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 tumor, are also provided. Additionally, an option for combination with chemotherapeutic and radiotherapeutic modalities to enhance overall tumor cell kill is provided. Methods for the detection of cellular hypoxia, both in vivo and in vitro, are additionally provided.
##STR00001##
|
##STR00014##
wherein each Y is independently selected from the group consisting of hydrogen, hydroxy, halogeno, amino, C1-4 alkoxy and C2-8 alkanoyloxy.
29. A method of detecting hypoxic cells in vitro or in vivo in a group of cells, the method comprising:
exposing a compound of formula v:
##STR00016##
wherein each Y is independently selected from the group consisting of hydrogen, hydroxy, halogeno, amino, C1-4 alkoxy and C2-8 alkanoyloxy,
to the group of cells;
analyzing the cells for the presence of a corresponding reduced compound of formula III:
##STR00017##
determining the hypoxic cells based on the presence of the corresponding reduced compound.
##STR00015##
3. The compound according to
4. The pharmaceutical composition comprising the compound according to
6. The kit according to
7. 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
8. The method according to
9. The method according to
10. The method according to
11. The method according to
12. The method according to
13. The method according to
14. The method according to
15. The method according to
16. 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
17. The method according to
18. The method according to
19. The method according to
20. The method according to
21. The method according to
22. The method according to
23. The method according to
24. The method according to
25. A process for making the compound according to
26. The process according to
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28. The process according to
30. The method according to
31. The method according to
32. The method according to
surgically excising cells identified as being hypoxic.
33. The method according to
34. The method according to
|
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The present application claims priority to Great Britain Patent Application No. 1214169.3, filed Aug. 8, 2012, incorporated herein in its entirety.
The present invention relates to novel anthraquinone compounds and uses of the same, for example in the treatment of cancer.
The therapeutic advantage of an anticancer drug depends primarily on the extent to which the agent shows selective activity for tumour cells and the limiting toxicity towards non-target tissues. Frequently the poor quality of the vasculature within the growing tumour mass compromises the delivery of drugs, nutrients and oxygen. It is recognised that tumours can have significantly lower median oxygen levels (approximately 1% oxygen; pO2 7.5 mmHg) compared to normal tissues (˜5.5% oxygen; 42 mmHg) (summarised from data presented by Brown and Wilson, 2004). In addition, oxygenation levels can vary throughout the tumour due to intermittent opening and closing of tumour blood vessels; poor vascularisation, especially in the tumour core, contributes to oxygen levels often being below 0.1% oxygen (1 mm Hg). Tumour cells experiencing varying degrees of hypoxia, relative to normally perfused tissues, can compromise treatment effectiveness and contribute to the malignancy. Hypoxia-selective agents (e.g. bioreductive drugs) comprise one class of agents that can be used to target tumour cells in very low oxygen environments by virtue of a selective activation to a cytotoxic form under reduced oxygenation, addressing the problems of non-target tissue toxicity, hypoxic cell drug resistance and cancer progression.
Poor oxygenation results in a relative state of hypoxia when compared with normoxic conditions in which oxygenation has not been compromised. Poor oxygenation within tumours can modify the responses to treatment modalities and contribute to cancer progression. Cells in such hypoxic areas are particularly resistant to treatment with many of the conventionally used anticancer drugs; this is attributed to poor drug delivery and/or lack of intrinsic tumour cell sensitivity of viable but quiescent cells. Radiotherapy is also less effective at very low oxygen levels since the cytotoxicity of ionising radiation is enhanced by the presence of oxygen (Radiobiology For The Radiologist, Hall E J, Giaccia A J, Lippincott Williams & Wilkins, (2005)). Recent evidence shows that tumour cells can adapt to low oxygen conditions and change the pharmacodynamic responses to anticancer agents through the induction of active cellular protective mechanisms (Vaupel and Mayer 2007, Cancer Metastasis Rev 26(2): 225-239). Additionally, it is recognized that tumour cells that survive hypoxic stress often show a more malignant metastatic phenotype (Vaupel P, Metabolic microenvironment of tumor cells: a key factor in malignant progression, Exp Oncol 2010; 32, 125-127); this has significant consequences for the patient. Following treatment with modalities that target predominantly the better-oxygenated cells, the stress-resistant hypoxic cells often repopulate the tumour with cells that have an enhanced potential to spread to distant tissues. The development of more malignant metastatic tumours is often the precursor to a more significant disease-related morbidity and the death of the patient.
An attractive approach is the use of a hypoxia activated prodrug that is non-toxic towards adequately oxygenated cells found in systemic tissues, but becomes activated or converted to a cytotoxic form under reduced oxygenation conditions. N-oxide derivatives of cytotoxic alkylaminoanthraquinones provide anthraquinone pro-drugs that show almost no cytotoxicity. Importantly these prodrugs are capable of being converted in vivo under the anaerobic/hypoxic conditions found within neoplastic tissue. Specificity for the tumour is ensured since systemic tissues, except for tumours, almost never experience oxygen levels low enough to facilitate the production of the cytotoxic drug.
The anthraquinone N-oxide AQ4N (CAS#136470-65-0) is a prodrug that is selectively bioreduced to AQ4, a potent DNA topoisomerase II inhibitor, in hypoxic tumour cells. Previous publications have taught the fundamental properties and in-vitro/in-vivo characteristics of the prodrug AQ4N (for example, see U.S. Pat. No. 5,132,327).
The invention seeks to address the need for improved cancer treatments by providing novel anthraquinone compounds with a combination of preferable pharmacological and hypoxia-sensing properties.
