US 20150307441A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2015/0307441 A1 Ogrodzinski et al. (43) Pub. Date: Oct. 29, 2015

(54) NEW COMPOUNDS AND USES THEREOF A 6LX3L/277 (2006.01) A63L/36 (2006.01) (71) Applicant: Biostatus Limited. Shepshed, A6II 45/06 (2006.01) Leicestershire (GB) (52) U.S. Cl. CPC ...... C07C 225/36 (2013.01); A61 K3I/136 (72) Inventors: Stefan Ogrodzinski, Shepshed, (2013.01); A61K 45/06 (2013.01); A61 K Leicestershire (GB); Paul Smith, 3I/277 (2013.01); G0IN 31/225 (2013.01) Shepshed, Leicestershire (GB); Stephanie McKeown, Shepshed, Leicestershire (GB); Laurence (57) ABSTRACT Patterson, Shepshed, Leicestershire (GB); Rachel Jane Errington, An anthraquinone compound of formula I (Such as the com Shepshed, Leicestershire (GB) pounds of formulae II to X) and processes for making the (73) Assignee: Biostatus Limited. Shepshed, same are provided. Pharmaceutical compositions for use in the treatment of cancer, optionally in combination with an Leicestershire (GB) agent capable of reducing the level of oxygenation of a (21) Appl. No.: 14/420,184 tumour, are also provided. Additionally, an option for com bination with chemotherapeutic and radiotherapeutic modali (22) PCT Filed: Aug. 7, 2013 ties to enhance overall tumour cell kill is provided. Methods for the detection of cellular hypoxia, both in vivo and in vitro, (86). PCT No.: PCT/GB2O13/O52106 are additionally provided. S371 (c)(1), (2) Date: Feb. 6, 2015 Formula I O (30) Foreign Application Priority Data X4 X e sas Aug. 8, 2012 (GB) ...... 12141693 e as

Publication Classification S. 1. X3 X2 (51) Int. Cl. O CD7C 225/36 (2006.01) GOIN3L/22 (2006.01) Patent Application Publication Oct. 29, 2015 Sheet 1 of 20 US 2015/0307441 A1

FGRE AQ4 x 4 days air OC100 x 4 days air

Equivalent arrest of cells in G2-M as a function of drug dose in air Patent Application Publication Oct. 29, 2015 Sheet 2 of 20 US 2015/0307441 A1

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O 50 OO Drug dose (ny x 4 days) Patent Application Publication Oct. 29, 2015 Sheet 4 of 20 US 2015/0307441 A1

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FGRE 5(A)

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O 5 OO Drug conc. (nM) Patent Application Publication Oct. 29, 2015 Sheet 6 of 20 US 2015/0307441 A1

FGRESS)

-- SU---4 16 OCOO2 -i-Su-DH-4 1% AC4N N. --S-OH-4.3%OCTOO2 N. - & - S -OH,-43% y AC4N -Q- SJ-3H-4 air OCI 10O2 -8- S-O-3-4 air i AGN

.2 SO OO Drug conc. (nw) Patent Application Publication Oct. 29, 2015 Sheet 7 of 20 US 2015/0307441 A1

GRES

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200 s

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C 1% oxygen OCT1002

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5 ...--A non-Oxia 9

O 50 OO Drug dose (n\ x 4 days) Patent Application Publication Oct. 29, 2015 Sheet 8 of 20 US 2015/0307441 A1

FGRE

Air Control OCOO x 4. days, a

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far-red fluorescence trans ission

Fluorescence intensity (ch, no. Ex633 nm, AEm >695 nm) Patent Application Publication Oct. 29, 2015 Sheet 11 of 20 US 2015/0307441 A1

FGRE (

2. one vehicle only a8a Bicalutamide $2mg/kgiday) 18

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Patent Application Publication Oct. 29, 2015 Sheet 13 of 20 US 2015/0307441 A1

FGRE 2{A}

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3 Patent Application Publication Oct. 29, 2015 Sheet 16 of 20 US 2015/0307441 A1

FGURE 3(8)

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Patent Application Publication Oct. 29, 2015 Sheet 19 of 20 US 2015/0307441 A1

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Patent Application Publication Oct. 29, 2015 Sheet 20 of 20 US 2015/0307441 A1

