) and sulfuric acid (3.1 M, 0.12 mL) was treated with a suspension of 17 (7 mg), NaBH₄ (14 mg) and tetrahydrofuran (2 mL), with aqueous NaOH (2 N) until the pH was >9, and extracted with ether (20 mL). The extract was evaporated to dryness by rotary evaporation (room temperature), and the solid was chromatographed (basic Al₂O₃ III, CH₂Cl₂), vacuum dried (40 °C) and weighed (7 mg, 67% based on 17). UV-vis (CH₃OH) λ_max, nm (log ε): 495 (4.0). NMR (CDCl₃): δ 8.32 (s, 1H), 7.75 (d, J = 9.2 Hz, 1H), 7.43 (s, 1H), 7.21 (dd, J = 9.2, 2.5 Hz, 1H), 7.14 (s, 1H), 7.06 (s, 1H), 4.03 (s, 3H), 3.14 (s, 6H), 2.99 (s, 6H). HRMS-FAB [M + H]⁺ (calcd with M as C₁₈H₂₂N₃O): 296.1763: found 296.1756. The product was a yellow solid. It was soluble in dimethylformamide and CH₂Cl₂, and insoluble in H₂O and hexanes.

2-Methoxy-3-dimethylamino-9-methylmercapto-6-aminoacridine, 19. Under H₂, a suspension of 16 (36 mg), Pd/C (10%, 93 mg) and tetrahydrofuran (25 mL) was stirred for 24 h, filtered, and evaporated to dryness by rotary evaporation (room temperature). The solid was chromatographed (basic Al₂O₃ III, CHCl₃–CH₃OH solution, 50 : 1), vacuum dried (40 °C) and weighed (20 mg, 64%). NMR (CDCl₃): δ 8.51 (d, J = 9.2 Hz, 1H), 7.85 (s, 1H), 7.39 (s, 1H), 7.20 (d, J = 2.3 Hz, 1H), 7.03 (dd, J = 9.2, 2.4 Hz, 1H), 4.10 (s, 3H), 3.00 (s, 6H), 2.45 (s, 3H). The product was a yellow solid. It was soluble in dimethylformamide and CH₂Cl₂, and insoluble in H₂O and hexanes.

2-Methoxy-3,6-bis(dimethylamino)-9-methylmercaptoacridine, 20. A solution of aqueous formaldehyde (37%, 43 mg) and aqueous H₂SO₄ (24%, 132 mg) was treated with a suspension of 19 (20 mg), NaBH₄ (17 mg) and tetrahydrofuran (1 mL), then with aqueous NaOH (2 N) until the pH was >9, and sonicated with CHCl₃ (30 mL). The CHCl₃ extract was evaporated to dryness (room temperature), and the solid was chromatographed (basic Al₂O₃ III, CHCl₃), vacuum dried (40 °C), and weighed (8 mg, 31%). UV-vis (CH₃OH) λ_max, nm (log ε): 519 (4.2). NMR (CDCl₃): δ 8.53 (d, J = 9.5 Hz, 1H), 7.86 (s, 1H), 7.40 (s, 1H), 7.30 (dd, J = 9.6, 2.6 Hz, 1H), 7.15 (d, J = 2.5 Hz, 1H), 4.10 (s, 3H), 3.15 (s, 6H), 3.00 (s, 6H), 2.46 (s, 3H). HRMS-EI [M]⁺ (calcd with M as C₁₉H₂₃N₃OS): 341.1562; found 341.1562. The product was an orange-red solid. It was soluble in dimethylformamide and CH₂Cl₂, and insoluble in H₂O and hexanes.

2-Methoxy-3,9-bis(dimethylamino)-6-nitroacridine, 21. In a sealed pressure tube, a suspension of 15 (65 mg) and dimethylamine (1.0 mL) was stirred at room temperature for 24 h, treated with aqueous NaOH (2 N) until the pH was >9, and filtered. The solid was chromatographed (basic Al₂O₃ III, CH₂Cl₂–hexanes solution, 3 : 1), vacuum dried (40 °C) and weighed (22 mg, 32%). NMR (CDCl₃): δ 8.98 (dd, J = 2.3, 0.5 Hz, 1H), 8.26 (dd, J = 9.5, 0.5 Hz, 1H), 8.07 (dd, J = 9.5, 2.3 Hz, 1H), 7.40 (s, 1H), 7.32 (s, 1H), 4.08 (s, 3H), 3.35 (s, 6H), 3.04 (s, 6H). The product was an orange-red solid. It was soluble in dimethylformamide, and CH₂Cl₂, and insoluble in H₂O and hexanes.

2-Methoxy-3,9-bis(dimethylamino)-6-aminoacridine, 22. Under H₂, a suspension of 21 (41 mg), 10% Pd/C (50 mg) and tetrahydrofuran (25 mL) was stirred for 24 h, filtered, and concentrated to an oil by rotary evaporation (room temperature). The oil was chromatographed (silica gel, CH₂Cl₂–CH₃OH solution, 20 : 3; CH₃OH), vacuum dried (40 °C), and weighed (19 mg, 51%). NMR (CDCl₃): δ 8.01 (d, J = 9.2 Hz, 1H), 7.38 (s, 1H), 7.31 (s, 1H), 7.19 (d, J = 2.3 Hz, 1H), 6.89 (dd, J = 9.2, 2.4 Hz, 1H), 4.04 (s, 3H), 3.30 (s, 6H), 2.99 (s, 6H). The product was an orange solid. It was soluble in dimethylformamide and CH₂Cl₂, and insoluble in H₂O and hexanes.