The first aspect of the invention provides a compound of Formula I
##STR00002##
wherein X1, X2, X3 and X4 are each independently selected from the group consisting of hydrogen, hydroxy, halogeno, amino, C1-4 alkoxy, C2-8 alkanoyloxy, —NH-A-NHR, —NH-A-NR′R″ and —NH-A-N(O)R′R″
wherein A is an alkylene group with a chain length of at least two carbon atoms (between NH and NHR or N(O)R′R″),
wherein R, R′ and R″ are each independently selected from C1-4 alkyl groups and C2-4 hydroxyalkyl and C2-4 dihydroxyalkyl groups in which the carbon atom attached to the nitrogen atom does not carry a hydroxy group and no carbon atom is substituted by two hydroxy groups, or wherein R and R″ together are a C2-6 alkylene group which with the nitrogen atom to which R′ and R″ are attached forms a heterocyclic group having 3 to 7 atoms in the ring,
wherein at least one of X1, X2, X3 and X4 is selected from the group consisting of deuterated forms of —NH-A-NHR, —NH-A-NR′R″ and —NH-A-N(O)R′R″.
Thus, the invention provides novel deuterated anthraquinone compounds.
By “deuterated” we include that the compound comprises at least one atom of deuterium or heavy hydrogen (i.e. D or 2H). It will be appreciated by persons skilled in the art that the compound may be partially (i.e. selectively) or fully deuterated (i.e. containing hydrogen present only in the form of deuterium).
By “selectively”, in this context, we mean that some but not all conventional 1H hydrogen atoms are replaced with deuterium. For example, one or more of substituent groups X1, X2, X3 and X4 may be deuterated while the central anthraquinone ring may be free of deuterium.
In one embodiment, the compound of the invention is selectively deuterated within one or more of substituent groups —NH-A-NHR, —NH-A-NR′R″ and/or —NH-A-N(O)R′R″ at positions X1, X2, X3 and/or X4. Within each such substituent group, it will be appreciated that A, R, R′ and R″ may be fully deuterated (i.e. thus containing no 1H) or may be partially deuterated.
In a preferred embodiment, the compound is deuterated only within one or more of the terminal groups R, R′ and R″. For example, R, R′ and/or R″ may represent:
The term “C1-4 alkyl” is intended to include linear or branched alkyl groups comprising between one and four carbons. Preferred alkyl groups which R, R′ and/or R″ may independently represent include C1 and C2 alkyl.
The term “lower alkylene” is to be construed accordingly.
The terms “C2-4 hydroxyalkyl” and “C2-4 dihydroxyalkyl” are intended to include linear or branched alkyl groups comprising between two and four carbons, to which are attached one or two hydroxy groups, respectively. For example, R, R′ and/or R″ may independently represent:
The term “C1-4 alkoxy” is intended to include linear or branched C1-4 alkyl groups bound to the core anthraquinone (anthracene-9,10-dione) ring via oxygen. For example, R, R′ and/or R″ may independently represent:
The term “C2-8 alkanoyloxy” is intended to include linear or branched C2-8 alkanoyl groups bound to the core anthraquinone (anthracene-9,10-dione) ring via oxygen. For example, R, R′ and/or R″ may independently represent:
The term “hydroxy” is intended to represent —OH.
The term “halogeno” is intended to represent any halogen group, such as —Br, —Cl and —F.
The term “amino” is intended to include primary amine groups, such as —NH2.
It will be appreciated by persons skilled in the art that the anthraquinone ring of the compounds may be substituted by X1, X2, X3 and X4 at any of ring positions 1, 2, 3, 4, 5, 6, 7 or 8:
##STR00003##
In one embodiment of the first aspect of the invention, the compound is substituted at ring positions 1, 4, 5 and 8, in accordance with Formula II:
##STR00004##
In one embodiment, X1, X2, X3 and X4 are each separately selected from the group consisting of hydrogen, hydroxy, —NH-A-NHR, —NH-A-NR′R″, —NH-A-N(O)R′R″ and deuterated forms thereof.
In one embodiment, X1, X2, X3 and X4 are each separately selected from the group consisting of hydroxy, —NH-A-NR′R″, —NH-A-N(O)R′R″ and deuterated forms thereof.
In one embodiment, X1 and X2 are both hydroxy and X3 and X4 are both —NH-A-N(O)R′R″ or deuterated forms thereof.
In one embodiment, X1 and X2 are both hydroxy and X3 and X4 are both NH-A-NR′R″ or deuterated forms thereof.
In one embodiment, A is unbranched. For example, A may be ethylene.
In one embodiment, R, R′ and R″ are each independently selected from the group consisting of —CH3, —CH2CH3, —CH2CH2CH3, —CH2CH2OH, —CH2CH2CH2OH, —CH(CH3)CH2OH, —CH2CHOHCH2OH and deuterated forms thereof.
In one embodiment, one or two of X1, X2, X3 and X4 are independently selected from the group consisting of —NH—(CH2)2—N(O)(CH3)2, —NH—(CH2)2—N(O)(CH3)C2H5, —NH—(CH2)2—N(O)(C2H5)2, —NH—(CH2)2—N(O) (CH2CH2OH)2, —NH—(CH2)2—N(O)(CH2CH2CH2OH)2, —NH—(CH2)2—N(O)CH(CH3)OH, —NH—(CH2)2—N(O)(CH2CHOHCH2OH)2 and deuterated forms thereof.
In one embodiment, one or two of X1, X2, X3 and X4 are independently selected from the group consisting of —NH—(CH2)2—N(CH3)2, —NH—(CH2)2—N(CH3)C2H5, —NH—(CH2)2—N(C2H5)2, —NH—(CH2)2—N(CH2CH2OH)2, —NH—(CH2)2—N(CH2CH2CH2OH)2, —NH—(CH2)2—NCH(CH3)OH, —NH—(CH2)2—N(CH2CHOHCH2OH)2 and deuterated forms thereof.
In one embodiment, the compound of the invention comprises one group —NH-A-N(O)R′R″ and one group —NH-A-NHR, the —NH-A-NHR group being selected from —NH—(CH2)2—NHCH3, —NH—(CH2)2—NHC2H5, —NH—(CH2)2—NHCH2CH2OH, —NH—(CH2)2—NHCH2CH2CH2OH, —NH—(CH2)2—NHCH(CH3)CH2OH, —NH—(CH2)2—NHCH2CHOHCH2OH and deuterated forms thereof.
