METHOD

Abstract

The invention provides mitochondria-targeted chemiluminescent agents and their use in methods of photodynamic therapy (PDT). In particular, the invention provides compounds of general formula (I), and their pharmaceutically acceptable salts: (I) in which A represents a chemiluminescent moiety; each L, which may be the same or different, is either a direct bond or a linker; each B, which may be the same or different, represents a mitotropic moiety; n is an integer from 1 to 3, preferably 1; and x is an integer from 1 to 3, preferably 1. Such compounds find particular use in the treatment of deeply-sited tumours, e.g. glioblastoma multiforme (GBM), when used in combination with a photosensitizer or photosensitizer precursor.

ALB].sub.n].sub.X  (I)

Claims

1. A mitochondria-targeted chemiluminescent agent for use in photodynamic therapy.

2. An agent for use as claimed in claim 1, wherein said agent is a conjugate comprising at least one chemiluminescent moiety attached to or otherwise associated with at least one mitotropic moiety.

3. An agent for use as claimed in claim 2, wherein said conjugate is a compound of general formula (I), or a pharmaceutically acceptable salt thereof:
ALB].sub.n].sub.X   (I) in which A represents a chemiluminescent moiety; each L, which may be the same or different, is either a direct bond or a linker (e.g. an organic linker); each B, which may be the same or different, represents a mitotropic moiety; n is an integer from 1 to 3, preferably 1; and x is an integer from 1 to 3, preferably 1.

4. An agent for use as claimed in claim 3, wherein said conjugate is a compound of formula (II), or a pharmaceutically acceptable salt thereof:
A-L-B   (II) in which A, L and B are as defined in claim 3.

5. An agent for use as claimed in any one of the preceding claims, wherein said chemiluminescent agent or chemiluminescent moiety is selected from the group consisting of luminol, isoluminol, lucigenin, acridinium esters, oxalate esters, and derivatives thereof.

6. An agent for use as claimed in claim 5, wherein said chemiluminescent agent or chemiluminescent moiety is luminol, isoluminol, acridinium esters, or a derivative thereof.

7. An agent for use as claimed in any one of claims 2 to 6, wherein said mitotropic moiety is a phosphonium ion, dequalinium or a derivative thereof, guanidinium or a derivative thereof, Rhodamine 123 or Rhodamine 110.

8. An agent for use as claimed in claim 7, wherein said mitotropic moiety is a phosphonium ion, Rhodamine 123 or Rhodamine 110

9. An agent for use as claimed in any one of claims 3 to 8, wherein said linker L comprises an alkylene chain (preferably a C.sub.1-15 alkylene, e.g. a C.sub.2-11 alkylene) optionally substituted by one or more groups selected from C.sub.1-3 alkyl, —O(C.sub.1-3)alkyl, —OH, cycloalkyl and aryl groups; and in which one or more —CH.sub.2— groups of the alkylene chain may be replaced by a group independently selected from —O—, —CO—, —NR— (where R is —H or C.sub.1-6 alkyl, preferably C.sub.1-3 alkyl, e.g. methyl), cycloalkyl, heterocyclic, aryl and heteroaryl groups.

10. An agent for use as claimed in claim 9, wherein said linker L is selected from the group consisting of: —C.sub.3H.sub.6—, —C.sub.4H.sub.8—, —C.sub.6H.sub.12—, —C.sub.8H.sub.20—, —C.sub.10H.sub.20—, —CH.sub.22—, —CO—CH.sub.2—, —CO—C.sub.3H.sub.6, —CO—C.sub.5H.sub.10—, —CO—C.sub.6H.sub.12—, —CO—C.sub.10H.sub.20—, polyethylene glycol groups containing from 1 to 4 ethylene oxide units; ##STR00059## (where a is an integer from 1 to 6, e.g. from 2 to 5).

11. An agent for use as claimed in any one of the preceding claims which is a compound of formula (III), or a pharmaceutically acceptable salt thereof: ##STR00060## (where L.sup.1 is a linker, e.g. a linker as defined in claim 9 or 10; B.sup.1 is a mitotropic moiety, e.g. a mitotropic moiety as defined in claim 7 or 8; R.sup.3 is hydrogen, or an alkyl group such as C.sub.1-3 alkyl (e.g. methyl); each R.sup.4 is independently selected from C.sub.1-6 alkyl, and —NR.sup.5R.sup.6; R.sup.5 and R.sup.6 are independently selected from H and C.sub.1-6 alkyl, preferably from H and C.sub.1-3 alkyl (e.g. —CH.sub.3); and p is an integer from 0 to 3, preferably 0, 1 or 2, e.g. 0 or 1).

12. An agent for use as claimed in claim 11 which is a compound of formula (IIIa) or (IIIb): ##STR00061## where L.sup.1, B.sup.1, R.sup.3, R.sup.4 and p are as defined in claim 11.

13. An agent for use as claimed in claim 11 or claim 12, wherein L is selected from the group consisting of ##STR00062## (where a is an integer from 1 to 10, preferably from 3 to 10; and b is an integer from 1 to 4, e.g. 2).

14. An agent for use as claimed in any one of claims 11 to 13, wherein B, is the following group. ##STR00063## (where R.sup.1 is phenyl, toluene (e.g. o-toluene, m-toluene or p-toluene), or cyclohexyl; and X is a monovalent anion, e.g. a Cl, Br, or I anion).

15. An agent for use as claimed in any one of claims 1 to 10 which is a compound of formula (IV), or a pharmaceutically acceptable salt thereof: ##STR00064## (where L.sup.2 is a linker, e.g. a linker as defined in claim 9 or 10; B.sup.2 is a mitotropic moiety, e.g. a mitotropic moiety as defined in claim 7 or 8; each R.sup.6 is independently selected from halogen (e.g. F, Cl, Br, I), and C.sub.1-6 alkyl (e.g. tBu); q is an integer from 0 to 4, preferably 0 or 2; and Z is a monovalent anion, e.g. a Cl, Br, I, or CF.sub.3OSO.sub.2 anion).

16. An agent for use as claimed in claim 15, wherein L.sup.2 represents the following group: ##STR00065## (wherein a is an integer from 1 to 10, preferably 3, 4 or 5).

17. An agent for use as claimed in claim 15 or claim 16, wherein B2 is the following group: ##STR00066## (where R.sup.1 is phenyl, toluene (e.g. o-toluene, m-toluene or p-toluene), or cyclohexyl; and X is a monovalent anion, e.g. a Cl, Br, or I anion).

18. An agent for use as claimed in any one of claims 1 to 10 which is compound of formula (V), or a pharmaceutically acceptable salt thereof: ##STR00067## (where L.sup.1 is a linker, e.g. a linker as defined in claim 9 or 10; B.sup.3 is a mitotropic agent, e.g. a mitotropic agent as defined in claim 7 or 8; each R.sup.6 is independently selected from halogen (e.g. F, Cl, Br, I), —CO.sub.2R.sup.8 (where R.sup.8 is hydrogen or C.sub.1-6 alkyl), cyano, and C.sub.1-6 alkyl (e.g. tBu); r is an integer from 0 to 5, preferably 0 or 3; and Z is a monovalent anion, e.g. a Cl, Br, I, or CF.sub.3OSO.sub.2 anion).