2-Methoxy-3,6-bis(dimethylamino)-9-dimethylaminoacridine, 23. A solution of aqueous formaldehyde (37%, 46 mg) and aqueous H₂SO₄ (9%, 0.59 g) was treated with a suspension of 22 (19 mg), NaBH₄ (27 mg) and tetrahydrofuran (1 mL), then with aqueous NaOH (2 N) until the pH was >9, and extracted with ether. The extract was evaporated to dryness by rotary evaporation (room temperature), and the solid was chromatographed (basic Al₂O₃ III, CHCl₃; CHCl₃–C₂H₅OH solution, 100 : 1), vacuum dried (40 °C), and weighed (8 mg, 33%, a yellow solid). UV-vis (CH₃OH) λ_max, nm (log ε): 462 (4.5). NMR (CDCl₃): δ 8.04 (d, J = 10 Hz, 1H), 7.40 (s, 1H), 7.38 (s, 1H), 7.15 (dd, J = 7.9, 2.5 Hz, 1H), 4.04 (s, 3H), 3.30 (s, 6H), 3.12 (s, 6H), 2.98 (s, 6H). HRMS-EI [M]⁺(calcd with M as C₂₀H₂₆N₄O): 338.2109; found, 338.2116. The product was a yellow solid. It was soluble in dimethylformamide and CH₂Cl₂, and insoluble in H₂O and hexanes.

3-Nitro-1,8-naphthalic anhydride, 24. 24 was purchased from Fisher Scientific.

3-Nitro-1-naphthoic acid, 25. 25 was prepared according to the procedure of Leuck.²⁶

3-Nitro-1-naphthalylamine, 26. 26 was prepared according to the procedure of Blicke.²⁷

3-Nitro-1-dimethylaminonaphthalene, 27. A solution of aqueous formaldehyde (37%, 6.65 g) and aqueous H₂SO₄ (6.2 N, 11 mL) was treated with a suspension of 26 (2.58 g), NaBH₄ (3.66 g) and tetrahydrofuran (94 mL), then with aqueous NaOH (2 N) until the pH was >9 and sonicated with CHCl₃ (200 mL). The CHCl₃ extract was evaporated to dryness (room temperature), and the solid was vacuum dried (40 °C) and weighed (1.99 g, 67%). NMR (CDCl₃): δ 8.43 (d, J = 2.1 Hz, 1H), 8.24 (d, J = 7.8 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.75 (d, J = 2.2 Hz, 1H), 7.67 (ddd, J = 8.5, 6.9, 1.4 Hz, 1H), 7.60 (dd, J = 8.1, 6.9, 1.3 Hz, 1H), 2.95 (s, 6H). The product was a red solid. It was soluble in dimethylformamide and CH₂Cl₂ and insoluble in H₂O and hexanes.

3-Amino-1-dimethylaminonaphthalene, 28. Under H₂, a suspension of 27 (1.51 g), Pd/C (10%, 288 mg) and CH₃OH (70 mL) was stirred at room temperature for 2 days, filtered, and evaporated to an oil by rotary evaporation (room temperature). The oil was subjected to vacuum (40 °C) and weighed (1.07 g, 82%). NMR (CDCl₃): δ 8.06 (m, 1H), 7.57 (m, 1H), 7.30 (m, 2H), 6.70 (t, J = 3.3 Hz, 1H), 6.55 (d, J = 2.2 Hz, 1H), 2.89 (s, 6H). The product was yellow oil. It was soluble in dimethylformamide and CH₂Cl₂, and insoluble in H₂O and hexanes.

2-(4-Dimethylamino-2-naphthalenylamino)-4-nitrobenzoic acid, 29. A suspension of 28 (1.83 g), 2-chloro-4-nitrobenzoic acid (1.54 g), K₂CO₃ (1.99 g), copper powder (267 mg), and 1-pentanol (5 mL) was heated (reflux) for 6.5 h, subjected to steam distillation, and filtered. At 80–100 °C the filtrate was treated with aqueous HCl (2 N) until the pH was 4, filtered, allowed to stand overnight, and again filtered. The solid recovered was vacuum dried (40 °C) and weighed (307 mg, 8.9% based on 28). The product was a brown solid. It was slightly soluble in dimethylformamide, CH₂Cl₂ and H₂O, and insoluble in hexanes.

12-Chloro-5-dimethylamino-9-nitrobenz(a)acridine, 30. A suspension of 29 (307 mg) and POCl₃ (20 mL) was heated (reflux) for 3.5 h, cooled, and added to a slurry of NH₄OH (conc, 320 mL), CHCl₃ (700 mL) and ice (50 g). The organic phase was separated and evaporated to dryness by rotary evaporation (room temperature). The solid was washed (conc NH₄OH), chromatographed (basic Al₂O₃ III, CHCl₃), vacuum dried (40 °C), and weighed (20 mg, 7%). NMR (CDCl₃): δ 9.63 (dd, J = 8.2, 1.1 Hz, 1H), 9.00 (d, J = 1.9 Hz, 1H), 8.66 (d, J = 9.4 Hz, 1H), 8.28 (m, J = 9.5, 8.6, 2.1 Hz, 2H), 7.74 (m, 2H), 7.26 (d, J = 6.0 Hz, 1H), 3.06 (s, 6H). The product was a red-brown solid. It was soluble in CH₂Cl_2, slightly soluble in dimethylformamide, and insoluble in H₂O and hexanes.