In one embodiment, the compound of the invention comprises one group —NH-A-NR′R″ and one group —NH-A-NHR, the —NH-A-NHR group being selected from —NH—(CH2)2—NHCH3, —NH—(CH2)2—NHC2H5, —NH—(CH2)2—NHCH2CH2OH, —NH—(CH2)2—NHCH2CH2CH2OH, —NH—(CH2)2—NHCH(CH3)CH2OH, —NH—(CH2)2—NHCH2CHOHCH2OH and deuterated forms thereof.
In preferred, but non-limiting, compounds of the invention:
(a) X1=—NH-A-N(O)R′R″, X2=—H and X3=X4=—OH;
(b) X1=—NH-A-N(O)R′R″, X2=—OH, X3=—OH and X4=—H;
(c) X1=—NH-A-N(O)R′R″ and X2=X3=X4=—OH;
(d) X1=X4=—NH-A-N(O)R′R″ and X2=X3=—OH;
(e) X1=X2=—NH-A-N(O)R′R″ and X3=X4=—OH;
(f) X1=X3=—NH-A-N(O)R′R″ and X2=X4=—OH;
(g) X1=—NH-A-NR′R″, X2=—H and X3=X4=—OH;
(h) X1=—NH-A-NR′R″, X2=—OH at position 4, X3=—OH and X4=—H;
(i) X1=—NH-A-NR′R″ and X2=X3=X4=—OH;
(j) X1=X4=—NH-A-NR′R″ and X2=X3=—OH;
(k) X1=X2=—NH-A-NR′R″ and X3=X4=—OH;
(l) X1=X3=—NH-A-NR′R″ and X2=X4=—OH;
and deuterated forms thereof.
In further preferred, but non-limiting, compounds of the invention:
(a) X1=—NH-A-N(O)R′R″, X2=—NH-A-NHR, and X3=X4=—OH;
(b) X1=—NH-A-N(O)R′R″, X2=—OH, X3=—NH-A-NHR and X4=—OH;
(c) X1=—NH-A-N(O)R′R″, X2=X3=—OH and X4=—NH-A-NHR;
(d) X1=—NH-A-NR′R″, X2=—NH-A-NHR, and X3=X4=—OH;
(e) X1=—NH-A-NR′R″, X2=—OH, X3=—NH-A-NHR and X4=—OH;
(f) X1=—NH-A-NR′R″, X2=X3=—OH and X4=—NH-A-NHR;
and deuterated forms thereof.
In further preferred, but non-limiting, compounds of the invention:
(a) X1=X2=—NH-A-N(O)R′R″ and X3=X4=—OH;
(b) X1=X3=—NH-A-N(O)R′R″ and X2=X4=—OH;
(c) X1=X2=—NH-A-NR′R″ and X3=X4=—OH; and
(d) X1=X3=—NH-A-NR′R″ and X2=X4=—OH
wherein
both —NH-A-N(O)R′R″ are —NH—(CH2)2N(O)(CH3)2 or —NH—(CH2)2N(O)(CH2CH2OH)2, or deuterated forms thereof and
both NH-A-NR′R″ are —NH—(CH2)2N(CH3)2 or —NH—(CH2)2N(CH2CH2OH)2, or deuterated forms thereof.
In further preferred, but non-limiting, compounds of the invention:
(a) X1=—NH-A-N(O)R′R″, X2=—NH-A-NHR and X3=X4=—OH;
(b) X1=—NH-A-N(O)R′R″, X2=—OH, X3=—NH-A-NHR and X4=—OH;
(c) X1=—NH-A-NR′R″, X2=—NH-A-NHR and X3=X4=—OH; and
(d) X1=—NH-A-NR′R″, X2=—OH, X3=—NH-A-NHR and X4=—OH,
wherein
—NH-A-N(O)R′R″ is —NH—(CH2)2N(O)(CH3)2 or —NH—(CH2)2N(O)(CH2CH2OH)2 or a deuterated form thereof
—NH-A-NHR is NH—(CH2)2NHCH3 or NH(CH2)2NHCH2CH2OH or a deuterated form thereof
and NH-A-NR′R″ is —NH—(CH2)2N(CH3)2 or —NH—(CH2)2N(CH2CH2OH)2 or a deuterated form thereof.
The present invention relates to novel anthraquinone compounds and uses of the same, for example in the treatment of cancer.
The present invention relates to novel anthraquinone compounds and uses of the same, for example in the treatment of cancer.
In one embodiment, the compound is of Formula III or IV:
##STR00005##
wherein Y are each independently selected from the group consisting of hydrogen, hydroxy, halogeno, amino, C1-4 alkoxy and C2-8 alkanoxy, or a prodrug thereof.
By “prodrug”, in this context, is included compounds which may readily be converted in vivo to a compound of Formula III or IV. In one embodiment, the conversion is triggered by the prodrug entering an hypoxic environment, such as a solid tumour.
Examples of suitable prodrugs include N-oxide derivatives of the compounds of Formula III or IV.
Thus, in one embodiment, the prodrug is a compound of Formula V or VI:
##STR00006##
wherein Y are each independently selected from the group consisting of hydrogen, hydroxy, halogeno, amino, C1-4 alkoxy and C2-8 alkanoxy.
In one preferred embodiment, the compound is of Formula VII or VIII:
##STR00007##
or a prodrug thereof.
The present invention relates to novel anthraquinone compounds and uses of the same, for example in the treatment of cancer.
The present invention relates to novel anthraquinone compounds and uses of the same, for example in the treatment of cancer.
In a further preferred embodiment, the compound is prodrug of Formula IX or X:
##STR00008##
In the compounds of Formulae III to X, it will be appreciated by persons skilled in the art that one or more of the deuterium atoms in one or more of the methyl groups attached to the nitrogen of the terminal amino groups may be replaced by conventional hydrogen (i.e. 1H), provided that the compound comprises at least one deuterium atom. For example, one, two, three or four of the methyl groups may be —CH3, —CH2D or —CHD2. In one embodiment, the methyl groups in the compound are either —CH3 or —CD3.