F&SR is

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Treatment groups US 2015/0307441 A1 Oct. 29, 2015

NEW COMPOUNDS AND USES THEREOF distant tissues. The development of more malignant meta static tumours is often the precursor to a more significant CROSS REFERENCE TO RELATED disease-related morbidity and the death of the patient. APPLICATION 0005. An attractive approach is the use of a hypoxia acti 0001. The present application claims priority to Great vated prodrug that is non-toxic towards adequately oxygen Britain Patent Application No. 1214169.3, filed Aug. 8, 2012, ated cells found in Systemic tissues, but becomes activated or incorporated herein in its entirety. converted to a cytotoxic form under reduced oxygenation conditions. N-oxide derivatives of cytotoxic alkylaminoan TECHNICAL FIELD thraquinones provide anthraquinone pro-drugs that show almost no cytotoxicity. Importantly these prodrugs are 0002 The present invention relates to novel capable of being converted in vivo under the anaerobic/hy anthraquinone compounds and uses of the same, for example poxic conditions found within neoplastic tissue. Specificity in the treatment of cancer. for the tumour is ensured since systemic tissues, except for tumours, almost never experience oxygen levels low enough BACKGROUND to facilitate the production of the cytotoxic drug. 0003. The therapeutic advantage of an anticancer drug 0006. The anthraquinone N-oxide AQ4N (CAS#136470 depends primarily on the extent to which the agent shows 65-0) is a prodrug that is selectively bioreduced to AQ4, a selective activity for tumour cells and the limiting toxicity potent DNA topoisomerase II inhibitor, in hypoxic tumour towards non-target tissues. Frequently the poor quality of the cells. Previous publications have taught the fundamental vasculature within the growing tumour mass compromises properties and in-vitro I in-vivo characteristics of the prodrug the delivery of drugs, nutrients and oxygen. It is recognised AQ4N (for example, see U.S. Pat. No. 5,132.327). that tumours can have significantly lower median oxygen 0007. The invention seeks to address the need for levels (approximately 1% oxygen; pO2 7.5 mmHg) com improved cancer treatments by providing novel pared to normal tissues (~5.5% oxygen; 42 mmHg) (Sum anthraquinone compounds with a combination of preferable marised from data presented by Brown and Wilson, 2004). In pharmacological and hypoxia-sensing properties. addition, oxygenation levels can vary throughout the tumour due to intermittent opening and closing of tumour blood SUMMARY vessels; poor vascularisation, especially in the tumour core, contributes to oxygen levels often being below 0.1% oxygen 0008. The first aspect of the invention provides a com (1 mm Hg). Tumour cells experiencing varying degrees of pound of Formula I hypoxia, relative to normally perfused tissues, can compro mise treatment effectiveness and contribute to the malig Formula I nancy. Hypoxia-selective agents (e.g. bioreductive drugs) O comprise one class of agents that can be used to target tumour X4 X cells in very low oxygen environments by virtue of a selective NS activation to a cytotoxic form under reduced oxygenation, e as addressing the problems of non-targettissue toxicity, hypoxic cell drug resistance and cancer progression. Sa 1. 0004 Poor oxygenation results in a relative state of X3 X2 hypoxia when compared with normoxic conditions in which O oxygenation has not been compromised. Poor oxygenation within tumours can modify the responses to treatment wherein X, X, X and X are each independently selected modalities and contribute to cancer progression. Cells in Such from the group consisting of hydrogen, hydroxy, halogeno, hypoxic areas are particularly resistant to treatment with amino, Calkoxy, Cisalkanoyloxy, —NH-A-NHR, —NH many of the conventionally used anticancer drugs; this is A-NR'R" and NH-A-N(O) R'R" attributed to poor drug delivery and/or lack of intrinsic wherein A is an alkylene group with a chain length of at least tumour cell sensitivity of viable but quiescent cells. Radio two carbon atoms (between NH and NHR or N(O)RR"). therapy is also less effective at very low oxygen levels since wherein R, R and R" are each independently selected from the cytotoxicity of ionising radiation is enhanced by the pres Calkyl groups and Ca hydroxyalkyl and C dihydroxy ence of oxygen (Radiobiology For The Radiologist, Hall E.J. alkyl groups in which the carbon atom attached to the nitro Giaccia A. J. Lippincott Williams & Wilkins, (2005)). Recent gen atom does not carry a hydroxy group and no carbonatom evidence shows that tumour cells can adapt to low oxygen is substituted by two hydroxy groups, or wherein R and R" conditions and change the pharmacodynamic responses to together are a C- alkylene group which with the nitrogen anticancer agents through the induction of active cellular atom to which R" and R" are attached forms a heterocyclic protective mechanisms (Vaupel and Mayer 2007, Cancer group having 3 to 7 atoms in the ring, Metastasis Rev 26(2): 225-239). Additionally, it is recognized that tumour cells that survive hypoxic stress often show a wherein at