19. An agent for use as claimed in claim 18, wherein L.sup.3 is a C.sub.1-10 alkylene group, e.g. C.sub.1-6 alkylene.

20. An agent for use as claimed in claim 18 or claim 19, wherein B is the following group: ##STR00068## (where R.sup.1 is phenyl, toluene (e.g. o-toluene, m-toluene or p-toluene), or cyclohexyl; and X is a monovalent anion, e.g. a Cl, Br, or I anion).

21. An agent for use as claimed in any one of claims 1 to 10 which is a compound of formula (VI) or a pharmaceutically acceptable salt thereof: ##STR00069## (where L.sup.4 is a linker, e.g. a linker as defined in claim 9 or 10; and A.sup.1 is a chemiluminescent moiety, e.g. a chemiluminescent moiety as defined in claim 5 or 6).

22. An agent for use as claimed in claim 21, wherein L.sup.4 is selected from the group consisting of ##STR00070## (where a is an integer from 1 to 10, preferably 4, 5 or 6).

23. An agent for use as claimed in claim 21 or claim 22, wherein A.sup.1 is selected from any of the following: ##STR00071## (where R.sup.3 is hydrogen, or an alkyl group such as C.sub.1-3 alkyl (e.g. methyl); each R is independently selected from C.sub.1-6 alkyl, and —NR.sup.5R.sup.6; R.sup.5 and R are independently selected from H and C.sub.1-6 alkyl, preferably from H and C.sub.3 alkyl (e.g. —CH.sub.3); p is an integer from 0 to 3, preferably 0, 1 or 2, e.g. 0 or 1; Z is a monovalent anion, e.g. a Cl, Br, I, or CF.sub.3SO.sub.2 anion; each R.sup.9 is independently selected from halogen (e.g. F, Cl, Br, I) and C.sub.6 alkyl (e.g. tBu); and s is an integer from 0 to 4, preferably 0, 2 or 3).

24. An agent for use as claimed in any one of claims 1 to 10 which is a compound of formula (VII), or a pharmaceutically acceptable salt thereof: ##STR00072## (where L.sup.5 is a linker, e.g. a linker as defined in claim 9 or 10; B.sup.4 is a mitotropic agent, e.g. a mitotropic agent as defined in claim 7 or 8; each R.sup.10 is independently selected from C.sub.1-6 alkyl (e.g. methyl), and —NR.sup.11R.sup.12; R.sup.11 and R.sup.12 are independently selected from H and C.sub.1-6 alkyl, preferably from H and C.sub.1-3 alkyl (e.g. —CH.sub.3); and t is an integer from 0 to 3, preferably 1 or 2).

25. An agent for use as claimed in claim 24 which is a compound of formula (VIIa): ##STR00073## (where L.sup.5, B.sup.4, R.sup.11 and R.sup.12 are as defined in claim 24; and R.sup.13 is H or Ca alkyl).

26. An agent for use as claimed in claim 24 or claim 25, wherein L.sup.5 is C.sub.1-11 alkylene, preferably C.sub.2-8 alkylene, e.g. propylene.

27. An agent for use as claimed in any one of the preceding claims, wherein said photodynamic therapy comprises simultaneous or sequential use of a photosensitizer or a precursor thereof.

28. An agent for use as claimed in claim 27, wherein said photosensitizer or precursor is selected from 5-aminolevulinic acid (5-ALA) and derivatives of 5-ALA, protoporphyrins (e.g. protoporphyrin IX); phthalocyanines such as aluminium phthalocyanines which may be sulphonated (i.e. AlPcS), e.g. di-sulphonated aluminium phthalocyanines such as AIPcS.sub.2 or AIPcS.sub.2a, or aluminium phthalocyanine tetra-sulfonate (AIPcS.sub.4); sulphonated tetraphenylporphyrins (e.g. TPPS.sub.2a, TPPS.sub.4, TPPS.sub.1 and TPPS.sub.2o); chlorins such as tetra(m-hydroxyphenyl)chlorins (m-THPC) (e.g. temoporfin which is marketed under the tradename Foscan); chlorin derivatives including bacteriochlorins and ketochlorins; mono-L-aspartyl chlorin e6 (NPe6) or chlorin e6; natural and synthetic porpyhrins including hematoporphyrin and benzoporphyrins; anthraquinones and derivatives thereof (e.g. hypericin, hypocrellins [A, B], cercosporin, calphostin, elsinochromes [A, B, C]).

29. An agent for use as claimed in claim 28, wherein said photosensitizer precursor is 5-aminolevulinic acid (5-ALA), a derivative or pharmaceutically acceptable salt thereof.

30. An agent as claimed in any one of the preceding claims for use in the photodynamic treatment of any disorder or abnormality of cells or tissues in an animal body (e.g. a human) which are responsive to photodynamic therapy.

31. An agent for use as claimed in claim 30 in the treatment of cancer, preferably in the treatment of an internal cancer, e.g. a deeply-sited cancer.

32. An agent for use as claimed in claim 31, wherein said cancer is selected from the group consisting of gliomas and other brain cancers, hepatic and pancreatic cancers, breast, lung and prostate cancer, cholangiocarcinoma, stomach and colon cancers, bladder cancer, cervical cancers, head and neck cancers.

33. An agent for use as claimed in claim 32, wherein said cancer is GBM.

34. A pharmaceutical composition comprising an agent as defined in any one of claims 1 to 26, together with at least one pharmaceutically acceptable carrier or excipient.

35. A pharmaceutical composition comprising an agent as defined in any one of claims 1 to 26, and a photosensitizer or photosensitizer precursor as defined in any one of claims 27 to 29, together with at least one pharmaceutically acceptable carrier or excipient.

36. A pharmaceutical composition as claimed in claim 34 or claim 35 for use in photodynamic therapy, preferably for use in the treatment of an internal cancer, e.g. a deeply-sited cancer.

37. A product comprising an agent as defined in any one of claims 1 to 26, and a photosensitizer or photosensitizer precursor as defined in any one of claims 27 to 29 for simultaneous, separate or sequential use in a method of photodynamic therapy.

38. A kit comprising: (i) an agent as defined in any one of claims 1 to 26; and separately (ii) a photosensitizer or photosensitizer precursor as defined in any one of claims 27 to 29; and optionally (iii) instructions for the use of (i) and (ii) in a method of photodynamic therapy.

39. Use of an agent as defined in any one of claims 1 to 26 in the manufacture of a medicament for use in combination therapy with a photosensitizer or photosensitizer precursor as defined in any one of claims 27 to 29, e.g. for use in a method of photodynamic therapy.

40. Use of a photosensitizer or photosensitizer precursor as defined in any one of claims 27 to 29 in the manufacture of a medicament for use in combination therapy with an agent as defined in any one of claims 1 to 26, e.g. for use in a method of photodynamic therapy.

