DEUTERATED COMPOUNDS AND USES THEREOF

Abstract

An anthraquinone compound of formula I (such as the compounds of formulae II to X) and processes for making the same are provided. Pharmaceutical compositions for use in the treatment of cancer, optionally in combination with an agent capable of reducing the level of oxygenation of a tumour, are also provided. Additionally, an option for combination with chemotherapeutic and radiotherapeutic modalities to enhance overall tumour cell kill is provided. Methods for the detection of cellular hypoxia, both in vivo and in vitro, are additionally provided.

##STR00001##

Claims

1.-80. (canceled)

81. A compound of Formula IV or VI: ##STR00014## wherein Y are each independently selected from the group consisting of hydrogen, hydroxy, halogeno, amino, C.sub.1-4 alkoxy and C.sub.2-8 alkanoxy.

82. A compound according to claim 81, wherein the compound is of Formula VIII or X: ##STR00015##

83. A compound according to claim 81 wherein the compound is in the form of a halide salt, for example a chloride salt.

84. A pharmaceutical composition comprising a compound according to claim 81 together with pharmaceutically acceptable buffer, diluent, carrier, adjuvant or excipient.

85. A pharmaceutical composition according to claim 84 formulated for parenteral administration.

86. A process for making a compound according to claim 81 comprising reacting an anthracene-9,10-dione with a deuterated alkylenediamine under conditions suitable for the production of an alkylaminoalkyl-aminoanthraquinone.

87. A process according to claim 86 further comprising the step of reacting the alkylaminoalkylaminoanthraquinone with a monoperoxyphthalate to under conditions suitable for the production of an N-oxide derivative of the alkylamino-alkylaminoanthraquinone.

88. A process according to claim 86 comprising reacting 1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione, 281-005 with deuterated--N,N-dimethylethylene-diamine under conditions suitable for the production of 1,4-bis-{[2-(deuterated-d6-dimethylamino)ethyl]amino)-5,8-dihydroxyanthracene-9,10-dione.

89. A process according to claim 88 further comprising the step of reacting the 1,4-bis-{[2-(deuterated-d6-dimethylamino)ethyl]amino)-5,8-dihydroxyanthracene-9,10-dione with magnesium monoperoxyphthalate under conditions suitable for the production of 1,4-bis-{[2-(deuterated-d6-dimethylamino-N-oxide)ethyl]amino)-5,8-dihydroxy-anthracene-9,10-dione.

90. A method of treating cancer in a patient comprising administering to the patient a therapeutically effective amount of a compound according to claim 81.

91. A method according to claim 90 wherein the patient is human.

92. A method according to claim 90, wherein administering the therapeutically effective amount treats metastases or reduces metastatic spread.

93. A method according to claim 90 wherein the compound is a monotherapy.

94. A method according to claim 90 further comprising administering to the patient one or more additional cancer treatments.

95. A method according to claim 94 wherein the one or more additional cancer treatments is/are selected from the group consisting of anti-androgens (steroidal and non-steroidal), vascular disrupting agents, anti-angiogenic agents, anti-VEGFR agents, IL8 inhibitors, NO synthase inhibitors, vasoconstricting agents, vasodilating agents, and radiotherapeutic modalities.

96. A method according to claim 95 wherein the one or more additional cancer treatments is at least one anti-androgen.

97. A method according to claim 96 wherein the at least one anti-androgen is selected from the group consisting of flutamide, nilutamide, bicalutamide, finasteride, dutasteride, bexlosteride, izonsteride, turosteride, epristeride and abiraterone.

98. A method according to claim 97 wherein the at least one anti-androgen is bicalutamide.

99. A method of detecting hypoxic cells in vitro or in vivo in a group of cells, the method comprising: exposing a compound of Formula VI: ##STR00016## wherein Y are each independently selected from the group consisting of hydrogen, hydroxy, halogeno, amino, C.sub.1-4 alkoxy and C.sub.2-8 alkanoxy to the group of cells; analysing the cells for the presence of a corresponding reduced compound of Formula IV ##STR00017## and determining the hypoxic cells based on the presence of the corresponding reduced compound.

100. The method according to claim 99 wherein the cells are mammalian, e.g. human.

101. The method according to claim 99 in vitro.

102. The method according to claim 99 in vivo.

103. The method according to claim 102, further comprising: surgically excising cells identified as being hypoxic.

104. The method according to claim 99 wherein the compound is used in combination with a non-deuterated form of a compound of Formula VI.

105. The method according to claim 99 wherein the compound is detected using a method selected from the group consisting of mass spectrometry, nuclear magnetic resonance, infrared spectroscopy, colorimetrically, Raman spectroscopy, nuclear magnetic resonance, affinity capture methods, immunohistochemistry, flow cytometry, microscopy and antibody-based detection methods.

106. A kit of parts for use in detecting the oxygenation level of cells comprising a compound according to claim 81.

107. A kit according to claim 106 further comprising a non-deuterated form of a compound of Formula VI.

108. The method according to claim 94 wherein the one or more additional cancer treatments decreases tumour oxygenation in vivo.

109. The method according to claim 108 wherein the one or more additional cancer treatments lowers the median oxygen level of the tumour to below 3%.

110. The compound according to claim 81 wherein each Y is independently selected from the group consisting of hydrogen, hydroxy and halogeno.

111. A method of treating a pancreatic cancer tumour in a patient comprising administering to the patient a therapeutically effective amount of the compound according to claim 81.

112. A method of treating a prostate cancer tumour in a patient comprising administering to the patient a therapeutically effective amount of the compound according to claim 81.

113. The method according to claim 112, wherein administering the therapeutically effective amount treats metastases or reduces metastatic spread.

114. The method according to claim 112 further comprising administering to the patient one or more additional cancer treatments in combination with the therapeutically effective amount of the compound.

115. The method according to claim 114 wherein the one or more additional cancer treatments is/are selected from the group consisting of anti-androgens (steroidal and non-steroidal), vascular disrupting agents, anti-angiogenic agents, anti-VEGFR agents, IL8 inhibitors, NO synthase inhibitors, vasoconstricting agents, vasodilating agents, and radiotherapeutic modalities.

116. The method according to claim 115 wherein the one or more additional cancer treatments is at least one anti-androgen.