least one of X, X, X and X is selected from the more malignant metastatic phenotype (Vaupel P. Metabolic group consisting of deuterated forms of —NH-A-NHR, microenvironment of tumor cells: a key factor in malignant NH-A-NR'R'' and NH-A-N(O) R'R". progression, Exp Oncol 2010: 32, 125-127); this has signifi 0009. Thus, the invention provides novel deuterated cant consequences for the patient. Following treatment with anthraquinone compounds. modalities that target predominantly the better-oxygenated 0010. By “deuterated we include that the compound cells, the stress-resistant hypoxic cells often repopulate the comprises at least one atom of deuterium or heavy hydrogen tumour with cells that have an enhanced potential to spread to (i.e. Dor H). It will be appreciated by persons skilled in the US 2015/0307441 A1 Oct. 29, 2015 art that the compound may be partially (i.e. selectively) or fully deuterated (i.e. containing hydrogen present only in the form of deuterium). 0011. By “selectively, in this context, we mean that some but not all conventional H hydrogenatoms are replaced with deuterium. For example, one or more of Substituent groups X, X, X and X may be deuterated while the central anthraquinone ring may be free of deuterium. 0012. 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 0040. In one embodiment of the first aspect of the inven (O)R'R'' at positions X, X, X and/or X. Within each such tion, the compound is Substituted at ring positions 1, 4, 5 and substituent group, it will be appreciated that A. R. R' and R" 8, in accordance with Formula II: may be fully deuterated (i.e. thus containing no 'H) or may be partially deuterated. 0013. In a preferred embodiment, the compound is deu Formula II terated only within one or more of the terminal groups R. R' and R". For example, R. R' and/or R" may represent: O014 CD: (0.015 CHCD: 0016 - CDCD: 10017 - CDCHCD; and 0018 – CDCDCDCD. X O X 0019. The term “Calkyl is intended to include linear or branched alkyl groups comprising between one and four car 0041. In one embodiment, X, X, X and X are each bons. Preferred alkyl groups which R, R and/or R" may separately selected from the group consisting of hydrogen, independently represent include C and C alkyl. hydroxy, NH-A-NHR, NH-A-NR'R", NH-A-N(O) 0020. The term “lower alkylene' is to be construed RR" and deuterated forms thereof. accordingly. 0042. In one embodiment, X, X, X and X are each 0021. The terms “Ca hydroxyalkyl and “C dihy separately selected from the group consisting of hydroxy, droxyalkyl are intended to include linear or branched alkyl - NH-A-NR'R", NH-A-N(O)RR" and deuterated forms groups comprising between two and four carbons, to which thereof. are attached one or two hydroxy groups, respectively. For 0043. In one embodiment, X and X are both hydroxy and example, R. R' and/or R" may independently represent: X and X are both —NH-A-N(O)R'R' or deuterated forms 0022 - CHCH-OH thereof. 0023 CHCH(OH)CH 0044) In one embodiment, X and X are both hydroxy and (0024 CHCHCH(OH)CHOH X and X are both NH-A-NR'R' or deuterated forms thereof. 0025. The term “Calkoxy” is intended to include linear 0045. In one embodiment, A is unbranched. For example, or branched C alkyl groups bound to the core A may be ethylene. anthraquinone (anthracene-9,10-dione) ring via oxygen. For 0046. In one embodiment, R, R and R" are each indepen example, R. R' and/or R" may independently represent: dently selected from the group consisting of —CH, (0026 – OCH, —CHCH —CH2CHCH —CHCH-OH, 0027 – OCHCH —CHCHCH-OH, -CH(CH,)CHOH, 0028 OCHCHCH - CHCHOHCH-OH and deuterated forms thereof. (0029 OCHCHCHCH, 0047. In one embodiment, one or two of X, X, X and X 0030 The term “Cs alkanoyloxy” is intended to include are independently selected from the group consisting of linear or branched Cls alkanoyl groups bound to the core —NH-(CH), N(O)(CH), NH-(CH2). N(O) anthraquinone (anthracene-9,10-dione) ring via oxygen. For (CH)CHs —NH-(CH), N(O)(CH), NH-(CH) example, R. R' and/or R" may independently represent: N(O) (CHCH-OH), - NH-(CH), N(O) (CHCHCH-OH), -NH-(CH), N(O)CH(CH)OH, 0031 - O(O)CCH, - NH-(CH), N(O)(CHCHOHCH-OH), and deuter 0032 - O(O)CCHCH ated forms thereof. 0033 – O(O)CCHCHCH 0048. In one embodiment, one or two of X, X, X andX, 0034 - O(O)CCHCHCHCH, are independently selected from the group consisting of 0035 - O(O)CCHCH-CH(CH)CH, —NH-(CH), N(CH), NH-(CH), N(CH) 0036. The term “hydroxy” is intended to represent –OH. CHs —NH-(CH2): NCCHs), —NH-(CH2) N 0037. The term “halogeno’ is intended to represent any (CHCH-OH), -NH-(CH), N(CH2CHCH-OH), halogen group. Such as —Br. —Cl and —F. NH-(CH), NCH(CH)OH, —NH-(CH), N 0038. The term “amino' is intended to include primary (CHCHOHCH-OH), and deuterated forms thereof. amine groups, such as —NH. 0049. In one embodiment, the compound of the invention 0039. It will be appreciated by persons skilled in the art comprises one group - NH-A-N(O)RR" and one group that the anthraquinone ring of the compounds may be substi —NH-A-NHR, the NH-A-NHR group being selected from tuted by X, X, X and X at any of ring positions 1, 2, 3, 4, - NH-(CH), NHCH, —NH (CH) NHCHs. 5, 6, 7 or 8: - NH-(CH), NHCHCH-OH, —NH-(CH2) -