41. Use of an agent as defined in any one of claims 1 to 26 together with a photosensitizer or photosensitizer precursor as defined in any one of claims 27 to 29 in the manufacture of a medicament for use in a method of photodynamic therapy.

42. A method of photodynamic therapy of cells or tissues of a patient (e.g. a human patient), said method comprising the step of administering to said cells or tissues: (a) an effective amount of an agent as defined in any one of claims 1 to 26 and, simultaneously, separately, or sequentially thereto, an effective amount of a photosensitizer or photosensitizer precursor as defined in any one of claims 27 to 29; or (b) an effective amount of a pharmaceutical composition as defined in claim 35.

43. A conjugate as defined in any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof.

Description

[0160] The invention will now be described further with reference to the following non-limiting Examples and the accompanying drawings in which:

[0161] FIG. 1—A schematic representation of the concept behind the invention in which a modified version of luminol is employed as a self-sustained, intracellular source of light and the target mitochondria is used as the power supply for “switching on the light” and activating the cytotoxic activity of the photosensitive drug (PpIX) from within the tumour cells. This representation is not to be construed as limiting in any way on the scope of the invention.

[0162] FIG. 2—Luminescence of mitotropic luminol derivatives in biomimetic conditions resembling the mitochondrial matrix environment.

[0163] FIG. 3—Luminescence from a cell layer after application of mitotropic luminol derivatives in propylene glycol.

[0164] FIG. 4—Energy transfer between luminol luminescence and various photosensitisers: A) luminescence of luminol in alkaline DMSO (KOH); B) addition of hypocrellin A (HYPA); C) addition of hypericin (HYP) instead of HYPA; D) Luminol in aqueous carbonate buffer (pH 10.3) with the addition of CuSO.sub.4 and urea peroxide in the presence of Rose Bengal (RB); E) 532 nm laser excitation; F) the luminol system in carbonate buffer as in D) but with the addition of TPPS4.

[0165] FIG. 5—Energy transfer between luminol luminescence and various photosensitisers: A) the spectrum of luminol in DMSO-tert-butoxide; B) Erythrosin B absorbs very strongly, especially around 500 and demonstrates very strong fluorescence around 580 nm; C) addition of tert butoxide to the DMSO-luminol solution.

[0166] FIG. 6—Confocal micrographs of mitotropic luminol derivatives and luminol. On the triple column (left) the mitochondrial localisation of two derivatives namely DZ160 and AP47 is shown in two different cell lines, the breast adenocarcinoma cell line MCF7 and the glioblastoma cell line U87. The three micrographs in each row of this triple column depict the localisation of the derivative, the localisation of the mitochondrial marker mitotracker green and the overlay of these two localisations in a merged image. The fluorescence of luminol is represented in the left column while mitotracker green fluorescence is shown in the middle column. Their overlay is shown in the third column. On the column in the right (fourth column) the cytosolic localisation of free luminol is shown on the top (with border) while in two cases the localisation of AP47 and DZ160 is depicted in cells not incubated with mitotracker green to exclude fluorescence spillover and hence cross talk between the mitotracker and luminol-derivative channels.

[0167] FIG. 7—LUMIBLAST experiments on U87 and MCF7 cells.

[0168] FIG. 8—Compound DZ68 in MCF7 and U87 cells in the presence of CuSO.sub.4 and with the use of HYPA as the photosensitizer.

[0169] FIG. 9—LUMIBLAST effect in U87 cells mediated by CuSO.sub.4 in the presence of HYPA as the photosensitizer and DZ167 triphenylphosphonium-luminol derivative.

[0170] FIG. 10—Subcellular localisation of cercosporin and erythrocin b (green in image) in T98G glioblastoma multiforme cells. The probe mitotracker deep red (red in image) was used to evaluate the colocalization of the two dyes with cell mitochondria.

[0171] FIG. 11—LUIMIBLAST effect with the use of the photosensitizer cercosporin and two luminol mitotropic derivatives namely DZ203 and DZ196, in U87G, BM cells. The absorption and emission properties of cercosporin are shown in the left top diagram whereas the emission of luminol is shown in the left lower graph.

[0172] FIG. 12—Metabolic analysis on MCF7 cells incubated with DZ167 and 5-ALA individually as well as in combination (first 5-ALA and then DZ167). Oxygen consumption rate (left) represents the effects of the compounds investigated on cellular respiration while the extracellular acidification rate (ECAR) shows the effect of the selected compounds on the process of glycolysis. Following compound injections, the cells were subsequently injected with oligomycin, FCCP and Antimycin A+Rotenone in the case of single compound administration (DZ167 or 5-ALA) and FCCP and Antimycin A+Rotenone in the case of combinatory administration (5-ALA and DZ167). These subsequent injections were performed in order to elucidate the effects on cellular respiration, and in the case of oligomycin also the glycolytic capacity of the cells.

[0173] FIG. 13—LUMIBLAST effect in MDA-MB-231 breast carcinoma cells in the absence (grey bars) or presence (white bars diagonal line pattern) of 100 μM CuSO.sub.4 and the co-administration of 5-ALA (1.2 mM) and DZ203 (400 μM).

EXAMPLES

Example 1—Synthesis of Phthalimides as Acylation Precursors (Intermediates)

[0174] ##STR00020##

[0175] Step 1: A mixture of nitrophthalic acid 12a or 12b (12.6 g, 0.06 mol) and acetic anhydride (11.15 mL, 0.12 mol) was refluxed for 1 hour. The mixture was brought to room temperature, toluene (300 mL) was added and the volatiles were removed in vacuo. The residue was washed several times with diethyl ether to afford the respective anhydride 13a or 13b (10.25 g, 90%) as a white solid.

[0176] Step 2: To a solution of nitrophthalic anhydride 13a or 13b (10 g, 0.052 mol) in acetic acid (90 mL) was added sec-butylamine (7.85 mL, 0.078 mol) and the mixture was refluxed for 5 hours. After cooling, the mixture was concentrated in vacuo, diluted with H.sub.2O (100 mL) and extracted with DCM (3×100 mL). The combined organic layer was washed with saturated aq. NaHCO.sub.3 and brine, dried over anhydrous Na.sub.2SO.sub.4, filtered and concentrated in vacuo to give the respective phthalimide 14a or 14b (6.45 g, 50%) as a white solid.

[0177] Step 3: 10% Pd/C (10 mol %) was added to a degassed (Ar purged) solution of nitrophthalimide 14a or 14b (9 g, 0.04 mol) in MeOH (250 mL) and the mixture was stirred in a hydrogen atmosphere (20 bar) for 24 hours. Then, the solution was filtered through a celite pad and the solvent was distilled off, yielding the respective phthalimide 15a or 15b (6.1 g, 70%) as a yellow solid.