12-Methylmercapto-5-dimethylamino-9-nitrobenz(a)acridinethione, 31. A solution of 30 (28 mg), thiourea (75 mg) and C₂H₅OH (5 mL) was heated (reflux) for 4.5 h, and filtered. The solid was vacuum dried (40 °C).

12-Methylmercapto-5-dimethylamino-9-nitrobenz(a)acridine, 32. 31 was dissolved in acetone (3 mL) and the solution was treated with a suspension of CH₃I (210 mg) and K₂CO₃ (35 mg), heated (reflux) for 1 h, concentrated by rotary evaporation (room temperature), diluted with CHCl₃ (20 mL), and filtered. The filtrate was evaporated to dryness by rotary evaporation (room temperature), and the solid was vacuum dried (40 °C) and weighed (28 mg, 96%). NMR (CDCl₃): δ 9.48 (dd, J = 8.1, 1.1 Hz, 1H), 8.98 (dd, J = 2.4, 0.5 Hz, 1H), 8.82 (dd, J = 9.4, 0.5 Hz, 1H), 8.26 (dd, J = 9.4, 2.4 Hz, 1H), 8.18 (dd, J = 7.6, 1.7 Hz, 1H), 7.69 (m, 2H), 7.15 (s, 1H), 3.06 (s, 6H), 2.37 (s, 3H). The product was a brown solid. It was soluble in dimethylformamide and CH₂Cl₂, and insoluble in H₂O and hexanes.

5-Dimethylamino-9-aminobenz(a)acridine, 33. Under H₂, a suspension of 32 (28 mg), Raney Ni (100 mg) and CH₃OH (15 mL) was stirred for 48 h, filtered, and evaporated to dryness by rotary evaporation (40 °C). The solid was chromatographed (basic Al₂O₃ III, CH₂Cl₂), vacuum dried (40 °C) and weighed (5 mg, 26%). NMR (CDCl₃): δ 9.12 (s, 1H), 8.67 (dd, J = 8.1, 1.3 Hz, 1H), 8.26 (dd, J = 8.1, 1.2 Hz, 1H), 7.86 (d, J = 8.8 Hz, 1H), 7.65 (m, 2H), 7.45 (s, 1H), 7.28 (d, J = 2.2 Hz, 1H), 7.01 (dd, J = 8.8, 2.2 Hz, 1H), 3.02 (s, 6H). The product was an orange solid. It was soluble in dimethylformamide and CH₂Cl₂, and insoluble in H₂O and hexanes.

5,9-Bis(dimethylamino)benz(a)acridine, 34. A solution of aqueous formaldehyde (37%, 56 mg) and sulfuric acid (6%, 0.032 mL) was treated with a suspension of 33 (5 mg), NaBH₄ (8 mg) and tetrahydrofuran (2 mL), treated with aqueous NaOH (2 N) until the pH was >9 and extracted with ether (20 mL). The extract was evaporated to dryness by rotary evaporation (room temperature), and the solid was chromatographed (basic Al₂O₃ III, CH₂Cl₂), vacuum dried (40 °C) and weighed (2.8 mg, 45%). UV-vis (CH₃OH) λ_max, nm (log ε): 495 (3.8). NMR (CDCl₃): δ 9.11 (s, 1H), 8.67 (dd, J = 8.1, 1.3 Hz, 1H), 8.26 (dd, J = 8.2, 1.3 Hz, 1H), 7.89 (d, J = 9.1 Hz, 1H), 7.64 (m, 2H), 7.45 (s, 1H), 7.24 (d, J = 6.6 Hz, 1H), 7.20 (s, 1H), 3.18 (s, 6H), 3.01 (s, 6H). HRMS-FAB [M + H]⁺ (calcd with M as C₂₁H₂₁N₃): 316.1814; found, 316.1805. The product was a yellow solid. It was soluble in dimethylformamide and CH₂Cl₂, and insoluble in H₂O and hexanes.

Rhodamine B, 35. 35 was purchased from Sigma-Aldrich.

2-Aminoethyl-3′,6′-bis(dimethylamino)-spiro[1H-isoindole-1,9′-[9H]xanthen]-3(2H)-one, 36. In a sealed pressure tube, a mixture of 35 (311 mg) and ethylenediamine (2.10 g) was stirred at room temperature for 24 h, diluted with CH₂Cl₂ (20 mL) and filtered. The filtrate was evaporated to dryness by rotary evaporation (room temperature), vacuum dried (40 °C), and weighed (300 mg, 85%). NMR (CDCl₃): δ 7.90 (dd, J = 6.0, 2.9 Hz, 1H), 7.44 (dd, J = 5.6, 3.1 Hz, 2H), 7.09 (dd, J = 5.6, 2.6 Hz, 1H), 6.43 (d, J = 8.8 Hz, 2H), 6.37 (d, J = 2.6 Hz, 2H), 6.27 (dd, J = 8.9, 2.6 Hz, 2H), 3.33 (q, J = 7.1 Hz, 8H), 3.18 (t, J = 6.7 Hz, 2H), 2.40 (t, J = 6.7 Hz, 2H), 1.16 (t, J = 7.1 Hz, 12H). The product was a pink solid. It was soluble in CH₂Cl₂, and insoluble in dimethylformamide, H₂O and hexanes.