US 2015/0307441 A1 Oct. 29, 2015

0084. By “prodrug, in this context, is included com -continued pounds which may readily be converted in vivo to a com Formula VIII pound of Formula III or IV. In one embodiment, the conver sion is triggered by the prodrug entering an hypoxic CD3 environment, such as a Solid tumour. N OH O N1,N1 YCD, 0085 Examples of suitable prodrugs include N-oxide derivatives of the compounds of Formula III or IV. I0086 Thus, in one embodiment, the prodrug is a com pound of Formula V or VI:

CPin-N-NH O OH Formula V CD3 CD3 - O Y o HN1)-1 CD or a prodrug thereof. I0088. The present invention relates to novel anthraquinone compounds and uses of the same, for example in the treatment of cancer. I0089. The present invention relates to novel CD3 anthraquinone compounds and uses of the same, for example Y O HN n1N - O in the treatment of cancer. 0090. In a further preferred embodiment, the compound is CD3 prodrug of Formula IX or X: Formula VI CD3 Formula IX O P Y o HN1\1 N-O CD3 oH Q HN1N1 CD

f NH O Y OH O HN P o-r- n1n - O CD CD3 Formula X wherein Y are each independently selected from the group CD3 consisting of hydrogen, hydroxy, halogeno, amino, Ca alkoxy and C2-s alkanoxy. oH Q HN1N1 O 0087. In one preferred embodiment, the compound is of CD Formula VII or VIII:

Formula VII o-r-f NH O OH CD3

(0091. 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. 'H), provided that the compound comprises at least one deuterium atom. For example, one, two, three or four of the methyl groups may be —CH, —CHD or —CHD. In one embodiment, the methyl groups in the compound are either —CH or —CDs. US 2015/0307441 A1 Oct. 29, 2015