Example 2—Synthesis of 6-bromo-N-(2-(sec-butyl)-1,3-dioxoisoindolin-4-yl)hexanamide (Intermediate)

[0178] ##STR00021##

[0179] 6-bromohexanoic acid (2.14 g, 0.011 mol) was suspended in oxalyl chloride (ca. 10 mL), stirred at ambient temperature for 2 hours and the excess oxalyl chloride was distilled off under reduced pressure. The residue (acid chloride) was dissolved in dry dichloromethane (10 mL) and added dropwise to a solution of 15a (2.2 g, 0.01 mol) and pyridine (1.62 ml, 0.02 mol) in dry DCM (15 mL) at 0° C. under argon. After the addition, the mixture was warmed to ambient temperature and stirred for 24 hours. DCM (50 mL) was added, the solution was washed with H.sub.2O (50 mL), 1 M aq. HCl (50 mL), saturated aq. NaHCO.sub.3 (50 mL), and brine (50 mL), dried (Na.sub.2SO.sub.4) and the solvent evaporated under reduced pressure. Purification of the residue by flash chromatography (silica gel, PE/EtOAc 6:1) yielded the amide 11a (2.80 g, 71%) as a white solid.

Example 3—Synthesis of 11-bromo-N-(2-(sec-butyl)-1,3-dioxoisoindolin-4-yl)undecanamide (Intermediate)

[0180] ##STR00022##

[0181] 11-bromoundecanoic acid (1.17 g, 4.4 mmol) was suspended in oxalyl chloride (ca. 7 mL), stirred at ambient temperature for 2 hours and the excess oxalyl chloride was distilled off under reduced pressure. The residue (acid chloride) was dissolved in dry dichloromethane (5 mL) and added dropwise to a solution of 15a (0.9 g, 4 mmol) and pyridine (0.65 ml, 8 mmol) in dry DCM (15 mL) at 0° C. under argon. After the addition, the mixture was warmed to ambient temperature and stirred for 24 hours. DCM (50 mL) was added, the solution was washed with H.sub.2O (50 mL), 1 M aq. HCl (50 mL), saturated aq. NaHCO.sub.3 (50 mL), and brine (50 mL), dried (Na.sub.2SO.sub.4) and the solvent evaporated under reduced pressure. Purification of the residue by flash chromatography (silica gel, PE/EtOAc 6:1) yielded the amide 11a (1.3 g, 70%) as a white solid.

Example 4—Synthesis of 6-bromo-N-(2-(sec-butyl)-1,3-dioxoisoindolin-5-yl)hexanamide (Intermediate)

[0182] ##STR00023##

[0183] 6-bromohexanoic acid (1.29 g, 7 mmol) was suspended in oxalyl chloride (ca. 10 mL), stirred at ambient temperature for 2 hours and the excess oxalyl chloride was distilled off under reduced pressure. The residue (acid chloride) was dissolved in dry dichloromethane (5 mL) and added dropwise to a solution of 15b (1.3 g, 6 mmol) and pyridine (0.98 ml, 12 mmol) in dry DCM (10 mL) at 0° C. under argon. After the addition, the mixture was warmed to ambient temperature and stirred for 24 hours. DCM (50 mL) was added, the solution was washed with —H.sub.2O (50 mL), 1 M aq. HC (50 mL), saturated aq. NaHCO.sub.3 (50 mL), and brine (50 mL), dried (Na.sub.2SO.sub.4) and the solvent evaporated under reduced pressure. Purification of the residue by flash chromatography (silica gel, 4% MeOH/DCM) yielded the amide 21a (1.12 g, 47%) as a white solid.

Example 5—Synthesis of 11-bromo-N-(2-(sec-butyl)-1,3-dioxoisoindolin-5-yl)undecanamide (Intermediate)

[0184] ##STR00024##

[0185] 11-bromoundecanoic acid (1.74 g, 7 mmol) was suspended in oxalyl chloride (ca. 7 mL), stirred at ambient temperature for 2 hours and the excess oxalyl chloride was distilled off under reduced pressure. The residue (acid chloride) was dissolved in dry dichloromethane mL) and added dropwise to a solution of 15b (1.3 g, 6 mmol) and pyridine (0.98 ml, 12 mmol) in dry DCM (15 mL) at 0° C. under argon. After the addition, the mixture was warmed to ambient temperature and stirred for 24 hours. DCM (50 mL) was added, the solution was washed with H.sub.2O (50 mL), 1 M aq. HCl (50 mL), saturated. aq. NaHCO.sub.3 (50 ml), and brine (50 mL), dried (Na.sub.2SO.sub.4) and the solvent evaporated under reduced pressure. Purification of the residue by flash chromatography (silica gel, 4% MeOH/DCM) yielded the amide 21b (1.62 g, 58%) as a white solid.

Example 6—Synthesis of (6-((2-(sec-butyl)-1,3-dioxoisoindolin-4-yl)amino)-6-oxohexyl)triphenylphosphonium Bromide (Intermediate)

[0186] ##STR00025##

[0187] A solution of 11a (429 mg, 1.09 mmol) and triphenylphosphine (570 mg, 2.17 mmol) in dry CH.sub.3CN (15 mL) was refluxed for 72 hours. The mixture was concentrated in vacuo and the residue was purified by flash chromatography (10% MeOH/DCM) to afford the title compound 22a (362 mg, 51%) as a white solid.

Example 7—Synthesis of (6-((2-(sec-butyl)-1,3-dioxoisoindolin-4-yl)amino)-6-oxohexyl)tri-p-tolylphosphonium Bromide (Intermediate)

[0188] ##STR00026##

[0189] A solution of 11a (146 mg, 0.37 mmol) and tri(p-tolyl)phosphine (225 mg, 0.74 mmol) in dry CH.sub.3CN (6 mL) was refluxed for 72 hours. The mixture was concentrated in vacuo and the residue was purified by flash chromatography (7% MeOH/DCM) to afford the title compound 22b (256 mg, 99%) as a white solid.

Example 8—Synthesis of (11-((2-(sec-butyl)-1,3-dioxoisoindolin-4-yl)amino)-11-oxoundecyl)triphenylphosphonium Bromide (Intermediate)

[0190] ##STR00027##

[0191] A solution of 11b (400 mg, 0.86 mmol) and triphenylphosphine (678 mg, 2.58 mmol) in dry CH.sub.3CN (5 mL) was refluxed for 72 h. The mixture was concentrated in vacuo and the residue was purified by flash chromatography (7% MeOH/DCM) to afford the title compound 22C (321 mg, 37%) as a white solid.

Example 9—Synthesis of (11-((2-(sec-butyl)-1,3-dioxoisoindolin-4-yl)amino)-11-oxoundecyl)tri-p-tolylphosphonium Bromide (Intermediate)

[0192] ##STR00028##

[0193] A solution of 11b (400 mg, 0.86 mmol) and tri(p-tolyl)phosphine (785 mg, 2.58 mmol) in dry CH.sub.3CN (7 mL) was refluxed for 72 hours. The mixture was concentrated in vacuo and the residue was purified by flash chromatography (50% MeOH/DCM) to afford the title compound 22d (636 mg, 96%) as a white solid.

Example 10—Synthesis of (11-((2-(sec-butyl)-1,3-dioxoisoindolin-4-yl)amino)-11-oxoundecyl)tricyclohexylphosphonium Bromide (Intermediate)

[0194] ##STR00029##

[0195] A solution of 11b (400 mg, 0.86 mmol) and tricyclohexylphosphine (723 mg, 2.58 mmol) in dry CH.sub.3CN (5 mL) was refluxed for 72 hours. The mixture was concentrated in vacuo and the residue was purified by flash chromatography (5% MeOH/DCM) to afford the title compound 22e (406 mg, 63%) as a white solid.