Lactam conjugate of 36 and acridine, 37. A solution of 5 (31 mg) and lactam 36 (54 mg) and 2-butanol (5 mL) was heated (reflux) for 6.5 h, and evaporated to dryness by rotary evaporation (40 °C). The solid was chromatographed (silica gel, CH₂Cl₂–CH₃OH solution, 10 : 1), washed with aqueous NaOH (2 N), filtered, vacuum dried (40 °C) and weighed (38 mg, 50% based on 5). UV-vis (CH₃OH) λ_max, nm (log ε): 424 (4.5). NMR (CDCl₃): δ 8.00 (m, 1H), 7.84 (d, J = 9.5 Hz, 2H), 7.44 (m, 2H), 7.06 (m, 1H), 6.91 (d, J = 2.6 Hz, 2H), 6.83 (dd, J = 9.5, 2.7 Hz, 2H), 6.31 (d, J = 2.6 Hz, 2H), 6.25 (d, J = 8.9 Hz, 2H), 5.90 (dd, J = 8.9, 2.6 Hz, 2H), 3.63 (t, 2H), 3.36 (t, J = 5.3 Hz, 2H), 3.22 (m, 8H), 3.06 (s, 12H), 1.08 (t, J = 7.1 Hz, 12H). HRMS-FAB [M + H]⁺ (calcd with M as C₄₇H₅₃N₇O₂): 748.4339; found, 748.4349. The product was a yellow solid. It was soluble in dimethylformamide and CH₂Cl_2, and insoluble in H₂O and hexanes.

9-Ethanolamino-3,6-bis(dimethylamino)acridine, 38. A mixture of 5 (63 mg), ethanolamine (78 mg) and CH₃OH (5 mL) was heated (reflux) for 73 h, and evaporated to dryness by rotary evaporation (40 °C). The solid was washed (aqueous NaOH, 2 N) until the washings were pH >8, further washed (H₂O), vacuum dried (40 °C), and weighed (65 mg, 95% based on 5). NMR (CDCl₃): δ 7.93 (d, J = 9.3 Hz, 2H), 6.95 (s, 2H), 6.93 (d, 2H), 3.85 (t, J = 5.1 Hz, 2H), 3.81 (t, J = 4.6 Hz, 2H), 3.05 (s, 12H). The product was a yellow solid. It was soluble in dimethylformamide and CH₂Cl₂, slightly soluble in H₂O, and insoluble in hexanes.

Ester conjugate of 38 and rhodamine B, 39. At 0 °C, a mixture of 38 (44 mg), 4-dimethylaminopyridine (3 mg), N,N-diisopropylcarbodiimide (15 mg) and CH₂Cl₂ (5 mL) was treated slowly with rhodamine B (30 mg), stirred for 96 h, and evaporated to dryness by rotary evaporation (40 °C). The solid was chromatographed (silica gel, CH₂Cl₂–CH₃OH solution, 10 : 1), vacuum dried (40 °C) and weighed (16 mg, 22%). UV-vis (CH₃OH) λ_max, nm (log ε): 418, 556 (4.0, 4.4). NMR (CDCl₃): δ 8.25 (d, J = 9.8 Hz, 2H), 8.16 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 3.5 Hz, 2H), 7.26 (s, 2H), 7.16 (d, J = 8.7 Hz, 1H), 6.95 (d, J = 9.4 Hz, 2H), 6.69 (m, 4H), 6.47 (d, J = 2.1 Hz, 2H), 4.58 (t, 2H), 4.08 (t, 2H), 3.57 (m, 8H), 3.13 (s, 12H), 1.26 (t, J = 7.1 Hz, 12H). HRMS-FAB [M + H]⁺ (calcd with M as C₄₇H₅₃N₆O₃): 750.4257; found, 750.4243. The product was a red solid. It was soluble in dimethylformamide, CH₂Cl₂ and H₂O, and insoluble in hexanes.

NAO, 3,6-bis(dimethylamino)-10-nonylacridinium bromide (acridine orange 10-nonyl bromide), 40. A mixture of acridine orange (69 mg, 0.26 mmol), 1-bromononane (0.99 g, 4.8 mmol) and toluene (5 mL) was heated (reflux) for 4 h and evaporated nearly to dryness by rotary evaporation (40 °C). The wet solid was chromatographed (basic Al₂O₃ III, CH₂Cl₂–ethanol solution, 80 : 1), washed (ether), vacuum dried (40 °C) and weighed (81 mg, 65%). UV-vis (CH₃OH) λ_max, nm (log ε): 495 (5.1). NMR (CDCl₃): δ 8.80 (s, 1H), 7.96 (d, J = 9.5 Hz, 2H), 7.05 (dd, J = 9.3, 2.1 Hz, 2H), 6.62 (d, J = 2.0 Hz, 2H), 4.83 (t, J = 9.0, 7.7 Hz, 2H), 3.34 (s, 12H), 1.95 (m, 2H), 1.62 (m, 2H), 1.40 (m, 2H), 1.26 (m, 8H), 0.86 (t, J = 7.2, 7.2 Hz, 3H). The product was a brown solid. It was soluble in dimethylformamide and CH₂Cl₂, slightly soluble in H₂O, and insoluble in hexanes.