117. The method according to claim 116 wherein the at least one anti-androgen is selected from the group consisting of flutamide, nilutamide, bicalutamide, finasteride, dutasteride, bexlosteride, izonsteride, turosteride, epristeride, abiraterone and combinations thereof.

118. The method according to claim 117 wherein the at least one anti-androgen is bicalutamide.

119. The method according to claim 114 wherein the one or more additional cancer treatments decreases tumour oxygenation in vivo.

120. The method according to claim 119 wherein the one or more additional cancer treatments lowers the median oxygen level of the tumour to below 3%.

Description

DESCRIPTION OF THE DRAWINGS

[0186] FIG. 1: The metabolites AQ4 and OCT1001 have similar cell cycle arresting actions, under normal oxygenation conditions, indicating that selective deuteration has not modified intrinsic biological activity.

[0187] See Example B

[0188] FIG. 2: Similar hypoxia-enhanced cytotoxicity for AQ4N and OCT1002

[0189] See Example B

[0190] FIG. 3: Exemplification of that the bioactivity of AQ4N and OCT1002 is dependent upon the degree of hypoxia

[0191] See Example B

[0192] FIG. 4: Hypoxia-dependent growth inhibition by AQ4N and OCT1002 arises from a similar mechanism of cell cycle arrest and is dependent on the degree of hypoxia

[0193] See Example B

[0194] FIGS. 5 (A & B): Exemplification of shared bioactivity of AQ4N and OCT1002 under hypoxic conditions for functional p53 (DoHH2) and mutant p53 (SU-DHL-4) human B cell lymphoma cells

[0195] See Example B

[0196] FIG. 6: Intracellular accumulation of the OCT1001 far-red fluorescent chromophore under hypoxia is responsive to OCT1002 pro-drug dose and oxygenation level

[0197] See Example B

[0198] FIG. 7: Deuteration does not affect the intrinsic capacity of the metabolite (AQ4 or OCT1001) to accumulate within a cell

[0199] See Example B

[0200] FIG. 8: Accumulation of converted pro-drug OCT1001 correlates with growth arrest

[0201] See Example B

[0202] FIGS. 9 (A & B): Demonstration of intracellular fluorescence following exposure to OCT1002 under hypoxic conditions and that prodrug deuteration reduces intracellular accumulation but increases persistence of the metabolite.

[0203] See Example B

[0204] FIG. 10: Effect of bicalutamide on the oxygenation of 22Rv1 prostate tumours grown as xenografts

[0205] See Example C

[0206] FIG. 11 (A-H): Effect of bicalutamide on blood vessels in 22Rv1 tumour xenografts. Tumour fragments were imaged before treatment began (A) vehicle and (E) bicalutamide pre-treatment groups and then after 7, 14 and 21 days of treatment, (B-D) vehicle only and (F-H) bicalutamide (10 magnification).

[0207] See Example C

[0208] FIGS. 12 (A & B): Effect of bicalutamide only or AQ4N single dose or OCT1002 single dose on 22Rv1 xenografts in mice

[0209] See Example C

[0210] FIGS. 13 (A & B): Combined effect of AQ4N single dose or OCT1002 single dose on 22Rv1 xenografts in mice treated daily with bicalutamide

[0211] See Example C

[0212] FIG. 14: Effect of OCT1002 on LNCaP xenografts in mice treated with/without bicalutamide

[0213] See Example C

[0214] FIG. 15 (A-C): OCT1002 is reduced in hypoxic LNCaP tumour cells in vivo In mice treated with vehicle+OCT1002 at day 7: the converted compound OCT1001 (blue) is in a few areas where vascularisation is poor (FIG. 15A). Mice treated with bicalutamide (2 mg/kg/day in vehicle): vascularisation was reduced at days 7. On day 7, two hours after intraperitoneal injection of a single dose of OCT1002 (50 mg/kg) large quantities of converted compound (OCT1001; blue) can be seen across the whole tumour fragment (FIG. 15B). OCT1001 (blue) is still localised to the tumour at day 14; by day 21 the amount of compound was considerably lower (FIG. 15C).

[0215] See Example C

[0216] FIG. 16: OCT1002 reduces the metastatic spread of LNCaP tumours to the lungs

[0217] See Example C

DETAILED DESCRIPTION OF THE EMBODIMENTS

Examples

Example A: Synthesis of Alkylaminoalkylaminoanthraquinones and their N-Oxides

(a) Preparation of 1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione

[0218] ##STR00009##

[0219] A mixture of 4,7-difluoroisobenzofuran-1,3-dione (8.50 g, 46.2 mmol), hydroquinone (5.64 g, 51.3 mmol), aluminium trichloride (36.9 g, 277 mmol) and sulfolane (10 mL) was stirred together for 16 hours at 165 C. The reaction was effectively a melt as the mixture does not become a viscous red syrup until 150 C. To minimise the risk of a sudden exotherm and evolution of HCl gas, the reaction was stirred in portions, cooled in an ice bath and stirred again until mixing was sufficient. Only then was the mixture heated.

[0220] The mixture was poured carefully into ice and 2M HCl added (50 mL). The mixture was stirred, then filtered, washing the resultant slurry with further 2M HCl. The solid was re-slurried a further 3 times with 2M HCl to reduce the aluminium content of the product. A final slurry was washed with ether twice; drying in a round bottom flask at 60 C. until constant weight afforded 1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione (9.82 g, 35.6 mmol, 77% yield).

[0221] .sup.1H NMR (DMSO-d.sup.6) was clean and consistent with the desired material.

(b) Preparation of 1,4-bis-{[2-(deuterated-d6-dimethylamino)ethyl]amino)-5,8-dihydroxy-anthracene-9,10-dione

[0222] ##STR00010##

[0223] A suspension of deuterated-d6-dimethylamine hydrochloride (18.4 g, 210 mmol) and 2-bromoacetonitrile (14.63 ml, 210 mmol) in anhydrous THF (250 mL) in a round bottom flask was cooled to 10 C. with vigorous stirring and treated portion-wise with potassium carbonate (58.1 g, 420 mmol). After addition of the base, the reaction was fitted with a reflux condenser and balloons and allowed to warm slowly to 5 C. over 2 hours. TLC (1:1 EtOAc/Iso-Hexanes) indicated the presence of product. The mixture was stirred at room temperature over a weekend.