Example 11—Synthesis of (6-((2-(sec-butyl)-1,3-dioxoisoindolin-4-yl)amino)-6-oxohexyl)tri-p-tolylphosphonium Bromide (Intermediate)

[0196] ##STR00030##

[0197] A solution of 21a (350 mg, 0.89 mmol) and tri(p-tolyl)phosphine (539 mg, 1.77 mmol) in dry CH.sub.3CN (4 mL) was refluxed for 72 hours. The mixture was concentrated in vacuo and the residue was purified by flash chromatography (5% MeOH/DCM) to afford the title compound 23a (448 mg, 72%) as a white solid.

Example 12—Synthesis of (6-((2-(sec-butyl)-1,3-dioxoisoindolin-5-yl)amino)-6-oxohexyl)tricyclohexylphosphonium Bromide (Intermediate)

[0198] ##STR00031##

[0199] A solution of 21a (350 mg, 0.89 mmol) and tricyclohexylphosphine (497 mg, 1.77 mmol) in dry CH.sub.3CN (4 mL) was refluxed for 72 hours. The mixture was concentrated in vacuo and the residue was purified by flash chromatography (5% MeOH/DCM) to afford the title compound 23b (529 mg, 88%) as a white solid.

Example 13—Synthesis of (11-((2-(sec-butyl)-1,3-dioxoisoindolin-5-yl)amino)-11-oxoundecyl)tri-p-tolylphosphonium Bromide (Intermediate)

[0200] ##STR00032##

[0201] A solution of 21b (523 mg, 1.72 mmol) and tri(p-tolyl)phosphine (523 mg, 1.72 mmol) in dry CH.sub.3CN (5 mL) was refluxed for 72 hours. The mixture was concentrated in vacuo and the residue was purified by flash chromatography (5% MeOH/DCM) to afford the title compound 23c (449 mg, 68%) as a white solid.

Example 14—Synthesis of Mitotropic 5-N-Acylated Luminol Derivatives 1a-e from Hydrazinolysis of the Respective Phthalimides 22a-e

[0202] ##STR00033##

[0203] Hydrazine hydrate (0.18 mL, 3.04 mmol) was added in a stirring solution of the given phthalimide (22a-e, 0.3 mmol) in absolute EtOH (6 mL) and the mixture was refluxed for 2 hours. The solvent was subsequently removed in vacuo and the residue was dissolved in H.sub.2O (10 mL), acidified with 1 M aq. HC and extracted with DCM (3×30 mL). The combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4, filtered and concentrated. The crude residue was purified by flash chromatography (5-15% MeOH/DCM) to afford the respective phthalhydrazide 1a (60%), 1b (32%), 1c (49%), 1d (60%) or 1e (30%) as a white solid.

Example 15—Synthesis of Mitotropic 6-N-Acylated Isoluminol Derivatives 2a-c from Hydrazinolysis of the Respective Phthalimides 23a-c

[0204] ##STR00034##

[0205] Hydrazine hydrate (037 mL, 6.4 mmol) was added in a stirring solution of the given phthalimide (23a-c, 0.64 mmol) in absolute EtOH (15 mL) and the mixture was refluxed for 3 hours. The solvent was subsequently removed in vacuo and the residue was dissolved in H.sub.2O (20 mL), acidified with 1 M aq. HCl and extracted with DCM (3×50 mL). The combined organic layers were washed with brine, dried over Na.sub.2SO.sub.1 filtered and concentrated. The crude residue was purified by flash chromatography (15% MeOH/DCM) to afford the respective phthalhydrazide 2a (78%), 2b (62%) or 2c (37%) as a white solid.

Example 16—Synthesis of 1,2-bis-(2-iodoethoxy)ethane (Intermediate)

[0206] ##STR00035##

[0207] Step 1: Following a published procedure, triethylene glycol di-(P-toluenesulfonate) 39 was prepared (see Bonger et al., Bioorg. Med. Chem. 15: 4841-4856, 2007). Potassium hydroxide (3 g, 53.47 mmol) was added portion wise to a stirred solution of triethylene glycol (1 g, 6.66 mmol) and tosyl chloride (2.54 g, 13.32) in dry dichloromethane (25 mL) at 0° C. under argon and left stirring overnight at room temperature. DCM (25 mL) was then added, the mixture was poured onto ice/water, phases were separated, the aqueous phase washed with DCM (2×40 mL) and the combined organic layers were washed with water (40 mL) and dried (Na.sub.2SO.sub.4). Evaporation of the solvent left 39 (2.44 g, 80%) as white dust.

[0208] Step 2: Following a published procedure, 1,2-bis-(2-iodoethoxy)ethane 40 was prepared (see Lee et al., Bull. Korean Chem. Soc. 36: 1654, 2015). Sodium iodide (9.5 g, 0.06 mol) was added to a solution of tosylate 39 (10 g, 0.02 mmol) in acetone (150 mL) and the mixture was stirred at 60° C. overnight. The remaining precipitate was filtered off and the filtrate was concentrated to dryness. The residue was partitioned between DCM and water, the aqueous phase washed with DCM and the combined organic layer was washed with water, dried (Na.sub.2SO.sub.4) and concentrated to dryness, leaving 40 (6 g, 78%) as pale yellow solid.

Example 17—Synthesis of Phosphonium Iodides (Intermediates)

[0209] ##STR00036##

[0210] Following a published procedure (Lin et al., J. Biol. Chem. 277: 17048, 2002), the diiodo compound (5 mmol) and the respective phosphine (1 mmol) (diiodo-propane, -hexane, -decane or -1,2-bis(ethylenoxy)ethane 40) were mixed in a flask, heated at 100° C. and the resulting melt was stirred for 3 hours in the dark. After cooling, diethyl ether was added to the reaction mixture, the precipitate was filtered and washed with ether. The produce was re-dissolved in dichloromethane and precipitated again with the addition of ether, yielding the respective phosphonium iodide 34 (87%), 35 (45%), 36 (83%), 37 (60%), 38 (85%) or 41 (87%) as a brown solid.

Example 18—Synthesis of Mitotropic 5-N-Alkylated Luminol Derivatives 3a-f

[0211] ##STR00037##

[0212] Luminol (400 mg, 2.26 mmol) was added to a solution of phosphonium iodide (34-38 or 41, 2.26 mmol) in N-methylpyrrolidone (2.5 mL) and the resulting solution was stirred at 110° C. for 24 hours. After cooling to room temperature, water (5 mL) was added and the precipitate thus formed was filtered and washed with aq. Na.sub.2S.sub.2O.sub.3 and water. The residue was chromatographed (silica gel, DCM, EtOAc/DCM, MeOH/EtOAc/DCM, MeOH/DCM up to 40%), yielding pure 3a (24%), 3b (15%), 3c (25%), 3d (21%), 3e (20%) or 3f (3%).