It will be further appreciated by skilled persons that certain compounds of formulae I to X above may be counterbalanced by counter-anions. Exemplary counter-anions include, but are not limited to, halides (e.g. fluoride, chloride and bromide), sulfates (e.g. decylsulfate), nitrates, perchlorates, sulfonates (e.g. methane sulfonate) and trifluoroacetate. Other suitable counter-anions will be well known to persons skilled in the art. Thus, pharmaceutically, and/or veterinarily, acceptable derivatives of the compounds of formulae I to X, such as salts and solvates, are also included within the scope of the invention. Salts which may be mentioned include: acid addition salts, for example, salts formed with inorganic acids such as hydrochloric, hydrobromic, sulfuric and phosphoric acid, with carboxylic acids or with organo-sulfonic acids; base addition salts; metal salts formed with bases, for example, the sodium and potassium salts.
In one embodiment, the compound is in the form of a halide salt, for example a chloride salt.
It will be further appreciated by skilled persons that certain compounds of formulae I to X may exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention.
Compounds of formulae I to X may also contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively, the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation, or by derivatisation, for example with a homochiral acid followed by separation of the diastereomeric esters by conventional means (e.g. HPLC, chromatography over silica). All stereoisomers are included within the scope of the invention.
Various routes are available for the synthesis of the compounds of the invention. One very convenient procedure for the preparation of compounds having a group —NH-A-NR′R″ at the 1 and 4 positions uses the appropriately substituted 2,3-dihydro(leuco)-1,4-dihydroxyanthracene-9,10-dione which is condensed with the appropriate amine R″R′N—A—NH2, the 1,4 positions being activated in the leuco compound for reaction with the amine. Such a condensation may conveniently be effected at a temperature in a range of about 25° C. or 35° C. to 50° C. or 60° C. for one or more hours using a solvent such as methanol, ethanol, water, dimethylformamide, 2-methoxyethanol, acetonitrile, nitrobenzene, N,N,N′N′-tetra-methylenediamine or mixtures thereof. In some instances a higher temperature and shorter reaction time may be appropriate, for example with the compounds containing cyclic groups NR′R″. The leuco derivative is then oxidized to the fully aromatic anthracene-9,10-dione, conveniently using air oxidation or oxidation with hydrogen peroxide, chloranil, sodium perborate or manganese dioxide.
Although leuco compounds are primarily of interest for the preparation of compounds substituted by two —NH-A-NHR′R″ groups, it is possible to use them to prepare compounds containing more than two such groups. Thus, by using 2,3-dihydro(leuco)-1,4,5,8-tetrahydroxyanthracene-9,10-dione and a large excess of an amine —NH-A-NHR′R″ an 8-hydroxyanthracene-9,10-dione having three groups —NH-A-NHR′R″ at the 1, 4 and 5 positions may be prepared.
The leuco derivatives themselves are obtainable by heat treatment of the corresponding fully aromatic 1,4-dihydroxyanthracene-9,10-dione, conveniently by heating at above 90° C. for 1 hour or more in a stream of nitrogen and, if necessary, in the presence of a suitable reducing agent such as sodium dithionite or zinc dust. Various anthracene-9,10-diones, particularly hydroxyanthracene-9,10-diones, are commercially available and various syntheses for such compounds are also reported in the literature. One suitable procedure for their preparation involves the reaction of an appropriately substituted phthalic anhydride with hydroquinone in the presence of aluminium chloride and sodium hydroxide at 180° C. for one hour or more. Anthracene-9,10-diones containing one form of substituent group can be modified to provide other forms of substituent group so that, for example, a dione containing an amino group can be treated with sodium hydroxide/dithionite to yield the corresponding hydroxy substituted compound.
Other suitable procedures for the preparation of intermediates for oxidation to the N-oxide compounds of the invention include the reaction of the appropriate chloro or fluoro substituted anthracene-9,10-dione with the appropriate amine R″R′N—A—NH2, for example by heating with a excess of the amine at its reflux temperature for one or more hours. Certain of these chloro- and fluoro anthracene-9,10-diones are known and various syntheses for such compounds are also reported in the literature. Thus, for example, a KF—NaF-mediated conversion of 3,6-dichlorophthalic anhydride to 3,6-difluorophthalic anhydride as a precursor to making 1,4-difluoro-4,8-dihydroxyanthracene-9,10-dione (see Lee & Denny, 1999, J. Chem. Soc., Perkin Trans. 1:2755-2758. Additionally, for example, 1,5-dichloro-4,8-dihydroxyanthracene-9,10-dione may be prepared by selective chlorination of 1,4,5,8-tetrahydroxyanthracene-9,10-dione using a stoichiometric amount of sulphuryl chloride and controlled temperature. This precursor may then be used to prepare an intermediate having groups —NH-A-NR′R″ at the 1 and 5 positions and hydroxy groups at the 4 and 8 positions, the hydroxy groups conveniently being protected during the reaction with the amine R″R′N—A—NH2. A similar approach is suitable for the preparation of other chlorohydroxyanthracene-9,10-dione intermediates.
Where the compound of the invention contains one or more groups —NH-A-NHR in addition to the one or more groups —N-A-NR′R″ the compound may conveniently be produced by reacting a suitable precursor as discussed above with a mixture of amines RN—A—NH.sub.2 and R″R′N—A—NH.sub.2, the resultant mixture of products then being separated, for example by chromatography. Thus, for example, 2,3-dihydro(leuco)-1,4-dihydroxyanthracene-9,10-dione on reaction with a mixture of 2-(2-hydroxyethylamino)ethylamine and 2-(diethylamino)ethylamine will yield a mixture of 1,4-bis{[2-(diethylamino)-ethyl]amino}anthracene-9,10-dione, 1,4-bis{[2-(2-hydroxyethyl-amino)-ethyl]amino}-anthracene-9,10-dione and 1-(2-(diethylamino)ethyl]amino)-4-{[2-(2-hydroxyethylamino)-ethyl]amino}anthracene-9,10-dione from which the last mentioned compound may be separated, for example by chromatography. On oxidation, only the tertiary nitrogen atom of the [2-(diethylamino)ethyl)] amino group will be converted to N-oxide form.