0092. It will be further appreciated by skilled persons that (0098. The leuco derivatives themselves are obtainable by certain compounds of formulae I to X above may be counter heat treatment of the corresponding fully aromatic 1.4-dihy balanced by counter-anions. Exemplary counter-anions droxyanthracene-9,10-dione, conveniently by heating at include, but are not limited to, halides (e.g. fluoride, chloride above 90° C. for 1 hour or more in a stream of nitrogen and, and bromide), Sulfates (e.g. decylsulfate), nitrates, perchlor if necessary, in the presence of a suitable reducing agent Such ates, Sulfonates (e.g. methane Sulfonate) and trifluoroacetate. as sodium dithionite or zinc dust. Various anthracene-9,10 Other suitable counter-anions will be well known to persons diones, particularly hydroxyanthracene-9,10-diones, are skilled in the art. Thus, pharmaceutically, and/or veterinarily, commercially available and various syntheses for Such com acceptable derivatives of the compounds of formulae I to X, pounds are also reported in the literature. One suitable pro Such as salts and Solvates, are also included within the scope cedure for their preparation involves the reaction of an appro of the invention. Salts which may be mentioned include: acid priately substituted phthalic anhydride with hydroquinone in addition salts, for example, salts formed with inorganic acids the presence of aluminium chloride and Sodium hydroxide at Such as hydrochloric, hydrobromic, Sulfuric and phosphoric 180° C. for one hour or more. Anthracene-9,10-diones con acid, with carboxylic acids or with organo-Sulfonic acids; taining one form of Substituent group can be modified to base addition salts; metal salts formed with bases, for provide otherforms of substituent group so that, for example, example, the Sodium and potassium salts. a dione containing anamino group can be treated with sodium 0093. In one embodiment, the compound is in the form of hydroxide/dithionite to yield the corresponding hydroxy sub a halide salt, for example a chloride salt. stituted compound. 0094. It will be further appreciated by skilled persons that (0099. Other suitable procedures for the preparation of certain compounds of formulae I to X may exhibit tautomer intermediates for oxidation to the N-oxide compounds of the ism. All tautomeric forms and mixtures thereof are included invention include the reaction of the appropriate chloro or within the scope of the invention. fluoro substituted anthracene-9,10-dione with the appropriate 0095 Compounds of formulae I to X may also contain one amine R"RN--A--NH2, for example by heating with a excess or more asymmetric carbon atoms and may therefore exhibit of the amine at its reflux temperature for one or more hours. optical and/or diastereoisomerism. Diastereoisomers may be Certain of these chloro- and fluoro anthracene-9,10-diones separated using conventional techniques, e.g. chromatogra are known and various syntheses for Such compounds are also phy or fractional crystallisation. The various stereoisomers reported in the literature. Thus, for example, a KF-NaF-me may be isolated by separation of a racemic or other mixture of diated conversion of 3,6-dichlorophthalic anhydride to 3.6- the compounds using conventional, e.g. fractional crystalli difluorophthalic anhydride as a precursor to making 1,4-dif sation or HPLC, techniques. Alternatively, the desired optical luoro-4,8-dihydroxyanthracene-9,10-dione (see Lee & isomers may be made by reaction of the appropriate optically Denny, 1999, J. Chem. Soc., Perkin Trans. 