Example 19—Synthesis of Mitotropic Luminol Derivatives of General Structure 56

[0213] ##STR00038##

[0214] Compounds according to general structure 56 can be prepared by alkylation of known compounds of formula XX (see Griesbeck et al., Chem. Eur. J. 21: 9975, 2015) in a method analogous to the synthesis of compounds 3a-d.

Example 20—Synthesis of Mitotropic Luminol Derivatives of General Structure 57

[0215] ##STR00039##

[0216] Compounds according to general structure 57 (R Me) can be prepared according to the following reaction scheme:

##STR00040##

Example 21—Synthesis of Mitotropic Acridinium Ester Derivatives

[0217] ##STR00041##

[0218] Mitotropic acridium ester derivatives A1-5 bearing a phosphonium moiety on an alkyl chain can be synthesised using a method similar to that used to prepare compounds 1-3. Starting from acridinic acid ACA, esterification is carried out using commercially available substituted phenols. This is followed by alkylation either directly to A1-5 (as in the synthesis of compounds 3), or via ACC (as in the synthesis of compounds 1 and 2).

Example 22—Synthesis of Mitotropic Acridinium Ester Derivatives

[0219] Mitotropic acridium ester derivatives B1-2 bearing a phosphonium moiety on an alkyl chain can be synthesised according to the following scheme:

##STR00042##

[0220] In this synthesis, the mitotropic chain is attached to the phenolic moiety, and the acridinium moiety is finally formed with methylation.

Example 23—Synthesis of a Luminol-Rhodamine Conjugate RLum

[0221] ##STR00043##

[0222] Excess adipic acid chloride is reacted with phthalimide 15a to yield acid R1 after aqueous work-up. R1 chloride is reacted with Boc-protected piperazine to form R2 which, after hydrazinolysis and subsequent hydrolysis, results in luminol derivative R4. This is then coupled with rhodamine 110 to yield the desired luminol-rhodium conjugate RLum.

Example 24—Synthesis of an Acridinium-Rhodamine Conjugate RhAC

[0223] ##STR00044##

[0224] Acridinium ester C1 is prepared from ACA through coupling with a phenol and subsequent methylation. The carboxylic acid derivative C2 is prepared by hydrolysis of CI in a procedure similar to a published one (see Natrajan et al., RSC Adv., 4: 21852-21863, 2014). Rhodamine derivative C4 is prepared from rhodamine 110 through coupling with Boc-protected piperazine and subsequent hydrolysis. C2 and C4 are coupled to yield the desired acridinium-rhodamine conjugate RhAC.

Examples 25 to 34

[0225] In Examples 25 to 34 the following codes are used to refer to the mitotropic conjugates according to the invention:

TABLE-US-00001 Compound Code Number Chemical Structure AP47 1a [00045] Molecular Weight: 616.50 (6-((1,4-dioxo-1,2,3,4-tetrahydrophthalazin-5-yl)amino)-6- oxohexyl)triphenylphosphonium bromide AP52 1b [00046] Molecular Weight: 658.58 (6-((1,4-dioxo-1,2,3,4-tetrahydrophthalazin-5-yl)amino)-6- oxohexyl)tri-p-tolylphosphonium bromide AP53 1c [00047] Molecular Weight: 686.63 (11-((1,4-dioxo-1,2,3,4-tetrahydrophthalazin-5-yl)amino)- 11-oxoundecyl)triphenylphosphonium bromide AP54 1d [00048] Molecular Weight: 728.71 (11-((1,4-dioxo-1,2,3,4-tetrahydrophthalazin-5-yl)amino- 11-oxoundecyl)tri-p-tolylphosphonium bromide AP55 1e [00049] Molecular Weight: 704.77 tricyclohexyl(11-((1,4-dioxo-1,2,3,4-tetrahydrophthalazin- 5-yl)amino)-11-oxoundecyl)phosphonium bromide AP71 2c [00050] Molecular Weight: 728.71 (11-((1,4-dioxo-1,2,3,4-tetrahydrophthalazin-6-yl)amino)- 11-oxoundecyl)tri-p-tolylphosphonium bromide AP72 2a [00051] Molecular Weight: 658.58 (6-((1,4-dioxo-1,2,3,4-tetrahydrophthalazin-6-yl)amino-6- oxohexyl)tri-p-tolylphosphonium bromide AP74 2b [00052] Molecular Weight: 634.64 tricyclohexyl(6-((1,4-dioxo-1,2,3,4-tetrahydrophthalazin-6- yl)amino)-6-oxohexyl)phosphonium bromide DZ163 3d [00053] Molecular weight: 747.70 (10-((1,4-dioxo-1,2,3,4-tetrahydrophthalazin-5- yl)amino)decyl)tri-p-tolylphosphonium iodide DZ160 3b [00054] Molecular Weight: 667.66 tricyclohexyl(6-((1,4-dioxo-1,2,3,4-tetrahydrophthalazin-5- yl)amino)hexyl)phosphonium iodide DZ167 3a [00055] Molecular Weight: 649.51 (6-((1,4-dioxo-1,2,3,4-tetrahydrophthalazin-5- yl)amino)hexyl)triphenylphosphonium iodide DZ168 3c [00056] Molecular Weight: 705.62 (10-((1,4-dioxo-1,2,3,4-tetrahydrophthalazin-5- yl)amino)decyl)triphenylphosphonium iodide DZ203 3e [00057] Molecular Weight: 607.43 (3-((1,4-dioxo-1,2,3,4-tetrahydrophthalazin-5- yl)amino)propyl)triphenylphosphonium iodide DZ196 3f [00058] Molecular Weight: 681.51 (2-(2-(2-((1,4-dioxo-1,2,3,4-tetrahydrophthalazin-5- yl)amino)ethoxy)ethoxy)ethyl)triphenylphosphonium iodide

Example 25—Luminescence of Mitotropic Luminol Derivatives in Conditions Resembling the Mitochondrial Matrix

[0226] Luminescence of the mitotropic luminol derivatives was investigated in biomimetic conditions mimicking those found in the mitochondrial matrix environment. The high protein content of the mitochondrial matrix and the abundance of hemes and metal-containing enzymes was represented in the model by 10% Fetal Bovine Serum (FBS).

[0227] Tris buffer (200 μL, 50 mM) set to pH 7.9 was added to a test tube for use in the ABEL meter (portable luminescence meter available from Knight Scientific). This was then supplemented with 10% Fetal Bovine Serum (FBS). Each compound to be investigated was added at 100 mM, followed by urea-H.sub.2O.sub.2 (10 mM). After the initial steady-state profile of the luminescence was obtained, CuSO.sub.4 was injected in real time at 2 mM. The results are presented in FIG. 2.

Example 26—Luminescence of Mitotropic Luminol Derivatives in MCF7 Cells

[0228] Chemiluminescence from MCF7 cells was recorded upon application of various luminol derivatives. In order to achieve this, three strategies were followed: 1) compounds dissolved in DMSO were applied to a monolayer of cells (˜5 million) pelleted at the U-shaped bottom of the luminometer test-tube; 2) compounds dissolved in propylene glycol and applied to the same cell monolayers as above; and 3) compounds dissolved in DMSO and applied to a cell concentrate (5 million cells) in 200 μl of PBS. A characteristic example of the experimental results is provided in FIG. 3 for DZ163 (compound 3d).