Where one or more substituent groups is present it may be appropriate, depending on the route of synthesis, to have these present throughout in their final form or to generate the desired groups at a later stage in the synthesis. Ether and ester groups X may of course readily be prepared by modification of hydroxy groups according to known procedures, precursors containing a hydroxy group X more often being described in the literature than those containing a corresponding ether or ester substituent.
It will be appreciated, however, that various alternative methods for the preparation of the compounds and intermediates therefor may be used as will be apparent in particular from the literature relating to such intermediates. Further details of the preparation of intermediates for the preparation of the compounds of the present invention are to be found in U.S. Pat. No. 4,197,249 and GB 2,004,293B (the disclosures of which are incorporated herein by reference).
Thus, a second aspect of the invention provides a process for making a compound according to the first aspect of the invention comprising reacting an anthracene-9,10-dione with a deuterated alkylenediamine under conditions suitable for the production of an alkylaminoalkylaminoanthraquinone.
Optionally, the process further comprises 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.
In one embodiment, the process comprises 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.
In a further embodiment, the process comprises 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.
A third aspect of the invention provides a pharmaceutical composition comprising a compound according to the first aspect of the invention together with pharmaceutically acceptable buffer, diluent, carrier, adjuvant or excipient.
By “pharmaceutically acceptable” we include a non-toxic material that does not decrease the therapeutic effectiveness of the compound of the invention. Such pharmaceutically acceptable buffers, carriers or excipients are well-known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R Gennaro, Ed., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000), the disclosures of which are incorporated herein by reference).
The term “buffer” is intended to mean an aqueous solution containing an acid-base mixture with the purpose of stabilising pH. Examples of buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazolelactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES.
The term “diluent” is intended to mean an aqueous or non-aqueous solution with the purpose of diluting the agent in the pharmaceutical preparation. The diluent may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil).
The term “adjuvant” is intended to mean any compound added to the formulation to increase the biological effect of the compound of the invention. The adjuvant may be one or more of zinc, copper or silver salts with different anions, for example, but not limited to fluoride, chloride, bromide, iodide, thiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate, and acetates of different acyl composition. The adjuvant may also be cationic polymers such as cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationic synthetic polymers such as poly(vinyl imidazole), and cationic polypeptides such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids.
The excipient may be one or more of carbohydrates, polymers, lipids and minerals. Examples of carbohydrates include lactose, glucose, sucrose, mannitol, and cyclodextrines, which are added to the composition, e.g., for facilitating lyophilisation. Examples of polymers are starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates, carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polysulphonate, polyethylenglycol/polyethylene oxide, polyethyleneoxide/polypropylene oxide copolymers, polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, and polyvinylpyrrolidone, all of different molecular weight, which are added to the composition, e.g., for viscosity control, for achieving bioadhesion, or for protecting the lipid from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, mono-, di-, and triglycerides, ceramides, sphingolipids and glycolipids, all of different acyl chain length and saturation, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin, which are added to the composition for reasons similar to those for polymers. Examples of minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain benefits such as reduction of liquid accumulation or advantageous pigment properties.
The compounds of the invention may be formulated into any type of pharmaceutical composition known in the art to be suitable for the delivery thereof.
In one preferred embodiment, the pharmaceutical compositions are administered parenterally, for example, intravenously, intracerebroventricularly, intraarticularly, intraarterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intramuscularly or subcutaneously, or they may be administered by infusion techniques. The pharmaceutical compositions may also administered intra-tumourally and/or peri-tumourally.
Such pharmaceutical compositions are conveniently used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
In a further embodiment, the pharmaceutical compositions of the invention may be in the form of a liposome, in which the agent is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids, which exist in aggregated forms as micelles, insoluble monolayers and liquid crystals. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Suitable lipids also include the lipids above modified by poly(ethylene glycol) in the polar headgroup for prolonging bloodstream circulation time. Preparation of such liposomal formulations is can be found in for example U.S. Pat. No. 4,235,871, the disclosures of which are incorporated herein by reference.
The pharmaceutical compositions of the invention may also be in the form of biodegradable microspheres. Aliphatic polyesters, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA) or poly(caprolactone) (PCL), and polyanhydrides have been widely used as biodegradable polymers in the production of microspheres. Preparations of such microspheres can be found in U.S. Pat. No. 5,851,451 and in EP 0 213 303, the disclosures of which are incorporated herein by reference.
In a further embodiment, the pharmaceutical compositions of the invention are provided in the form of polymer gels, where polymers such as starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates, carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polyvinyl imidazole, polysulphonate, polyethylenglycol/polyethylene oxide, polyethyleneoxide/polypropylene oxide copolymers, polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, and polyvinylpyrrolidone are used for thickening of the solution containing the agent. The polymers may also comprise gelatin or collagen.
Alternatively, the compounds may simply be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil), tragacanth gum, and/or various buffers.
It will be appreciated that the pharmaceutical compositions of the invention may include ions and a defined pH for potentiation of action of the active agent. Additionally, the compositions may be subjected to conventional pharmaceutical operations such as sterilisation and/or may contain conventional adjuvants such as preservatives, stabilisers, wetting agents, emulsifiers, buffers, fillers, etc.
The pharmaceutical compositions according to the invention may be administered via any suitable route known to those skilled in the art. Thus, possible routes of administration include parenteral (intravenous, subcutaneous, and intramuscular), topical, ocular, nasal, pulmonar, buccal, oral, parenteral, vaginal and rectal. Also administration from implants is possible.