9-Methylmercapto-3,6-bis(dimethylamino)-10-hexylacridinium bromide(9-methylmercaptoacridine orange 10-hexyl bromide), 41. A mixture of acridine 3 (133 mg, 0.427 mmol), 1-bromohexane (1.20 g, 7.27 mmol) and CHCl₃ (3 mL) was heated (reflux) for 10 days and evaporated nearly to dryness by rotary evaporation (40 °C). The product was chromatographed (basic Al₂O₃ III, CH₂Cl₂–ethanol solution, 30 : 1), and the solid obtained was washed (ether), vacuum dried (40 °C), and weighed (49 mg, 23%). UV-vis (CH₃OH) λ_max, nm (log ε): 523 (4.8). NMR (CDCl₃):δ 8.58 (d, J = 9.7 Hz, 2H), 7.19 (dd, J = 9.7, 5.0 Hz, 2H), 6.68 (d, J = 2.1 Hz, 2H), 4.88 (t, J = 9.2, 8.0 Hz, 2H), 3.38 (s, 12H), 2.52 (s, 3H), 1.99 (m, 2H), 1.70 (m, 2H), 1.40 (m, 4H), 0.91 (t, J = 7.8, 7.2 Hz, 3H). HRMS-FAB [M − Br]⁺ (calcd with M as C₂₄H₃₄N₃SBr): 396.2473; found, 396.2474. The product was a brown solid. It was soluble in dimethylformamide and CH₂Cl₂, slightly soluble in H₂O, and insoluble in hexanes.

9-Methylmercapto-3,6-bis(dimethylamino)-10-nonylacridinium bromide(9-methylmercaptoacridine orange 10-nonyl bromide), 42. A mixture of acridine 3 (101 mg, 0.324 mmol), 1-bromononane (685 mg, 3.31 mmol) and CHCl₃ (5 mL) was heated (reflux) for 6 days and evaporated nearly to dryness by rotary evaporation (40 °C). The product was chromatographed (basic Al₂O₃ III, CH₂Cl₂–ethanol solution, 20 : 1), and the solid obtained was washed (ether), vacuum dried (40 °C), and weighed (38 mg, 23%). UV-vis (CH₃OH) λ_max, nm (log ε): 524 (4.7). NMR (CDCl₃): δ 8.60 (d, J = 9.2 Hz, 2H), 7.20 (dd, J = 9.7, 2.1 Hz, 2H), 6.72 (d, J = 1.8 Hz, 2H), 4.92 (t, J = 9.4, 7.6 Hz, 2H),3.40 (s, 12H), 2.51 (s, 3H), 2.03 (m, 2H), 1.70 (m, 2H), 1.45 (m, 2H), 1.25 (m, 8H), 0.91 (t, J = 7.2, 6.5 Hz, 3H). HRMS-FAB [M − Br]⁺ (calcd with M as C₂₇H₄₀N₃SBr): 438.2943; found, 438.2931. The product was a brown solid. It was soluble in dimethylformamide and CH₂Cl₂, slightly soluble in H₂O, and insoluble in hexanes.

9-Cyano-3,6-bis(dimethylamino)-10-hexylacridinium bromide(9-cyanoacridine orange 10-hexyl bromide), 43. A mixture of acridine 9 (52 mg, 0.18 mmol), 1-bromohexane (1.08 g, 6.54 mmol) and toluene (5 mL) was heated (reflux) for 9 h, and evaporated nearly to dryness by rotary evaporation (40 °C). The product was chromatographed (basic Al₂O₃ III, CH₂Cl₂–CH₃OH solution, 100 : 1), and the solid obtained was washed (ether), vacuum dried (40 °C) and weighed (24 mg, 29%). UV-vis (CH₃OH) λ_max, nm (log ε): 578 (4.8). NMR (CDCl₃): δ 7.98 (d, J = 9.5 Hz, 2H), 7.31 (dd, J = 9.5, 2.1 Hz, 2H), 6.82 (d, J = 2.0 Hz, 2H), 5.09 (t, J = 9.2, 7.9 Hz, 2H), 3.44 (s, 12H), 1.95 (m, 2H), 1.68 (m, 2H), 1.40 (m, 2H), 1.36 (m, 2H), 0.87 (t, J = 7.2, 7.1 Hz, 3H). HRMS-FAB [M − Br]⁺ (calcd with M as C₂₄H₃₁N₄Br): 375.2549; found, 375.2549. The product was a purple solid. It was soluble in dimethylformamide and CH₂Cl₂, slightly soluble in H₂O, and insoluble in hexanes.