[0224] The residue was diluted with DCM (250 mL) and filtered, washing with copious amounts of DCM. The mother liquors were degassed with N.sub.2 for 1 hour, then reduced in volume by half on the rotavap. Then a 4M dioxane solution of hydrogen chloride (52.5 ml, 210 mmol) was added, precipitating a white solid and the mixture allowed to stand for 10 minutes before being filtered, washing with DCM to afford deuterated-d6-dimethylacetonitrile (21.73 g, 172 mmol, 82% yield).

[0225] .sup.1H NMR (400 MHz, d.sub.6-DMSO) : 4.47 (2H, s) was consistent with the desired material.

(c) Preparation of deuterated-d6-N,N-dimethylethylenediamine

[0226] ##STR00011##

[0227] .sup.To a stirred suspension of deuturated-d6-dimethylacetonitrile (21.72 g, 172 mmol) in Et.sub.2O (200 mL) at 0 C. was added d/w a 1M ether solution of lithium aluminium hydride (515 ml, 515 mmol) via dropping funnel over 1.5 hours. After the addition, the cooling bath was removed. After a further 1.5 hr, the reaction was quenched at 15 C. (no higher than 18 C.) with sodium sulfate decahydrate (0.5 eq rel. to LiAlH4, 80 g) cautiously (delayed reaction) over 1.5 hours. The mixture was left to stir for 1 hour and subsequently filtered, washing with ether. The filtrate was stored overnight in the dark. The ether was removed on the rotavap at 40 C. with no vacuum to afford deuterated-d6-N,N-dimethylethylenediamine (15.89 g, 160 mmol, 93% yield was clean and consistent with the desired material but contained 0.25 eq ether).

[0228] .sup.1H NMR (400 MHz, CDCl.sub.3) : 2.76 (2H, t), 2.33 (2H, t)

(d) Preparation of 1,4-bis-{[2-(deuterated-d6-dimethylamino)ethyl]amino)-5,8-dihydroxy-anthracene-9,10-dione (OCT1001)

[0229] ##STR00012##

[0230] A solution of 1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione, (4.9 g, 17.74 mmol) in pyridine (35 mL) was treated with deuterated-d6-N,N-dimethylethylenediamine, (16.57 ml, 142 mmol) as a steady stream. The mixture was warmed to 40 C. and allowed to stir for 24 hours under a flow of nitrogen. The reaction was taken off heat and cooled in an ice-bath. A chilled mixture of ammonium hydroxide (30%, 30 mL) and brine (30 mL) were added and the mixture stirred in an ice-bath for 2 hours. After this time the mixture was filtered washing with a 10% ammonium hydroxide solution (130 mL). The solid was air-dried for 30 minutes, then transferred to a tared flask and dried under vacuum at 60 C. until constant weight (2 h).

[0231] The bulk material was purified by flash chromatography (Biotage, 120 g) loading in DCM (through cotton wool plug) eluting with 6 then 10% MeOH (containing 1% NH3)/DCM to give 1,4-bis-{[2-(deuterated-d6-dimethylamino)ethyl]amino)-5,8-dihydroxyanthracene-9,10-dione (2.01 g, 4.73 mmol, 26.7% yield).

[0232] The product was analysed by LCMS (m/z 425.3 (M+H).sup.+ (ES.sup.+); 423.2 (MH) (ES), at 0.90 and 1.03 min (product smears on column), 100%.

[0233] .sup.1H NMR (CDCl.sub.3) was clean and consistent with the desired material .sup.1H NMR (400 MHz, CDCl.sub.3) : 13.51 (2H, s), 10.40 (2H, br t), 7.17 (2H, s), 7.11 (2H, s), 3.47 (4H, q), 2.66 (4H, t).

(e) Preparation of 1,4-bis-{[2-(deuterated-d6-dimethylamino-N-oxide)ethyl]amino)-5,8-di-hydroxyanthracene-9,10-dione (OCT1002)

[0234] ##STR00013##

[0235] A suspension containing magnesium monoperoxyphthalate, MMPP (3.10 g, 6.27 mmol) in methanol (8 mL) was added dropwise to a stirred solution of 281-041 (1.90 g, 4.48 mmol), AQ4 in methanol (8 mL) and DCM (30 mL) cooled to 11 C. After the addition was complete, the reaction solution was allowed to warm to 0 C. and stirred overnight in the dark (warmed to room temperature during this time). Pre-cooled EtOAc (30 mL) and EtOH (6 mL) were added the reaction mixture at 0 C. This mixture was allowed to stir for 30 minutes then a 4M solution of hydrogen chloride (4.48 ml, 17.90 mmol) in dioxane was added dropwise at approximately 10 to 15 C. The resulting slurry was then stirred for 10 minutes then filtered, washing with EtOH/Water (9:1, 100 mL), MeOH/EtOAc (1:1, 100 mL) and EtOAc (60 mL) and dried under vacuum (on rotavap) at 40 C. for 2 hours (constant weight) to afford 1,4-bis-{[2-(deuterated-d6-dimethylamino-N-oxide)ethyl]-amino)-5,8-di-hydroxyanthracene-9,10-dione (2.15 g, 3.99 mmol, 89% yield) as a dark blue powder.

[0236] The product was analysed by LCMS (standard 4 min. method, agilent), m/z 458.2 (M+H).sup.+ (ES.sup.+), at 3.07 min, 98.3% purity @ 254 nm. .sup.1H NMR (400 MHz, D.sub.2O) : 6.73 (2H, br s), 6.43 (2H, br s), 3.76 (4H, br s), 3.58 (4H, br s).

[0237] .sup.1H NMR (D.sub.2O) was consistent with the desired material.