Alternatively, the pharmaceutical compositions may be administered intranasally or by inhalation (for example, in the form of an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoro-methane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas). In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active polypeptide, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.
The pharmaceutical compositions will be administered to a patient in a pharmaceutically effective dose. A ‘therapeutically effective amount’, or ‘effective amount’, or ‘therapeutically effective’, as used herein, refers to that amount which provides a therapeutic effect for a given condition and administration regimen. This is a predetermined quantity of active material calculated to produce a desired therapeutic effect in association with the required additive and diluent, i.e. a carrier or administration vehicle. Further, it is intended to mean an amount sufficient to reduce and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a host. As is appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity. Suitable dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluent. In the methods and use for manufacture of compositions of the invention, a therapeutically effective amount of the active component is provided. A therapeutically effective amount can be determined by the ordinary skilled medical or veterinary worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art. The administration of the pharmaceutically effective dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administrations of subdivided doses at specific intervals. Alternatively, the dose may be provided as a continuous infusion over a prolonged period.
It will be appreciated that the compositions of the invention may be formulated in unit dosage form, i.e. in the form of discrete portions containing a unit dose or a multiple or sub-unit of a unit dose.
Whilst the dosage of the compound used will vary according to the activity of the particular compound and the condition being treated, it may be stated by way of guidance that a dosage selected in the range from 0.1 to 20 mg/kg per body weight per day, particularly in the range from 0.1 to 5 mg/kg of body weight per day, will often be suitable although higher doses than this, for example in the range from 0.1 to 50 mg/kg of body weight per day (or possibly even as high as described in U.S. Pat. No. 4,197,249) may be considered in view of the lower level of toxic side effects obtained with the compounds. This dosage regime may be continued for however many days is appropriate to the patient in question, the daily dosages being divided into several separate administrations if desired. Thus, for example, in the case of conditions such as advanced breast cancer, non-Hodgkin's lymphoma and hepatoma, treatment for one day followed by a repeated dose after an interval, such as 21 days, may be appropriate whilst for the treatment of acute non-lymphocytic leukaemia, treatment over 5 consecutive days may be more suitable.
A fourth aspect of the invention provides a compound according to the first aspect of the invention for use in medicine (clinical and/or veterinary).
A fifth aspect of the invention provides a compound according to the first aspect of the invention for use as a cytotoxin, or a hypoxia activated prodrug thereof.
In one embodiment, the compound is for use in vivo as a cytotoxin, or a hypoxia activated prodrug thereof.
By “hypoxia activated prodrug thereof” we include that the compound is preferentially cytotoxic under, or following exposure to, hypoxic conditions (i.e. exhibits greater cytotoxicity under, or following exposure to, hypoxic conditions). For example, N-oxide compounds of the invention, such as those of formulae V, VI, IX and X, are relatively non-cytotoxic under normoxic conditions but are readily reduced under hypoxic conditions to generate cytotoxic compounds, such as those of formulae III, IV, VII and VIII.
In this context, “hypoxia” may be regarded as an oxygenation level of 4% or lower (or ≤23 mmHg) when measured directly by electrode methods. For example, the level of oxygenation may be lower than 3.0%, 2.5%, 2%, 1.5%, 1% or 0.5 or 0.1%.
It will be appreciated by persons skilled in the art that the hypoxia-induced activation of a compound's cytotoxic activity may be determined either in vitro or in vivo.
For example, cytotoxicity may be determined in vitro at various oxygenation levels measured by direct electrode methods.
Alternatively, the level of oxygenation in a tissue may be measured indirectly, for example using histological sections probed with an enzyme detection assay or by gene expression analysis.
For confirmation of hypoxia-activated cytotoxicity in vivo, oxygenation levels in living tissue may be determined using both the Helzel and OxyLite systems (for example, see Wen et al., 2008, Radiat. Res. 169:67-75).
The results of blood flow and perfusion analyses may also infer the existence of hypoxia in a given tissues. The application of agents that modify blood flow or compromise blood vessel formation would also on first principles be expected to reduce oxygenation in affected tissues.
In particular, the invention provides a compound according to the first aspect of the invention for use in the treatment of cancer in mammals (most notably in humans).
For example, the compound may be for use in the treatment of a cancer selected from the group consisting of bladder cancer, breast cancer, bone cancer (primary and secondary, such as osteosarcoma and Ewings sarcoma), brain cancer (including glioblastoma multiforme and astrocytoma), cervical cancer, choriocarcinoma, colon and rectal cancer, endometrial cancer, eye cancer, gallbladder cancer, gastric cancer, gestational tumours, head and neck cancer, kidney (renal cell) cancer, laryngeal cancer, leukaemias (such as ALL, AML, CLL, CML and hairy cell leukaemias), liver cancer, lung cancer, lymphomas (such as Hodkin's lymphoma and non-Hodkin's lymphoma), melanoma, mesothelioma, mouth cancer, myeloma, nasal and sinus cancers, nasopharyngeal cancer, oesophageal cancer, ovarian cancer, pancreatic cancer, penile cancer, prostate cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, vaginal cancer, vulvar cancer and womb cancer.
In one embodiment, the compound is for use in the treatment of a solid tumour, such as various forms of sarcoma and carcinoma.
The compounds of the invention may be of particular use in the treatment of a tumour that is naturally hypoxic, at least in part (for example, having a median oxygen level of below 3%, e.g. lower than 2.5%, 2%, 1.5%, 1% or 0.5%). An example of such tumours are pancreatic cancer and prostate cancer, both typically exhibiting low oxygen levels and a propensity for malignant progression.
The hypoxia-activated cytotoxicity of the prodrug compounds of the invention allows the cytotoxicity to be targeted to the tumour cells, reducing the risk of damage to healthy cells.
It is believed that hypoxia may play a role in facilitating the malignant progression of certain cancers (for example, see Rudolfsson & Bergh, 2009, Exp. Opin. Ther. Tar. 13:219-225). By exerting a cytotoxic effect preferentially within the regions of tumour hypoxia, the compounds of the invention may be able to target cancer cells that are otherwise resistant to treatment, e.g. by radiotherapy or conventional chemotherapeutic agents. Eradication of such resistant cells may, in turn, lead to a reduction in metastasis.