9-Cyano-3,6-bis(dimethylamino)-10-nonylacridinium bromide(9-cyanoacridine orange 10-nonyl bromide), 44. A mixture of acridine 9 (70 mg, 0.24 mmol), 1-bromononane (312 mg, 1.51 mmol) and toluene (5 mL) was heated (reflux) for 88 h, and evaporated nearly to dryness by rotary evaporation (40 °C). The product was chromatographed (basic Al₂O₃ III, CHCl₃–CH₃OH solution, 100 : 1), and the solid obtained was washed (ether), vacuum dried (40 °C) and weighed (26 mg, 22%). UV-vis (CH₃OH) λ_max, nm (log ε): 577 (4.5). NMR (CDCl₃): δ 7.97 (d, J = 9.6 Hz, 2H), 7.31 (dd, J = 9.5, 2.0 Hz, 2H), 6.79 (d, J = 2.2 Hz, 2H), 5.09 (t, J = 9.7, 7.7 Hz, 2H), 3.43 (s, 12H), 1.98 (m, 2H), 1.75 (m, 2H), 1.40 (m, 2H), 1.25 (m, 8H), 0.84 (t, J = 7.2, 7.1 Hz, 3H). HRMS-FAB [M − Br]⁺ (calcd with M as C₂₇H₃₇N₄Br): 417.3018; found, 417.3023. The product was a purple solid. It was soluble in dimethylformamide and CH₂Cl₂, slightly soluble in H₂O, and insoluble in hexanes.

Nonyl trifluoromethanesulfonate, 45. With the synthesis of related compounds as a guide,^28,29 a solution of 1-nonanol (314 mg, 2.18 mmol), pyridine (255 mg, 3.22 mmol) and CH₂Cl₂ (5 mL) at 0 °C was treated slowly with trifluoromethanesulfonic anhydride (553 mg), stirred for 1.5 h, and concentrated to an oil by rotary evaporation (room temperature). The oil was chromatographed (silica gel, hexanes–ethyl acetate solution, 10 : 1), vacuum dried (room temperature) and weighed (144 mg, 0.521 mmol, 24%). NMR (CDCl₃): δ 4.55 (t, J = 6.7, 6.5 Hz, 2H), 1.84 (m, 2H), 1.30 (m, 12H), 0.95 (t, J = 7.4, 7.0 Hz, 3H). The product was colorless oil. It was soluble in dimethylformamide, CH₂Cl₂ and hexanes, and immiscible in H₂O.

9-Phenylethynyl-3,6-bis(dimethylamino)-10-nonylacridiniumtrifluoromethanesulfonate (9-phenylethynylacridine orange 10-nonyltrifluoromethanesulfonate), 46. A solution of acridine 8 (21 mg, 0.057 mmol), sulfonate 45 (144 mg, 0.522 mmol), 2,6-di-tert-butyl-4-methylpyridine (112 mg, 0.546 mmol), and CH₂Cl₂ (10 mL) was stirred for 10 days, and concentrated to an oil by rotary evaporation (room temperature). The concentrate was chromatographed (silica gel, CH₂Cl₂–CH₃OH solution, 50 : 1), and the solid obtained was vacuum dried (room temperature) and weighed (27 mg, 74%). UV-vis (CH₃OH) λ_max, nm (log ε): 549 (4.9). NMR (CDCl₃): δ 8.32 (d, J = 9.5 Hz, 2H), 7.78 (m, 2H), 7.54 (m, 3H), 7.19 (dd, J = 9.5, 2.1 Hz, 2H), 6.63 (d, J = 2.1 Hz, 2H), 4.72 (t, J = 9.6, 7.8 Hz, 2H), 3.34 (s, 12H), 2.00 (m, 2H), 1.64 (m, 2H), 1.42 (m, 2H), 1.26 (m, 8H), 0.86 (t, J = 7.0, 6.8 Hz, 3H). HRMS-FAB [M − OSO₂CF₃]⁺ (calcd with M as C₃₅H₄₂N₃SO₃F₃): 492.3379; found, 492.3374. The product was a golden-yellow solid. It was soluble in dimethylformamide, slightly soluble in CH₂Cl₂, and insoluble in H₂O and hexanes.

Treatment of MCF-7c3 cells with Pc 4 and acridine orange salts 40–44 and 46

In 60 mm tissue culture dishes, 6 × 10⁵ MCF-7c3 cells per dish were plated in 5 mL of complete growth medium, and the cultures were incubated for enough time (24–48 h) to obtain 80% confluent cells. The incubated cultures were treated with a solution of Pc 4 in complete growth medium (200 nM, 3 mL), and incubated for ∼17 h. The medium was removed, and the cultures were then treated with solutions of either 40–44 or 46 in complete growth medium (200 nM, 3 mL) and incubated for 1 h. The cells from the cultures were washed with 1 mL phenol red-free Hanks' Balanced Salt Solution (HBSS), harvested by trypsinization, resuspended in 3 mL of phenol red-free HBSS, washed, and resuspended in 2 mL phenol red-free HBSS containing glucose (5 mM).

Fluorescence measurements

The fluorescence spectra of 40–44 and 46 in CH₃OH at selected excitation wavelengths were measured. In addition, the fluorescence spectra of the treated cell suspensions of 40–44 and 46 were measured at selected excitation wavelengths.