[0229] Cells were grown in a T175 flask and grown to confluence. The cells were then detached by trypsin, centrifuged into a big pellet and re-suspended in 12 ml medium (RPMI 1640 without phenol red). 1 mL of this suspension was introduced into each of 12 flow cytometry tubes and these were centrifuged so that the cells formed a film at their U-shaped bottoms. The tubes were placed in the ABEL meter and scanned. The luminol moiety (100 μM in propylene glycol) was then added and finally, where necessary, DMSO was injected in real time.

[0230] In all cases, the luminescence in the cell layer was only induced by the introduction of DMSO, either as a solvent of the luminol derivatives or as a subsequent additive. No additional oxidants such as hydrogen peroxide or any catalysts such as metals were used. It has to be noted that pure DMSO will eventually kill the cells but luminescence registration was instantaneous (seconds after injection). Even in the eventuality of cell death, however, DMSO facilitated the luminescence of the mitotropic compounds. The level of luminescence was substantially decreased in the dispersed cells, most probably due to increased volume.

Example 27—Energy Transfer Between Luminol Luminescence and Various Photosensitizers

[0231] The feasibility of energy transfer from luminescent luminol to various photosensitizers was investigated. The chemiluminescence emission profiles were recorded with the use of a Horiba iHR320 f/4.1 imaging spectrometer equipped with a Synapse CCD head. The emission of luminol (100 μM) was excited in DMSO, with the addition of base (either KOH or potassium tert.butoxide, 100 mM) and the photosensitizers were added. In the case of TPPS4 which was dissolved in water, luminol luminescence was excited by the addition of urea-H.sub.2O.sub.2 (10 mM) and catalyst (CuSO.sub.4 at 2 mM). The singlet oxygen registration was facilitated by the use of a Hamamatsu 5509-73 NIR PMT detector. Two filters were placed in front of the detector—a long-pass filter with a cut on at ˜1000 nm and a bandpass filter centred at 1270 nm, which is the central wavelength of singlet oxygen phosphoresence. The results are presented in FIGS. 4 and 5.

[0232] FIG. 4 shows the energy transfer between luminol luminescence and various photosensitisers: A) luminescence of luminol in alkaline DMSO (KOH); B) addition of hypocrellin A (HYPA)—the characteristic HYPA fluorescence can be seen (around 600 nm); C) addition of hypericin (HYP) instead of HYPA—again the characteristic HYP fluorescence double peak at 600 and 650 can be seen; D) luminol in aqueous carbonate buffer (pH 10.3) with the addition of CuSO.sub.4 and urea peroxide in the presence of Rose Bengal (RB)—the characteristic fluorescence of RB is obvious as verified in E) by 532 nm laser excitation; and F) the luminol system in carbonate buffer as in D) but with the addition of TPPS4, a porphyrin spectrally similar to 5-ALA-derived PpIX—the characteristic fluorescence of TPPS4 is not present. Luminol emission in the aqueous system shows a hump at around 400 nm in addition to the main peak around 490 nm. In the presence of TPPS4 (F) this hump is severely depleted which shows that TPPS4 absorbs strongly at this region in contrast to the other photosensitizers which seem to deplete the main peak at 490 nm.

[0233] In FIG. 5, the photosensitiser erythrosin B is added to luminol in DMSO alkalinised by potassium tert.butoxide.: A) the spectrum of luminol in DMSO-tert-butoxide; B) erythrosin B absorbs very strongly, especially around 500 and demonstrates very strong fluorescence around 580 nm; and C) addition of tert.butoxide to the DMSO-luminol solution triggered strong and long-lived luminol luminescence and also luminescence around 1270 nm indicating the presence of singlet oxygen since this luminescence was quenched by histidine.

[0234] These experiments demonstrate that many photosensitizers can receive energy from luminol luminescence.

Example 28—Micrographs of Luminol Derivatives and Luminol

[0235] The sub-cellular localisation of the luminol derivatives was investigated. Cells were inoculated into Petri dishes with glass bottoms and left to grow overnight. The cells were treated with the luminol moieties for 4 hours and then 15 mins prior to imaging, mitotracker green FM was added (100 nm). The cells were washed with PBS and mounted onto a Zeiss LSM 710 confocal microscope. The luminol fluorescence was excited at 405 nm, while the mitotracker green FM fluorescence was excited at 488 nm. The luminol emission was collected between 420-490 nm (red channel) while the mitotracker green FM fluorescence was collected at the FITC channel (green channel). The green and red channel images were subsequently superimposed in Photoshop to yield the overlay images. In these images, yellow in each case indicated co-localisation of the luminol derivative with mitotracker green FM (and hence with cell mitochondria).

[0236] The representative micrographs in FIG. 6 give an overview of the subcellular localisation of the mitotropic compounds versus that of free luminol. On the triple column (left) the mitochondrial localisation of two derivatives namely DZ160 and AP47 is shown in two different cell lines, the breast adenocarcinoma cell line MCF7 and the glioblastoma cell line U87. The three micrographs in each row of this triple column depict the localisation of the derivative, the localisation of the mitochondrial marker mitotracker green and the overlay of these two localisations in a merged image. The fluorescence of luminol is represented in the left column while mitotracker green fluorescence is shown in the middle column. Their overlay is shown in the third column. On the column in the right (fourth column) the cytosolic localisation of free luminol is shown on the top (with border) while in two cases the localisation of AP47 and DZ160 is depicted in cells not incubated with mitotracker green to exclude fluorescence spillover and hence cross talk between the mitotracker and luminol-derivative channels. This confirms the dissimilar localisation of the luminal derivatives (mitochondria) and free luminol (cytosolic).

Example 29-5—ALA/Hypocrellin A and HD92 (AP47) on U87 and MCF7 Cells

[0237] Efficiency of the luminol derivatives was investigated. The initial derivative tested was HD92 (also referred to herein as “AP47”). U87 cells were inoculated in 96 well plates. The cells were then divided into the following groups (at least 6 parallels per group): CTRL (media only), ALA CTRL (2 mM), HD92 CTRL (500 μM) and the LUMIBLAST group (2 mM 5-ALA and 500 μM HD92). Correspondingly, MCF7 cells were inoculated in 96 well plates and left to grow overnight. The cells were then divided into the following groups (at least 6 parallels per group): CTRL (media only), Hypocrellin A CTRL (HYPA 7 μM), HD92 CTRL (500 μM) and the LUMIBLAST group (7 μM HYPA and 500 μM HD92). These four groups were incubated in media with and without 10 μM FeSO.sub.4 in the case of experiments with HYPA. Following incubation of the cells with their respective drug strategies overnight (˜20 hours), the cell groups were tested for their viability using a standard MTT assay. In brief, all cell groups were incubated with 0.5 mg/mL MTT for 3 hours. The MTT media were subsequently replaced with DMSO (100 μL) to solubilise the formazan. The wells were read for absorbance at 562 nm using a Tecan Spark OM plate reader. The cytotoxicity was determined as the percentage of control (media only) absorbance following subtraction of blank values from wells without cells.