Thus, in one embodiment, the compounds are for use in the treatment or prevention of metastases (which may arise from the aetiology of the cancer or as a consequence of treatment).
It will be appreciated by persons skilled in the art that the compounds of the invention may be used on their own or in combination with other cancer treatments (such as radiotherapeutic modalities, e.g. radioisotopes and external beam radiation, and chemotherapeutic agents; see below).
In one embodiment, the compounds are for use as a monotherapy (i.e. without any other cancer treatments). However, it will be appreciated that the cancer patient may also be receiving different types of beneficial medication (such as a painkiller, sedative, antidepressant, antibiotic, etc).
However, the compounds of the invention may alternatively be for use in combination with one or more additional cancer treatments. For example, the compounds may be used in combination with one, two, three, four, five or more additional cancer treatments.
By “in combination” we include that the compound is administered to a subject who is receiving one or more additional cancer treatments in the same course of therapy. Thus, the term covers not only the concomitant administration of the compound with one or more additional cancer treatments (either as bolus doses or infusions) but also the temporally separate administration of these cancer treatments. For example, the compound may be administered within a treatment schedule/cycle as defined by the patient's oncologist to include one or more additional cancer treatments, administered either before, concomitantly with or after the compound; for example within ten weeks, nine weeks, eight weeks, seven weeks, six weeks, five weeks, four weeks, three weeks, two week, ten days, one week, five days, four days, three days, two days, one day, 12 hours, 10 hours, 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, 10 minutes or five minutes. Each treatment cycle may be repeated on several occasions, normally up to 6 cycles, but could be more or less than this number depending on the nature of the cancer and its response to treatment.
It will be appreciated by persons skilled in the art that the one or more additional cancer treatments may be chemotherapeutic agents or radiotherapeutic modalities.
In one embodiment, however, the one or more additional cancer treatments comprise or consist of one or more chemotherapeutic and/or radiotherapeutic modality.
Given the hypoxia-activated cytotoxicity of the prodrug compounds of the invention, it is 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. For example, the one or more chemotherapeutic agents and/or radiotherapeutic modalities may be capable of lowering the median oxygen level of the tumour to below 3%, for example below 2.5%, 2%, 1.5%, 1%, 0.5%, 0.4%, 0.3%, 0.2% or below 0.1%.
It will be appreciated by skilled persons that a reduction in tumour oxygenation levels may be achieved by a number of different means, for example by the disruption of established tumour vasculature, prevention of angiogenesis (new blood vessel formation) and/or vasoconstriction.
Suitable cancer treatments may be 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 radiotherapy.
By “steroidal anti-androgens” we include cyproterone acetate.
By “anti-angiogenic agents” we include:
By “vascular disrupting agents” we include small molecules (such as taxanes, taxol, paclitaxel combretastatins, CA4P, Oxi4503, aurostatins, dolostatins, colchine, azacolchicinol, ZD6126I, MMP-activated colchicines, ICT2588, DMXAA, TZT1027 and AVE8062) and biologicals (such as ADEPT, GDEPT and antibody drug-conjugates that target the tumour vasculature).
By “IL8 inhibitors” we include repertaxin.
By “NO synthase inhibitors” we include NG-methyl-1-arginine hydrochloride (546C88; 1-NMMA), NG-nitro-L-arginine (L-NNA), L-nitroarginine methyl ester (L-NAME), LG-nitro-L-arginine (L-NO-Arg) and 7-Nitro-Indazole (7-NI).
By “vasoconstricting agents” we include alpha 1 adrenoceptor agonists (e.g. methoxamine, phenylephrine, oxymetazoline, tetrahydralazine, xylometazoline), alpha 2 adrenoceptor agonists (e.g. clonidine, guanabenz, guanfacine, α-methyldopa) and vasopressin analogues (e.g. arginine vasopressin and triglycyl lysine vasopressin).
By “vasodilating (‘vascular steal’) agents” we include alpha-adrenoceptor antagonists (alpha-blockers), angiotensin converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), beta2-adrenoceptor agonists (β2-agonists), calcium-channel blockers (CCBs), centrally acting sympatholytics, direct acting vasodilators, endothelin receptor antagonists, ganglionic blockers, nitrodilators, phosphodiesterase inhibitors, potassium-channel openers and renin inhibitors.
By “radiotherapy modalities” we include conventional external beam radiation therapy (2DXRT), stereotactic radiosurgery (SRS), stereotactic body radiation therapy (SBRT) and particle therapy such as proton therapy; brachytherapy such as SAVI™, MammoSite™, Contura™, Proxcelan™, TheraSeed™ and I-Seed™; radioisotope therapy such as metaiodobenzylguanidine (MIBG), iodine-131, hormone-bound lutetium-177 and yttrium-90 (peptide receptor radionuclide therapy).
In one preferred embodiment, the one or more cancer treatments is/are non-steroidal anti-androgens, such as flutamide, nilutamide, bicalutamide, finasteride, dutasteride, bexlosteride, izonsteride, turosteride, epristeride and abiraterone.
Thus, in one embodiment, a compound according to the first aspect of the invention is used in combination with bicalutamide in the treatment of cancer, e.g. the prevention or reduction of metastasis.
Thus, in one embodiment, a compound according to the first aspect of the invention is used in combination with cancer chemotherapeutic agents and/or radiotherapeutic modalities and/or methods to reduce or increase the air being breathed by the patients e.g. carbogen (with or without nicotinamide).
A related, sixth aspect of the invention provides the use of a compound of the first aspect of the invention in the preparation of a medicament for treating cancer.
Preferred embodiments of the sixth aspect of the invention are described above in relation to the fifth aspect of the invention.
A seventh aspect of the invention provides a method of treating cancer in a patient comprising administering to the patient a therapeutically effective amount of a compound of the first aspect of the invention.