Example B: In Vitro Properties of 1,4-bis-{[2-(deuterated-d6-dimethylamino-N-oxide)ethyl]amino)-5,8-di-hydroxyanthracene-9,10-dione and its Active Metabolite

[0238] (a) The metabolites AQ4 and OCT1001 have similar cell cycle arresting actions, under normal oxygenation conditions, indicating that selective deuteration has not modified intrinsic biological activity. [0239] A549 human lung cancer cells were cultured using conventional methods for adherent cells and exposed for 4 days to 0, 1, 3 or 10 nM agents under standard cell culture conditions of 5% carbon dioxide in air at 37 deg C. Harvested cells were permeabilised and stained with the DNA fluorescent dye ethidium bromide and cell cycle distributions determined by conventional flow cytometry. [0240] FIG. 1 (flow cytometry) shows similar increases in the G2 peaks of the DNA content distributions between 3-10 nM (indicating cell cycle arrest) for cells exposed to exogenous metabolites 1,4-bis-{[2-(dimethylamino)ethyl]amino)-5,8-dihydroxy-anthracene-9,10-dione (AQ4) and 1,4-bis-{[2-(deuterated-d6-dimethylamino)-ethyl]amino)-5,8-dihydroxy-anthracene-9,10-dione (OCT1001). [0241] (b) Similar hypoxia-enhanced cytotoxicity for AQ4N and OCT1002 [0242] Human T cell leukaemia cells (Jurkat) were cultured using conventional methods for suspension cultures in air or under 1% oxygen conditions for 4 days in the presence of a range of concentrations of either AQ4N or OCT1002. The relative cell number was determined using a conventional Coulter Counter particle counting method. [0243] FIG. 2 shows that the compounds tested require hypoxic conditions for the inhibition of cell proliferation. Thus, 1,4-bis-{[2-(dimethylamino-N-oxide)ethyl]amino)-5,8-di-hydroxyanthracene-9,10-dione (AQ4N) and 1,4-bis-{[2-(deuterated-d6-dimethyl-amino-N-oxide)ethyl]amino)-5,8-di-hydroxy-anthracene-9,10-dione (OCT1002) both exhibit pronounced cytostatic activity under conditions of hypoxia (1% oxygen). [0244] As a control it is shown that hypoxia does not modify the cytostatic action of a direct acting DNA topoisomerase inhibitor (VP-16), achieving similar levels of prolonged cytostatic action. [0245] (c) Exemplification of that the bioactivity of AQ4N and OCT1002 is dependent upon the degree of hypoxia [0246] A549 human lung cancer cells were cultured using conventional methods for adherent cells and exposed for 4 days to varying concentrations of either AQ4N and OCT1002 agents under standard cell culture conditions of 5% carbon dioxide in air (normoxia) at 37 deg C., or under conditions of reduced oxygen (1% and 3%). [0247] Data are plotted as relative population doublings determined by cell detachment and Coulter Counter particle counting of cell densities at the start and end of the exposure period. [0248] FIG. 3 shows that for the compounds tested, namely 1,4-bis-{[2-(dimethylamino-N-oxide)ethyl]amino)-5,8-di-hydroxy-anthracene-9,10-dione (AQ4N) and 1,4-bis-{[2-(deuterated-d6-dimethyl-amino-N-oxide)ethyl]amino)-5,8-di-hydroxyanthracene-9,10-dione (OCT1002), growth inhibition is dependent upon the degree of hypoxia and drug concentration, with the two agents showing similar responses. [0249] (d) Hypoxic sensitisation by AQ4N and OCT1002 [0250] A549 human lung cancer cells were used in this experiment; culture conditions were as described in (c) above. [0251] Cell cycle analysis was performed as described in (a) above. [0252] FIG. 4 shows that the compounds tested, namely 1,4-bis-{[2-(dimethylamino-N-oxide)ethyl]amino)-5,8-di-hydroxy-anthracene-9,10-dione (AQ4N) and 1,4-bis-{[2-(deuterated-d6-dimethyl-amino-N-oxide)ethyl]amino)-5,8-di-hydroxy-anthracene-9,10-dione (OCT1002), generate similar cell cycle arrest (determined by flow cytometry) within the bioactive drug dose range. [0253] The degree of late cell cycle arrest is increased as oxygenation levels are reduced. [0254] (e) Exemplification of shared bioactivity of AQ4N and OCT1002 under hypoxic conditions for p53 functional and mutant p53 human B cell lymphoma cell lines [0255] Human B cell lymphoma cells were cultured using conventional methods for suspension cultures in air, 1% or 3% oxygenation conditions for 4 days in the presence of a range of concentrations of either AQ4N or OCT1002. The relative cell numbers were determined using a conventional Coulter Counter particle counting method. [0256] FIG. 5(A) shows that the compounds tested are equally and selectively cytotoxic in hypoxic conditions against DoHH2 human B cell lymphoma cells (bcl2 overexpressing; p53 wt) grown in suspension and exposed to prodrugs for 4 days under 21% (circles), 3% (triangles) or 1% O.sub.2 (squares). Thus, 1,4-bis-{[2-(dimethylamino-N-oxide)ethyl]amino)-5,8-di-hydroxyanthracene-9,10-dione (AQ4N) and 1,4-bis-{[2-(deuterated-d6-dimethyl-amino-N-oxide)ethyl]amino)-5,8-di-hydroxy-anthracene-9,10-dione (OCT1002) both exhibit pronounced cytostatic activity under conditions of hypoxia (1% oxygen), with the growth inhibition being sensitive to the degree of hypoxia. [0257] Likewise, FIG. 5(B) shows that the prodrugs AQ4N and OCT1002 are equally selectively cytotoxic in hypoxic conditions against SU-DHL-4 human B cell lymphoma cells (bcl2 overexpressing; p53 mutant) grown in suspension and exposed to prodrugs for 4 days under 21% (circles), 3% (triangles) or 1% O.sub.2 (squares). Again, the growth inhibition is sensitive to the degree of hypoxia. [0258] (f) Reciprocity between an imposed pO.sub.2 level and the degree of end-product generation [0259] OCT1002 and AQ4N show reciprocity between an imposed pO.sub.2 level and the degree of end-product generation in the biologically relevant range of hypoxia with low or undetectable levels of conversion under normoxia (and undetectable levels of AQ4N or OCT1002 showing that the metabolites are the primary persistent anthraquinone forms) [0260] Relative to AQ4N, the deuterated variant OCT1002 shows a reduction in overall capacity for reduction/accumulation (HPLC analysis) within moribund cells, under protracted exposure conditions showing a reduction of redundant targeting in a human lung cancer cell line. In this case redundant targeting of a prodrug refers to the over-generation of the cytotoxic form beyond that required for cell inactivation since conversion of the prodrug can continue even when cell cycle arrest has occurred. The consequences of over-generation will be increased deleterious effects of the converted form when released from the initial target cell. This undesirable bystander effect on nearby tissue not initially subject to hypoxic conditions will comprise non-target normal and tumour cells. Damage to normal cells is clearly undesirable. Suboptimal exposure of non-target tumour cells through a bystander effect may compromise their responses to other agent(s) delivered in combination or generate selective conditions for the development of drug resistance. [0261] Table 1 shows a comparison of HPLC analysis of metabolite generation following exposure of human A549 cells to AQ4N and OCT1002 under varying degrees of hypoxia and concentration (data derived from two determinations) where 21% is taken to represent normal oxygenation conditions. [0262] Data show the consistent reduction in the generation of OCT1001 compared with AQ4 in cells exposed to the conditions indicated and washed prior to assay for the presence of prodrug or their metabolites. Data also shows that the molecular forms present in cells experiencing hypoxia are the metabolites and not parent prodrugs.