1:2755-2758. active starting materials under conditions which will not Additionally, for example, 1,5-dichloro-4,8-dihydroxyan cause racemisation or epimerisation, or by derivatisation, for thracene-9,10-dione may be prepared by selective chlorina example with a homochiral acid followed by separation of the tion of 1,4,5,8-tetrahydroxyanthracene-9,10-dione using a diastereomeric esters by conventional means (e.g. HPLC, stoichiometric amount of sulphuryl chloride and controlled chromatography over silica). All stereoisomers are included temperature. This precursor may then be used to prepare an within the scope of the invention. intermediate having groups —NH-A-NR'R'' at the 1 and 5 0096 Various routes are available for the synthesis of the positions and hydroxy groups at the 4 and 8 positions, the compounds of the invention. One very convenient procedure hydroxy groups conveniently being protected during the reac for the preparation of compounds having a group —NH-A- tion with the amine R"RN-A-NH. A similar approach is NR'R'' at the 1 and 4 positions uses the appropriately substi suitable for the preparation of other chlorohydroxyan tuted 2,3-dihydro(leuco)-1,4-dihydroxyanthracene-9,10-di thracene-9,10-dione intermediates. one which is condensed with the appropriate amine R"R"N-- 0100 Where the compound of the invention contains one A--NH2, the 1.4 positions being activated in the leuco or more groups —NH-A-NHR in addition to the one or more compound for reaction with the amine. Such a condensation groups —N-A-NR'R'" the compound may conveniently be may conveniently be effected at a temperature in a range of produced by reacting a suitable precursor as discussed above about 25° C. or 35° C. to 50° C. or 60° C. for one or more with a mixture of amines RN--A--NH. Sub.2 and R'R''N--A-- hours using a solvent such as methanol, ethanol, water, dim NH.sub.2, the resultant mixture of products then being sepa ethylformamide, 2-methoxyethanol, acetonitrile, nitroben rated, for example by chromatography. Thus, for example, Zene, N.N.N'N'-tetra-methylenediamine or mixtures thereof. 2,3-dihydro(leuco)-1,4-dihydroxyanthracene-9,10-dione on In some instances a higher temperature and shorter reaction reaction with a mixture of 2-(2-hydroxyethylamino)ethy time may be appropriate, for example with the compounds lamine and 2-(diethylamino)ethylamine will yield a mixture containing cyclic groups NR'R''. The leuco derivative is then of 1,4-bis(2-(diethylamino)-ethylaminoanthracene-9,10 oxidized to the fully aromatic anthracene-9,10-dione, conve dione, 1,4-bis(2-(2-hydroxyethyl-amino)-ethylamino-an niently using air oxidation or oxidation with hydrogen per thracene-9,10-dione and 1-(2-(diethylamino)ethylamino)- oxide, chloranil, Sodium perborate or manganese dioxide. 4-2-(2-hydroxyethylamino)-ethylaminoanthracene-9, 0097 Although leuco compounds are primarily of interest 10-dione from which the last mentioned compound may be for the preparation of compounds substituted by two —NH separated, for example by chromatography. On oxidation, A-NHR'R" groups, it is possible to use them to prepare com only the tertiary nitrogen atom of the 2-(diethylamino) pounds containing more than two Such groups. Thus, by using ethyl)amino group will be converted to N-oxide form. 2,3-dihydro(leuco)-1,4,5,8-tetrahydroxyanthracene-9,10-di 0101. Where one or more substituent groups is present it one and a large excess of an amine —NH-A-NHR'R'' an may be appropriate, depending on the route of synthesis, to 8-hydroxyanthracene-9,10-dione having three groups —NH have these present throughout in their final form or to gener A-NHR'R'' at the 1.4 and 5 positions may be prepared. ate the desired groups at a later stage in the synthesis. Ether US 2015/0307441 A1 Oct. 29, 2015