In one embodiment, the patient is mammalian (e.g. human).
Preferred embodiments of the seventh aspect of the invention are described above in relation to the fifth aspect of the invention.
An eighth aspect of the invention provides the use of a compound of the first aspect of the invention as a marker of the oxygenation level of cells. In particular, such compounds may be used as a cellular hypoxic marker, either in vitro or in vivo.
In one embodiment, the cells are mammalian (e.g. human).
Exposure of the N-oxide forms of the compounds of the invention (such as those of formulae V and VI) to hypoxic cells causes their reduction to the corresponding amine form (such as those of formulae III and IV), which can be readily detected by known means.
The presence of the reduced compound (such as those of formulae III and IV) can be used to detect hypoxic cells in vitro or in vivo. The innate fluorescence properties retained by the reduced compound(s) and the intracellular persistence of the reduced compound(s) are advantageous for the discrimination, quantification and localisation of cells that have been exposed to, or continue to be exposed to hypoxic conditions.
For example, when acting as a cellular marker for hypoxia, the reduced compound (such as those of formulae III and IV) maybe detected using method(s) that identify chemical composition or physical properties that include but are not limited to mass spectrometry, infrared spectroscopy, colorimetry, Raman spectroscopy, nuclear magnetic resonance or positron emission tomography. Affinity capture methods would exploit the high affinity binding potential of the reduced compound to DNA or synthetic polynucleotide sequences.
Optical properties of the reduced compound(s) may be used to detect compound in biological samples and include but are not limited to flow cytometry and microscopy utilising the innate fluorescent properties of the reduced compound. Secondary methods of detection of reduced compound include but are not limited to a combination with other molecular reporter compounds with the reduced compound participating in resonant energy transfer reactions as either an acceptor or donor. Other secondary methods of detection of reduced compound include but are not limited to methods using antibody based methods for molecular detection.
In one embodiment, the compounds of the invention are used to identify hypoxic tumour cells in vivo, which may then be visualised in situ or excised surgically.
In a further embodiment, a compound of the first aspect of the invention is used as a cellular hypoxic marker in combination with a non-deuterated form of a compound of the first aspect of the invention.
By “in combination” in this context this includes that the compounds may be applied to the cells (e.g. administered to a patient) either concomitantly or sequentially (for example, within 24 hours, 12 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 30 minutes, 30 minutes, 10 minutes or less).
Thus, in a preferred embodiment, a compound of formulae IX or X is used as a cellular hypoxic marker (in vivo or in vitro) in combination with a compound as disclosed in U.S. Pat. No. 5,132,327 (for example, AQ4N).
A related, ninth aspect of the invention provides a kit of parts for use in detecting the oxygenation level of cells comprising a compound according to the first aspect of the invention.
Optionally, the kit further comprises a non-deuterated form of a compound according to the first aspect of the invention (such as a compound as disclosed in U.S. Pat. No. 5,132,327, for example AQ4N).
Preferably, the compound(s) is/are provided in a sterile, pyrogen-free form.
It will be appreciated that the kits of the invention may further comprise one or more regents, control samples and/or instructions.
Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figures:
See Example B
See Example B
See Example B
See Example B
See Example B
See Example B
See Example B
See Example
See Example B
See Example C
See Example C
See Example C
See Example C
See Example C
See Example C
See Example C
##STR00009##
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.
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).
1H NMR (DMSO-d6) was clean and consistent with the desired material.
##STR00010##
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.
The residue was diluted with DCM (250 mL) and filtered, washing with copious amounts of DCM. The mother liquors were degassed with N2 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).
1H NMR (400 MHz, d6-DMSO) δ: 4.47 (2H, s) was consistent with the desired material.
##STR00011##
To a stirred suspension of deuterated-d6-dimethylacetonitrile (21.72 g, 172 mmol) in Et2O (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).
1H NMR (400 MHz, CDCl3) δ: 2.76 (2H, t), 2.33 (2H, t)
##STR00012##
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).
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).
The product was analysed by LCMS (m/z 425.3 (M+H)+ (ES+); 423.2 (M−H)− (ES)−, at 0.90 and 1.03 min (product smears on column), 100%.
1H NMR (CDCl3) was clean and consistent with the desired material 1H NMR (400 MHz, CDCl3) δ: 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).
##STR00013##
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.
The product was analysed by LCMS (standard 4 min. method, agilent), m/z 458.2 (M+H)+ (ES+), at 3.07 min, 98.3% purity @ 254 nm. 1H NMR (400 MHz, D2O) δ: 6.73 (2H, br s), 6.43 (2H, br s), 3.76 (4H, br s), 3.58 (4H, br s).
1H NMR (D2O) was consistent with the desired material.
TABLE 1
Dose of
Humidified
Relative
prodrug
oxygenation
pmoles metabolite generated
prodrug
(OCT1002
conditions
per 105 cellsa
reduction to
or AQ4N)
pO2 mm
range
range
metabolite
nM × days
% O2
Hg
AQ4
OCT1001
AQ4
OCT1001
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
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.
(g) Intracellular Accumulation of the OCT1001 Far-Red Fluorescent Chromophore Under Hypoxia is Responsive to OCT1002 Prodrug Dose and Oxygenation Level
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).
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
Day of
Mean p02 ± SD
Significance
Significance
Treatment
Treatment
(mmHg)
(to vehicle)
(to 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/
7
1.996 ± 1.989
<0.05
<0.05
day)
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.
(i) the first is an earlier effective tumour growth inhibition of OCT1002 on the bicalutamide treated tumours compared to AQ4N;
(ii) the second indicates a sustained tumour growth inhibition (indicated by a maintained linear response); that reflects a persistence OCT1002 and tumour growth inhibition.
TABLE 6
Time to 2x start volume
Growth
Treatment
(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
Smith, Paul, Errington, Rachel Jane, Patterson, Laurence, Ogrodzinski, Stefan, McKeown, Stephanie
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