TABLE-US-00001 TABLE 1 Relative Dose of Humidified prodrug prodrug oxygenation pmoles metabolite generated reduction to (OCT1002 conditions per 10.sup.5 cells.sup.a metabolite or AQ4N) pO.sub.2 mm range range OCT1001/ nM days % O.sub.2 Hg AQ4 OCT1001 AQ4 OCT1001 AQ4 30 1% 7.1 9.25 5.64 1.46 1.20 0.61 30 3% 21.4 0.78 0.49 0.05 0.06 0.62 30 21% 142.2 <0.10 0.10 0.03 0.02 1.02 100 1% 7.1 >42.95 16.17 6.59 8.16 <0.38 100 3% 21.4 5.58 1.93 1.13 0.16 0.35 100 21% 142.2 0.23 0.11 0.08 0.03 0.50 .sup.aNo AQ4N or OCT1002 detected in any sample indicating that either all prodrug forms are depleted by undergoing metabolism or that, by the method used, such forms are not readily retained within cells. [0263] (g) Intracellular accumulation of the OCT001 far-red fluorescent chromophore under hypoxia is responsive to OCT1002 prodrug dose and oxygenation level [0264] Adherent A549 cells were cultured by conventional methods and exposed to 0, 30 or 100 nM OCT1002 for 4 days in air, 1% or 3% oxygenation levels. Detached cells were analysed far red fluorescence intensity using conventional flow cytometry and 633 nm wavelength excitation (110.sup.4 cells analysed). [0265] FIG. 6 shows mean fluorescence intensity increases in a linear function of pro-drug dose and is dependent upon oxygenation levels. This provides a convenient fluorometric, single live cell analytical method for analyzing cell population experience of prevailing pO.sub.2 levels. [0266] (h) Deuteration does not affect the intrinsic capacity of the active metabolite (OCT1001) to accumulate [0267] A549 human lung cancer cells were used in this experiment, as described in (g) above. [0268] Under normoxia conditions, similar levels of accumulation of OCT1001 and AQ4 were observed within cells (see FIG. 7). Thus, the overlaid histograms for the population distribution of fluorescence in cells exposed to AQ4 or OCT1001 under normoxia shows similar cellular accumulation potential. [0269] (i) Accumulation of converted pro-drug OCT1001 correlates with growth arrest (increasingly moribund cells) [0270] A549 human lung cancer cells were used in this experiment, as described in (g) above, with the exception that light side scatter (488 nm wavelength) was collected versus fluorescence intensity (>695 nm wavelength). [0271] FIG. 8 shows collected flow cytometry data for A549 cells exposed to 0, 30 and 100 nM OCT1002 under 21%, 3% and 1% oxygen over 4 days. [0272] Plotting all data points reveals that increasing light side scatter parameter (reflecting the expansion of cell size and complexity associated with growth arrest) correlates with the increase in fluorescence intensity (indicating co-accumulation of OCT1001). [0273] (j) Demonstration of intracellular fluorescence following exposure to OCT1002 under hypoxic conditions and that prodrug deuteration reduces intracellular accumulation but increases persistence of the metabolite. [0274] A549 cells were cultured using conventional methods and allowed to attach to the glass substrate in chamber slides and exposed to OCT1002 under hypoxia. Fluorescence imaging of live cells used conventional confocal fluorescence microscopy using red-line laser excitation. [0275] FIG. 9(A) shows that the far red fluorescence detected in cells is intracellular (background fluorescence not detectable in control cultures) with evidence of regions of cytoplasmic accumulation. The data exemplify the single cell hypoxia sensing properties of the deuterated pro-drug at the single-cell level. [0276] Given the confirmation of intracellular fluorescence associated with conversion of OCT1002 to OCT1001 under hypoxia, A549 human lung cancer cells were further used to assess differential accumulation or retention of the metabolites using flow cytometry as described in (g) above. Following exposure to AQ4N or OCT1002 under 1% oxygen, cells were detached for analysis, or washed and incubated for 24 h in drug free medium and held under normal oxygenation conditions prior to detachment and analysis by flow cytometry [0277] Flow cytometry data in FIG. 9(B) shows the reduced cellular accumulation (after 4 day exposure) but also reduced loss (after 24 h post exposure recovery) of intracellular fluorescence attributable to the metabolite OCT1001, compared with the fluorescence attributable to the metabolite AQ4, following exposure of A549 cells to pro-drugs OCT1001 and AQ4 in A549 under hypoxia. Thus, deuteration changes the in situ intracellular compartment loading/retention of hypoxia converted forms of OCT1002.

Conclusions

[0278] The above studies demonstrate the in vitro properties of an exemplary deuterated compound of the invention (the N-oxide prodrug, OCT1002, and its active metabolite, OCT1001). [0279] (a) Evidence of primary biological activity following reduction of the prodrug in hypoxia that elicits growth arrest in different tumour cell types; [0280] (b) For an equally effective toxicity for the reduced drug (OCT1001) the toxicity of OCT1002 to cells in normoxia is significantly less. [0281] (c) Reciprocity between pO.sub.2 level and end-product generation in the biologically relevant range of hypoxia; [0282] (d) The ability of cellular fluorescence to report in situ generation of metabolite providing for the sensing and reporting of hypoxic environments; [0283] (e) A distinct molecular/atomic signature provided by site-specific deuteration that can be used to trace prodrug conversion and metabolism by physico-chemical methods; and [0284] (f) Prodrug deuteration results in reduced accumulation of the reduced form under hypoxia but increased persistence/retention of the reduced form upon removal of external drug and re-oxygenation. This property demonstrated in moribund cells confirms both reduced redundant targeting of the deuterated form and convenient signal persistence for hypoxia sensing applications.