Computations

All geometries were optimized at the B3LYP/6-31G(d) level with tight convergence criteria and no imposed symmetry. All optimized stationary points were confirmed to be energy minima on the corresponding potential energy surfaces through vibrational frequency analysis (there were no imaginary frequencies). The calculations were carried out with the Gaussian 09 package.³⁰ Note that our calculations give the molecular properties in vacuum while our experiments (e.g., UV-vis spectra and the condition in which a tautomer exists) were performed in condensed phases. There are two computational methods to simulate the solvent molecules. The use of explicit solvation models is very difficult because of a large number of possible chemical interactions and conformations. Implicit solvation models, on the other hand, cannot describe many important chemical interactions such as hydrogen bondings.

Results and discussion

Synthetic procedures

The eight target 9-substituted acridine orange analogues with simple substituents prepared, 3, 4, 5, 6, 7, 8, 9 and 10, have five different atoms (Cl, O, S, N and C) at the 9-position. The routes used are summarized in Scheme 1. 4 was alternatively made by treatment of 5 with CH₃OH, but the reaction was slow. 5 was made from 2 with only POCl₃, however the yield of this procedure was low. 7 probably could have been made with dimethylformamide as a solvent,^31,32 and 9 probably could have been prepared alternatively from 5 and KCN.³³

Scheme 1 Synthesis of 9-substituted acridine oranges.

The synthetic routes used for the three acridine oranges with 2-methoxy groups which were prepared, 18, 20 and 23, are shown in Scheme 2. Intermediate 14 was contaminated with considerable 2-chloro-4-nitrobenzoic acid. While no satisfactory method of purifying 14 was found, it was nevertheless suitable for preparing 15. Both 17 and 19 were made from the same reactants but a longer reaction time was used for 17.

Scheme 2 Synthesis of 2-methoxyacridine oranges.

The benzo substituted acridine orange, 34, was prepared in ten steps, Scheme 3. Literature precedents were used in the preparation of 25. Not surprisingly, 29 was contaminated with considerable 2-chloro-4-nitrobenzoic acid, but it was satisfactory for the preparation of 30. Intermediate 31 was assumed to be present as the thione tautomer.¹⁸

Scheme 3 Synthesis of expanded core acridine oranges.

Conjugates lactam 37 and rhodamine 39 were prepared as shown in Scheme 4.^34,35

Scheme 4 Synthesis of conjugated acridine orange derivatives.

Nonyl acridine orange and NAO analogues were prepared by quaternarization reactions, Scheme 5. The reactive reagent nonyl trifluoromethanesulfonate 45 was used for the preparation of analogue 46 because the nitrogen of the 9-phenylethynyl acridine orange, 8, is deactivated by the electron-withdrawing phenylethynyl group.

Scheme 5 Synthesis of nonyl acridine orange and some of its analogues.

Favored tautomers of acrdine oranges

The additional stabilities of the favored tautomers of compounds 2, 3 and 4 calculated at the B3LYP/6-31G(d) level are shown in Fig. 2. For comparison the additional stability of the favored tautomer of the hypothetical compound 47 is also shown. As expected on the basis of the experimental data, the tautomers with the chalogen carrying a hydrogen are calculated to be less stable while those with it carrying a methyl group are calculated to be more stable.

Fig. 2 Additional stability of favored acridine orange tautomers at the B3LYP/6-31G(d) level.

Band shifts of acridine oranges caused by solvent effect

The position of the visible band of the acridine oranges 1, 3, 8 and 9 in CH₃OH and DMF showed a solvent effect as expected, Table 1. No attempt was made to account for this effect by computation using the explicit solvent model (a model in which the solvent and solute molecules are treated as quantum particles) because of the large number of interactions and conformations that should be considered. Nor was one made with the implicit solvation model (a model in which the solvent molecules are treated as continuum medium) because it does not allow for consideration of types of bonding that should be taken into account, e.g., hydrogen bonding. In the remaining ultraviolet-visible work, CH₃OH was used as a solvent (Table 2).

Table 1 Band shifts caused by solvent effect

	9-Subs	CH3OH		DMF
		λmax (nm)	log ε	λmax (nm)	log ε
1		489	4.9	406	3.9
9	C≡N	488	4.6	488	4.2
3	SMe	516	4.8	445	4.3
8	C≡Ph	542	3.8	464	4.1

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Table 2 Frontier orbital changes and band shifts caused by substitution core expansion and conjugation

	Substituent			HOMO (eV)	LUMO (eV)	H–L (eV)	λmax (nm)	log ε	Shift (nm)
	2	2,3	9
a Due to the rhodamine group.
39			Rhoda				556^a	4.4	67
8			C≡CPh	−4.8	−1.9	2.9	542	3.8	53
20	OMe		SMe	−4.8	−1.5	3.2	519	4.2	30
3			SMe	−4.9	−1.6	3.3	516	4.8	18
5			Cl	−4.9	−1.6	3.3	505	4.5	16
10			C=ONH2	−4.9	−1.6	3.3	503	4.7	14
18	OMe			−4.7	−1.3	3.4	495	4.0	6
34		Benzo		−4.9	−1.4	3.5	495	3.8	6
1				−4.8	−1.4	3.4	489	4.9	0
9			C≡N	−5.1	−2.1	3.0	488	4.6	−1
4			OMe	−4.8	−1.4	3.4	476	4.1	−13
23	OMe		NMe2	−4.7	−1.3	3.3	462	4.5	−27
7			NMe2	−4.8	−1.4	3.4	450	4.4	−39
37			Lactam				424	4.5	−65
6			NH2	−4.6	−1.1	3.5	418	4.4	−71