[0238] The results are presented in FIG. 7. From the data obtained it can be seen that these results were obtained at very high concentrations of HD92, still sub-toxic but very near the margins of chemical toxicity. It can also be seen that 5-ALA is less efficient in producing LUMIBLAST effects than HYPA as a photosensitizer, however, the HYPA effects were achieved in the presence of metal catalyst, in the present case small concentration of FeSO.sub.4. The data also shows that since the combined experimental group survival values are shown with respect to the HD92 (LTPP1) control, the combinatory cytotoxic effect is profoundly significant and a result of photosensitizer (PS) and HD92 synergy.

Example 30—HYPA, DZ168 and CuSO.SUB.4 .in MCF7 and U87 Cells

[0239] MCF7 and U87 cells were inoculated in 96 well plates. The cells were then divided into the following groups (at least 6 parallels per group): CTRL (media only), HYPA CTRL (3 μM), DZ168 CTRL (5, 10, 20 and 30 μM), and the LUMIBLAST combinations (3 μM HYPA+DZ168 5-30 μM). These cell group incubations were also repeated in media containing 100 μM CuSO.sub.4. Following incubation of the cells with their respective drug strategies overnight (˜20 hours), the cell groups were tested for their viability using the MTT assay.

[0240] The results are presented in FIG. 8. When HYPA was employed as the photosensitizer, MCF7 cells showed enhanced toxicity in the HYPA+copper groups, while the U87 cell groups were not affected.

Example 31—HYPA, DZ167 and CuSO.SUB.4 .in MCF7 and U87 Cells

[0241] MCF7 and U87 cells were inoculated in 96 well plates. The cells were then divided into the following groups (at least 6 parallels per group):CTRL (media only), HYPA CTRL (5 μM), DZ167 CTRL (25-200 μM), and the LUMIBLAST combinations (5 μM HYPA+DZ167 25-200 μM). These incubations were performed in media containing 50 μM CuSO.sub.4. Following incubation of the cells with their respective drug strategies overnight (˜20 hours), the cell groups were tested for their viability using the MTT assay.

[0242] The results are presented in FIG. 9. At 200 μM concentration of DZ167, 5 μM HYPA and 50 μM CuSO.sub.4, there is a synergistic effect in U87 cells.

Example 32—Sub-Cellular Localisation of Cercosporin and Erythrocin B in T98G Glioblastoma Multiforme Cells

[0243] The subcellular localisation of cercosporin together with that of Erythrosin B was investigated. Cells were inoculated into Petri dishes with glass bottoms and left to grow overnight. The cells were treated with Erythrosin B (4 μM) and cercosporin (3 μM) for 4 hours and then 15 mins prior to imaging, mitotracker deep-red FM was added (100 nm). The cells were washed with PBS and mounted onto a Zeiss LSM 710 confocal microscope. The cercosporin and erythrosin B fluorescence was excited at 488 nm, while the mitotracker deep-red FM fluorescence was excited at 633 nm. The cercosporin and erythrosin B emission was collected emission was collected beyond 550 nm (green channel) while the mitotracker deep-red FM fluorescence was collected beyond 640 nm (red channel). The green and red channel images were subsequently superimposed in Photoshop to yield the overlay images. In these images, yellow in each case indicates co-localisation of the photosensitizers with mitotracker green FM (and hence cell mitochondria). From the micrographs in FIG. 10 it can be seen that cercosporin and erythrocin B partly co-localise with cell mitochondria and hence they can be used in LUMIBLAST.

Example 33—Cercosporin and DZ203/DZ196 in U87 GBM Cells

[0244] The subcellular localisation of cercosporin together with that of synthesised derivatives DZ203 (short alkyl linker) and DZ196 (oligoPEG linker) was investigated. U87 cells were inoculated in 96 well plates. The cells were then divided into the following groups (at least 6 parallels per group). CTRL (media only), Cercosporin CTRL (3 μM), DZ203 CTRL (200 μM), DZ196 CTRL (500 μM) and the LUMIBLAST combinations (3 μM cercosporin+DZ203 200 μM or 3 μM cercosporin DZ196 500 μM). These incubations were performed in media with and without 100 μM CuSO.sub.4. Following incubation of the cells with their respective drug strategies overnight (20 hours), the cell groups were tested for their viability using the MTT assay. The results are presented in FIG. 11 and show a very profound effect for cercosporin incubated with DZ203 (200 μM) for 24 hours in the presence of Cu (150 μM). The smaller effect with DZ196 (500 μM) was achieved without the catalytic effect of Cu.

Example 33—Metabolic Analysis of MCF7 Cells with DZ167 and 5-ALA

[0245] Metabolic analysis was performed to investigate the respiration and glycolysis of intact cells upon administration of the luminol derivatives and photosensitizer (exemplified here by AP47 and 5-ALA respectively). MCF7 cells were inoculated into XFe96 seahorse metabolic analyser 96-well plates and left overnight to incubate. The cells were incubated in un-buffered medium without FBS at a 37° C. incubator without CO.sub.2 for one hour prior to the experiments. The cells were then analysed for their oxygen consumption rates (OCR) corresponding to respiratory activity and extracellular acidification rates (ECAR) corresponding to the glycolytic activity of the cells. The measurements were then performed with the help of the XFe96 metabolic analyser, at 4 conditions (serial injections) as denoted on the graphs in FIG. 12. These injections included the luminol mitotropic derivative AP47 (200 μM) and 5-ALA (1 mM) to study their effect and the effect of their combination on the cellular metabolism. Also oligomycin (1 μM), FCCP (1 μM), and a combination of Antimycin A and Rotenone (1 μM each), were used to modulate the cellular respiration. Oligomycin inhibits ATP synthesis revealing the amount of respiration required for ATP production, FCCP collapses the mitochondrial proton gradient forcing maximal electron transport and hence maximal oxygen consumption. Finally, the mix of rotenone and antimycin A totally inhibits the electron transport to the mitochondrial complex III stopping all respiratory activity. In some instances, also 2-deoxy glucose (2DG) was also injected to the cells as it totally inhibits cell glycolysis, as a tool to study the effects of AP47 and 5-ALA on cell glycolysis.

Example 34—5-ALA and DZ203 in MDA-MB-231 Breast Carcinoma Cells

[0246] MDA-MB-231 cells were inoculated in 96 well plates. The cells were then divided into the following groups (at least 6 parallels per group): CTRL (media only), 5-ALA CTRL (1.2 mM), DZ203 CTRL (400 μM) and the LUMIBLAST combinations (1.2 mM 5-ALA+DZ203 400 μM). These incubations were performed in media in the presence and absence of a catalyst (100 μM CuSO.sub.4). Following incubation of the cells with their respective drug strategies overnight (˜20 hours), the cell groups were tested for their viability using the MTT assay. The results are presented in FIG. 13 and show a substantial synergy in the presence of Cu as catalyst.