Example CEffect of OCT1002 on Tumour Growth and Metastasis In Vivo

[0285] Given the hypoxia-activated cytotoxicity of the prodrug compounds of the invention, it may be advantageous to administer them as part of a combination treatment with one or more chemotherapeutic agents and/or radiotherapeutic modalities capable of decreasing (at least, transiently) tumour oxygenation levels in vivo. Bicalutamide (marketed as Casodex, Cosudex, Calutide, Kalumid) is an oral non-steroidal anti-androgen used in the treatment of prostate cancer including as monotherapy for the treatment of earlier stages of the disease. 22Rv1 is a human prostate carcinoma epithelial cell line (Sramkoski R M, Pretlow T G 2nd, Giaconia J M, Pretlow T P, Schwartz S, Sy M S, Marengo S R, Rhim J S, Zhang D, Jacobberger J W A new human prostate carcinoma cell line, 22Rv1. In Vitro Cell Dev Biol Anim. 1999 July-August; 35(7):403-9). The cell line expresses prostate specific antigen (PSA). Growth is weakly stimulated by dihydroxytestosterone and lysates are immunoreactive with androgen receptor antibody by Western blot analysis.

(i) Effect of Bicalutamide on the Oxygenation of 22Rv1 Prostate Tumours Grown as Xenografts

[0286] Male SCID mice (>8 weeks) bearing 22Rv1 prostate tumours of 100-150 mm.sup.3 were treated daily for 28 days by oral gavage with either vehicle (0.1% DMSO in corn oil) or bicalutamide (2 mg/kg/day in vehicle). [0287] Before commencement of treatment (day 0) pO2 (mmHg) was measured using an Oxylite oxygen electrode probe; this was repeated on the days indicated.

TABLE-US-00002 Mean Signifi- Signifi- Day of pO2 SD cance (to cance (to Treatment Treatment (mmHg) vehicle) day 0) Vehicle only 0 15.277 11.254 7 14.741 4.290 14 3.165 3.275 21 2.660 1.889 28 3.546 1.563 Bicalutamide 0 15.277 11.254 ns (2 mg/kg/day) 7 1.996 1.989 <0.05 <0.05 14 0.486 0.107 ns <0.05 21 1.291 0.291 ns <0.05 28 11.905 0.861 <0.01 ns Table 3 shows mean pO2 values SD. Also shown are statistical comparisons of the bicalutamide group compared to control and to day 0 values; ns = not significant. [0288] 22Rv1 cells grow as a solid tumour on the backs of SCID mice. [0289] Tumour oxygenation was measured over 28 days in vehicle and bicalutamide (2 mg/kg/day) treated mice (see Table 3 above). [0290] Bicalutamide caused a drop in tumour oxygenation (as shown in FIG. 10); from 15.3 mmHg (2% oxygen) to 2.0 mmHg (0.3% oxygen) at day 7 and to 0.5 mmHg (0.1% oxygen) at day 14. This drop persists for approximately 2 weeks before recovering to almost normal somewhere beyond 21 and 28 (at which time it is not significantly different from the starting level of oxygenation). [0291] The faster-growing, vehicle-treated, controls showed no significant drop in oxygen levels up to day 7. However, during the subsequent week (probably related to tumour size) the median oxygen levels drop to about 3 mmHg (0.4% oxygen) and do indicate recovery.

[0292] Conclusion [0293] Hypoxia exists in the 22Rv1 solid tumour model. The addition of bicalutamide alters the patterns of oxygen levels indicated by the tumour. Hypoxia is clearly relevant to the 22Rv1 model and the response of such a model to monotherapy (bicalutamide); and the potential role of OCT1002 in a combination treatment.

(ii) Effect of Bicalutamide on Blood Vessels in 22Rv1 Tumour Xenografts

[0294] Dorsal skin folds were secured using window chambers onto the backs of male SCID mice (>8 weeks). 22Rv1 tumour fragments were implanted and allowed to vascularise for 7 days before commencement of treatment. [0295] Animals were treated daily via oral gavage with either vehicle (0.1% DMSO in corn oil) or bicalutamide (2 mg/kg in vehicle). [0296] Anaesthetised mice were injected i.v. with FITC-labelled dextran immediately prior to imaging with a confocal microscope. [0297] Each image is representative of a minimum of 5 animals per treatment group. [0298] 22Rv1 tumours were grown in window chambers/dorsal skin flaps on the backs of SCID mice. Tumour fragments were imaged (see FIG. 11) before treatment began (A) vehicle and (E) bicalutamide pre-treatment groups and then after 7, 14 and 21 days of treatment, (B-D) vehicle only (F-H) bicalutamide (10 magnification). [0299] Within 7 days tumour fragments showed the development of extensive small vessels indicated as day 0 of the experimental period (see FIG. 11). [0300] In vehicle-treated tumours vessel density showed a slight change by day 14 and by day 21 the small vessel numbers were reduced. [0301] In bicalutamide-treated tumours, loss of small vessels was seen at days 7 and 14 with some recovery by day 21. This is consistent with oxygen electrode data i.e., fall and then recovery of oxygenation.