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Frontier orbital changes and band shifts caused by substitution

Examination of the orbital diagrams of 2,9-substituted acridine orange, 23, and acridine orange itself, 1, Fig. 3a and b, shows that the acridine orange core of 23 is the major contributor to its HOMO and LUMO and that its substituents make only small contributions. Consistent with this, the gaps between the HOMOs and LUMOs of the eight target acridine oranges which are substituted in the 9-position (3–10), the two in the 2- and 9-positions (20, 23) and the one in the 2-position (18) are not affected much by these substitutions, Table 2. From this it is concluded that in general the frontier orbitals of the substituted acridine oranges are dominated by their cores and little influenced by their substituents.

Fig. 3 HOMOs and LUMOs of acridine oranges (a) 1, (b) 23 and (c) 34.

Correspondingly the band positions of the eleven target substituted acridine oranges (3–10, 18, 20, 23) studied are not greatly affected by their substitution. As a result, the shift of the substituted acridine orange with the greatest red shift, 8, is moderate, 53 nm. Not surprisingly the extinction coefficients of all these bands are near that of acridine orange 1 itself.

Frontier orbital changes and band shifts caused by core expansion

Examination of the orbital diagrams of ring-expanded acridine orange 34 and of 1 shows that the acridine orange core of 34 is the main contributor to its HOMO and LUMO, Fig. 3a and c. As then expected, the wavelength of the band of 34 is not much different from that of 1 nor is its extinction coefficient much different, Table 2. On the basis of these results the core expansion approach is in general not expected to yield the sought after red-shifted band.

Band positions arising from acridine orange-chromophore conjugation

The conjugation of acridine orange with rhodamine B led to rhodamine 39, a compound having a band with a fairly long wavelength, 556 nm (although not a red band). This compound differs from those made by acridine orange substitution or core expansion in that its long wavelength visible band is provided by its rhodamine group not its acridine orange group. The acridine group provides only for its targeting potential. The acridine orange-chromophore conjugation approach used here greatly expands the number of potentially useful compounds available.

Another attempt to apply this acridine orange-chromophore approach to the synthesis of a compound like 39 except for the replacement of its ester linkage by an amide linkage led to a compound without an acridine orange group because of lactam formation, lactam 37.

This acridine orange-chromophore conjugation approach has not been fully exploited and further work with it may well be fruitful.

FRET between Pc 4 and 9-substituted acridine orange salts

In previous studies when untreated MCF-7c3 cells and when MCF-7c3 cells treated with Pc 4 were excited at 488 nm, their emission spectra were very similar and neither had an observable fluorescence between 500 and 800 nm.¹⁵ With PC-3 cells containing both NAO and Pc 4, excitation of the NAO at 488 nm resulted in the emission of Pc 4 at 675 nm and confirmed FRET between them.¹⁴

In this study, MCF-7c3 cells containing Pc 4 and 40 or an NAO analogue of 40 (41, 42, 43, 44 or 46) were excited at a selected wavelength, Fig. 4. The lack of an appreciable fluorescence peak at 675 nm in the case of the suspensions treated with both Pc 4 and 41, 42 or 46, Fig. 4, in spite of the significant overlap of the fluorescence spectra of 41, 42 and 46 with the absorption spectrum of Pc 4, Fig. 5, shows that little FRET occurred between these 9-substituted acridine orange salts and Pc 4 in cells. This is probably due to a lack of uptake of the acridine orange salts by the cells.

Fig. 4 The fluorescence spectra of cell suspensions treated with Pc 4 and with 40, 41, 42, 43, 44 or 46. The excitation wavelengths in nm are given next to the compound numbers.

Fig. 5 Fluorescence spectra of 40, 41, 42, 43, 44 and 46 in CH₃OH and the absorption spectrum of Pc 4 in CH₃OH.

The fluorescence peak of cells treated with Pc 4 and 40 at 675 nm is as expected on the basis of previous FRET work.¹⁴ The substantial fluorescence peak at 675 nm of cells treated with Pc 4 and 43 or 44 shows that FRET occurred between these two acridine orange salts and Pc 4. The occurrence of FRET with these acridine orange salts suggests that it may be possible to find acridine orange salts that are stable and give much stronger FRET with Pc 4 than these do. This would be important for mechanistic studies of Pc 4.

Summary and conclusions

Nonyl acridine orange analogues have been synthesized in an effort to find analogues with visible bands allowing them to act as FRET donors to Pc 4 in cells. The occurrence of Pc 4-FRET in MCF-7c3 cells with these NAO analogues has been explored. The results of the work done suggest that NAO analogues giving strong FRET with Pc 4 in cells can be found.

Acknowledgements

We thank Prof. Nancy L. Oleinick for many useful discussions and Mr James Faulk for help with the mass spectra. This work was supported by in part by allocations of computing resources from the Ohio Supercomputer Center and the Case High Performance Computing System.

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Footnote

† Electronic supplementary information (ESI) available: Calculated structures and IR data of acridine oranges. See DOI: 10.1039/c5ra28126a

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