Conclusions

[0302] Vehicle has no effect on blood vessels for at least 7 days. By day 14 there is a slight pruning of vessels which is clearly seen by day 21. This vessel loss, although not as dramatic as seen in the bicalutamide treated tumours (at days 7 and 14; Ming et al., 2007), may be due to vascular collapse and necrosis seen at this time in this fast growing vehicle-treated tumour. The oxygen levels drop somewhat earlier, i.e. sometime between days 7 and 14 (see FIG. 10). [0303] In bicalutamide-treated 22Rv1 tumours there is a marked early loss of tumour vasculature (by day 7). The data provide evidence that bicalutamide causes a profound drop in tumour oxygenation through an anti-vascular effect; this may be direct or alternatively it could be due to inhibition of the production of pro-angiogenic factors by the tumour cells. [0304] By day 21, the small vessels have returned which is consistent with the increased level of oxygenation seen in FIG. 10.
(iii) Effect of Bicalutamide Only or AQ4N Single Dose or OCT1002 Single Dose on 22Rv1 Xenografts in Mice. [0305] Male SCID mice (>8 weeks) bearing 22Rv1 xenograft tumours of 100-150 mm3 were treated for 28 days. Treatment included Vehicle (0.1% DMSO in corn oil) or bicalutamide (2 mg/kg/day in vehicle) both administered daily via oral gavage. Alternatively, at day 7 of the experimental period AQ4N or OCT1002 (50 mg/kg in sterile PBS) was administered intraperitoneally as a single dose. [0306] Tumour volumes were measured using callipers every other day. [0307] Data analysis to determine the time dependent effect of treatment(s) on tumour volume was performed. Tumour volume was normalised to day 6 (ie pre-prodrug addition). Time series and regression analysis was undertaken. [0308] Tumour growth is normalised to day 6, so that overall tumour growth, and patterns can be compared FIGS. 12 (A and B). [0309] Despite the lack of sensitivity to bicalutamide in vitro, the 22Rv1 tumours show a small but significant slowing of growth. Classical cross-sectional comparison of growth delay showed that mice treated with vehicle required 14.00.3 days to reach four times the volume at the start of treatment. Bicalutamide treatment (2 mg/kg/day) increased this to 18.50.8 days; thus this was a growth delay of 4.5 days. [0310] Graphical regression fits indicate that 22RV1 tumours treated with bicalutamide only show a delay in growth (during days 10-20), despite continuing daily exposure to bicalutamide; the tumours exhibit an overall exponential growth pattern (R.sup.2=0.9915) to day 24. [0311] Addition of AQ4N given as a single dose (50 mg/kg) on day 7, a different growth pattern was observed compared to that of the bicalutamide treatment alone, regression fitting showed a non-linear polynomial growth pattern (R.sup.2=0.9948). [0312] Addition of OCT1002 given as a single dose (50 mg/kg) on day 7; tumours treated with this single dose were capable of maintaining a polynomial (x.sup.2) growth rate pattern, this was also a non-linear pattern (R.sup.2=0.9978). [0313] OCT1002 treated tumours showed an overall reduced rate of growth over the remaining period of the experiment (beyond day 22) compared to the bicalutamide only and AQ4N only treated tumours. Cumulative growth over the entire period (progressive area under the curve), indicates this difference (FIG. 12B).
(iv) Combined Effect of AQ4N Single Dose or OCT1002 Single Dose on 22Rv1 Xenografts in Mice Treated Daily with Bicalutamide [0314] Male SCID mice (>8 weeks) bearing 22Rv1 xenograft tumours of 100-150 mm.sup.3 were treated for 28 days. Vehicle (0.1% DMSO in corn oil) and bicalutamide (2 mg/kg/day in vehicle) treatments were administered daily via oral gavage. [0315] AQ4N or OCT1002 (50 mg/kg in sterile PBS) was administered intraperitoneally as a single dose at day 7. [0316] Tumour volumes were measured using callipers every other day. [0317] Animals were culled once the tumour burden reached 800 mm.sup.3. [0318] Tumour growth is normalised to day 6, so that overall tumour growth, and patterns can be compared (FIGS. 13 (A and B)). [0319] Bicalutamide treatment alone (2 mg/kg/day) is discussed above; it exhibits a overall exponential growth pattern (R.sup.2=0.9915) to day 24. [0320] Bicalutamide treatment was combined with an AQ4N single dose (50 mg/kg) given on day 7, a modified growth pattern was observed compared to that of the bicalutamide treatment alone, regression fitting showed a non-linear polynomial growth pattern (R.sup.2=0.9982), with divergence of growth to bicalutamide alone apparent at beyond day 20. [0321] Bicalutamide treatment treatment was combined with an OCT1002 single dose (50 mg/kg) given on day 7; a different modified growth pattern was observed regression fitting showed a linear tumour growth response (R.sup.2=0.9955), with divergence of growth to bicalutamide alone apparent at beyond day 14.

Conclusions

[0322] The combined treatment indicates two critical features. [0323] (i) the first is an earlier effective tumour growth inhibition of OCT1002 on the bicalutamide treated tumours compared to AQ4N; [0324] (ii) the second indicates a sustained tumour growth inhibition (indicated by a maintained linear response); that reflects a persistence OCT1002 and tumour growth inhibition. [0325] Thus with OCT1002 administered at the time when hypoxia/low oxygen levels were achieved; an early and sustained effect was obtained. The combination of OCT1002 with bicalutamide was more effective at inhibiting tumour growth as compared to AQ4N with bicalutamide.
(v) Effect of OCT1002 on LNCaP Xenografts in Mice Treated with/without Bicalutamide [0326] Male SCID mice (>8 weeks) bearing LNCaP xenograft tumours of 100-150 mm.sup.3 were treated for 28 days. [0327] Vehicle (0.1% DMSO in corn oil) and bicalutamide (2 mg/kg/day in vehicle) treatments were administered daily via oral gavage. OCT1002 (50 mg/kg in sterile PBS) was administered intraperitoneally as a single dose at day 7. [0328] Tumour volumes were measured using callipers every other day. [0329] Growth curves are the mean of 5 animals in bicalutamide and vehicle treatment groups; bicalutamide+OCT1002 group (n=5 until day 14; then n=3) and vehicle+OCT1002 (n=5 until day 13; n=1)s.e. [0330] Table 6 below shows the growth delays calculated for the time to reach twice the treatment size. [0331] Bicalutamide causes a 5.1 day delay in LNCaP tumour growth compared to vehicle. [0332] When OCT1002 (50 mg/kg single dose on day 7) was given in combination with vehicle (daily administration) there was no appreciable effect on tumour growth (Table 6 below). [0333] Bicalutamide (daily for 28 days) initially slows tumour growth until day 12-14. Tumour growth then recovers and the tumours are the same size as the vehicle-treated tumours by day 28 (Table 6 below). [0334] Tumours treated with a single dose of OCT1002 reduced the growth rate in combination with bicalutamide and this was significantly different from control at all times between days 14 and days 28 at the termination of the experiment (FIG. 15).

Conclusions

[0335] Administration of OCT1002 at day 7 had no significant effect on LNCaP tumour growth. This shows that the better-oxygenated tumours (i.e. as compared to bicalutamide-treated tumours) there is low toxicity of OCT1002 and that this better-oxygenated fraction of cells is predominant in contributing to growth in vehicle-treated control tumours. [0336] Combination of a single dose of OCT1002 with bicalutamide blocked the increase in growth rate observed in the bicalutamide-treated group. OCT1002 is very effective at blocking tumour growth from 12 days onwards where, for bicalutamide alone, there is a delay and then recovery. [0337] The initial slowing and then recovery after day 14 of LNCaP tumour growth, during daily treatment with bicalutamide, is consistent with the drop and then recovery of tumour oxygenation and blood vessels (Ming et al., 2012, supra.).

TABLE-US-00003 TABLE 6 Time to 2x start Growth Treatment volume (days) Delay (days) Vehicle Only 11.2 1.88 Bicalutamide 16.2 1.94 5 3.82 OCT1002 only 13 0.89 1.8 2.77 OCT1002 + 25.5 3.22 14.3 5.1 Bicalutamide

(vi) OCT1002 Prodrug is Converted to Metabolites in Hypoxic LNCaP Tumour Cells In Vivo

Methods

[0338] A dorsal skin flap (window chamber) was attached to the dorsum of male SCID mice and a 1 mm.sup.3 LNCaP-Luc tumour fragment inserted; this was left to vascularise for 7 days. [0339] Mice were then treated orally for 21 days with either vehicle (0.1% DMSO in corn oil) or bicalutamide (2 mg/kg/day). [0340] Seven days after induction of (a) vehicle or (b) bicalutamide mice were dosed intraperitoneally with OCT1002 (50 mg/kg). [0341] Two hours after injection of OCT1002 mice were injected intravenously with FITC-dextran. [0342] Images were captured using a confocal laser scanning microscopy to show blood vessels (green) and OCT1001 (blue) patterns in the tumour. (Magnification 10 with 3 digital zoom) (pixel resolution). [0343] Images were also acquired at day 0 (i.e. 7 days after tumour fragment implantation), 14 and 21. [0344] Only FITC-dextran was administered on days 0, 14 and 21. (c) Full panel of images 0, 7, 14 and 21 days. [0345] Control mice were treated orally for 21 days with vehicle (0.1% DMSO in corn oil): vascularisation was maintained throughout. By 7 days the tumour fragment was vascularised (day 0 of experiment shown in FIG. 15C green). [0346] In mice treated with vehicle+OCT1002 at day 7: the converted compound OCT1001 (blue) is in a few areas where vascularisation is poor (FIG. 15A). [0347] Mice treated with bicalutamide (2 mg/kg/day in vehicle): vascularisation was reduced at days 7. On day 7, two hours after intraperitoneal injection of a single dose of OCT1002 (50 mg/kg) large quantities of converted compound (OCT1001; blue) can be seen across the whole tumour fragment (FIG. 15B). [0348] Mice treated with bicalutamide (2 mg/kg/day in vehicle): vascularisation was reduced at days 7 and 14, this recovered by day 21 (Ming et al., 2012, supra.). [0349] Tumours were re-examined at days 14 and 21. [0350] OCT 1001 (blue) is still localised to the tumour at day 14; by day 21 the amount of compound was considerably lower (FIG. 15C).

Conclusions

[0351] OCT1002, administered intraperitoneally, distributed widely throughout the tumour fragments localised in the skin fold on the backs of the mice. [0352] Distribution was extensive even when the vasculature was significantly decreased (i.e. by the bicalutamide treatment at days 7 and 14). [0353] OCT1001 was found predominantly where the oxygen levels are low (i.e. areas of poor vascularisation); small areas were seen in the control also (indicating that hypoxia can occur in untreated tumours but to a lesser extent. [0354] Extensive localisation of OCT1001 was still observed at day 14 of bicalutamide treatment showing that the compound remains for at least 7 days. [0355] By day 21, tumour blood vessels show some recovery and OCT1001 levels are lower although still above background. [0356] The persistence of the reduced product, OCT1001, for >7 days shows that the half-life of the converted compound is long. [0357] However it may be less than AQ4 since by day 21 the OCT1001 signal is very much decreased. [0358] This may be due to the different cellular binding properties of OCT1001 as compared to AQ4 and potentially will provide a rationale for less cumulative systemic toxicity which might be caused through persistence of small amount of reduced compound in marginally hypoxic peripheral tissues. This should not affect the primary efficacy of OCT1002/OCT1001 at the predominant site of metabolism (i.e. the hypoxic cells in tumours) since large amounts are seen throughout the hypoxic tumour fragment which persists for greater than 7 days.
(vii) OCT1002 Reduces the Metastatic Spread of LNCaP Tumours to the Lungs

Methods

[0359] Male SCID mice (>8 weeks) bearing LNCaP-luc xenograft tumours of 100-150 mm.sup.3 were treated for 28 days (the luciferase-expressing cells had similar characteristics to parental LNCaP cells; Ming et al., 2012, supra.). [0360] Vehicle (0.1% DMSO in corn oil) and bicalutamide (2 mg/kg/day in vehicle) treatments were administered daily via oral gavage. [0361] OCT1002 (50 mg/kg in sterile PBS) was administered intraperitoneally as a single dose at day 7. [0362] On day 28 of treatment, animals were injected i.p. with a solution of D-luciferin (150 mg/kg in PBS) 15 mins prior to imaging. [0363] Animals were then killed and a range of tissues were removed for the detection of bioluminescence using the IVIS imaging system (Xenogen, USA). [0364] Images were taken for 5 minutes and quantification of bioluminescence was achieved by drawing a region of interest around the area and measuring total flux in photons/second (ph/sec). [0365] A range of tissues were excised, however only the lungs and tumour showed measurable bioluminescence. The means.e of bioluminescence in the lungs is shown in FIG. 16; bicalutamide and vehicle treatment groups (n=10); bicalutamide+OCT1002 group (n=3). and vehicle+OCT1002 (n=1). * Bicalutamide vs bicalutamide+OCT1002 (p=0.024). Mice treated with vehicle showed some metastatic spread to the lung. OCT1002, single dose day 7, had no effect on this spread. [0366] Bicalutamide appeared to increase the extent of metastatic spread although the result did not reach significance. [0367] Combination of OCT1002 with bicalutamide showed that OCT1002 significantly reduces the metastatic spread to the lungs caused by bicalutamide. (P=0.024)

Conclusions

[0368] OCT1002 given as a single dose at day 7 was able to reduce significantly the metastatic spread to the lungs caused by bicalutamide treatment.