and ester groups X may of course readily be prepared by glycol, ethanol or oils (such as safflower oil, corn oil, peanut modification of hydroxy groups according to known proce oil, cottonseed oil or sesame oil). dures, precursors containing a hydroxy group X more often 0111. The term “adjuvant” is intended to mean any com being described in the literature than those containing a cor pound added to the formulation to increase the biological responding ether or ester Substituent. effect of the compound of the invention. The adjuvant may be 0102. It will be appreciated, however, that various alterna one or more of zinc, copper or silver salts with different tive methods for the preparation of the compounds and inter anions, for example, but not limited to fluoride, chloride, mediates therefor may be used as will be apparent in particu bromide, iodide, thiocyanate, Sulfite, hydroxide, phosphate, lar from the literature relating to such intermediates. Further carbonate, lactate, glycolate, citrate, borate, tartrate, and details of the preparation of intermediates for the preparation acetates of different acyl composition. The adjuvant may also of the compounds of the present invention are to be found in be cationic polymers such as cationic cellulose ethers, cat U.S. Pat. No. 4,197,249 and GB 2.004,293B (the disclosures ionic cellulose esters, deacetylated hyaluronic acid, chitosan, of which are incorporated herein by reference). cationic dendrimers, cationic synthetic polymers such as poly 0103) Thus, a second aspect of the invention provides a (vinyl imidazole), and cationic polypeptides such as polyhis process for making a compound according to the first aspect tidine, polylysine, polyarginine, and peptides containing of the invention comprising reacting an anthracene-9,10-di these amino acids. one with a deuterated alkylenediamine under conditions Suit 0112 The excipient may be one or more of carbohydrates, able for the production of an alkylaminoalkylaminoan polymers, lipids and minerals. Examples of carbohydrates thraquinone. include lactose, glucose. Sucrose, mannitol, and cyclodex 0104 Optionally, the process further comprises the step of trines, which are added to the composition, e.g., for facilitat reacting the alkylamino-alkylaminoanthraquinone with a inglyophilisation. Examples of polymers are starch, cellulose monoperoxyphthalate to under conditions suitable for the ethers, cellulose carboxymethylcellulose, hydroxypropylm production of an N-oxide derivative of the alkylaminoalky ethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl laminoanthraquinone. cellulose, alginates, carageenans, hyaluronic acid and deriva 0105. In one embodiment, the process comprises reacting tives thereof, polyacrylic acid, polysulphonate, polyethyl 1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione, 281-005 englycol/polyethylene oxide, polyethyleneoxide/polypropy with deuterated N,N-dimethylethylene-diamine under con lene oxide copolymers, polyvinylalcohol/polyvinylacetate of ditions suitable for the production of 1,4-bis-2-(deuterated different degree of hydrolysis, and polyvinylpyrrolidone, all d6-dimethylamino)ethylamino)-5,8-dihydroxyanthracene of different molecular weight, which are added to the com 9,10-dione. position, e.g., for viscosity control, for achieving bioadhe 0106. In a further embodiment, the process comprises the Sion, or for protecting the lipid from chemical and proteolytic step of reacting the 1,4-bis-2-(deuterated-d6-dimethy degradation. Examples of lipids are fatty acids, phospholip lamino)ethylamino)-5,8-dihydroxyanthracene-9,10-dione ids, mono-, di-, and triglycerides, ceramides, sphingolipids with magnesium monoperoxyphthalate under conditions and glycolipids, all of different acyl chain length and satura suitable for the production of 1,4-bis-(2-(deuterated-d6 tion, egg lecithin, Soy lecithin, hydrogenated egg and Soy dimethylamino-N-oxide)ethylamino)-5,8-dihydroxy-an lecithin, which are added to the composition for reasons thracene-9,10-dione. similar to those for polymers. Examples of minerals are talc, 0107. A third aspect of the invention provides a pharma magnesium oxide, Zinc oxide and titanium oxide, which are ceutical composition comprising a compound according to added to the composition to obtain benefits such as reduction the first aspect of the invention together with pharmaceuti of liquid accumulation or advantageous pigment properties. cally acceptable buffer, diluent, carrier, adjuvant or excipient. 0113. The compounds of the invention may beformulated 0108) By “pharmaceutically acceptable' we include a into any type of pharmaceutical composition known in the art non-toxic material that does not decrease the therapeutic to be suitable for the delivery thereof. effectiveness of the compound of the invention. Such phar 0114. In one preferred embodiment, the pharmaceutical maceutically acceptable buffers, carriers or excipients are compositions are administered parenterally, for example, well-known in the art (see Remington's Pharmaceutical Sci intravenously, intracerebroventricularly, intraarticularly, ences, 18th edition, A. R Gennaro, Ed., Mack Publishing intra-arterially, intraperitoneally, intrathecally, intraventricu Company (1990) and handbook of Pharmaceutical Excipi larly, intrasternally, intracranially, intramuscularly or Subcu ents, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000), taneously, or they may be administered by infusion tech the disclosures of which are incorporated herein by refer niques. The pharmaceutical compositions may also ence). administered intra-tumourally and/or peri-tumourally. 0109 The term “buffer is intended to mean an aqueous 0115 Such pharmaceutical compositions are conve Solution containing an acid-base mixture with the purpose of niently used in the form of a sterile aqueous Solution which stabilising pH. Examples of buffers are Trizma, Bicine, Tri may contain other Substances, for example, enough salts or cine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, glucose to make the Solution isotonic with blood. The aque phosphate, carbonate, acetate, citrate, glycolate, lactate, ous solutions should be suitably buffered (preferably to a pH borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, of from 3 to 9), if necessary. The preparation of suitable CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, parenteral formulations under Sterile conditions is readily glycine, HEPPSO, imidazole, imidazolelactic acid, PIPES, accomplished by standard pharmaceutical techniques well SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES. known to those skilled in the art. 0110. The term "diluent is intended to mean an aqueous 0116 Formulations suitable for parenteral administration or non-aqueous solution with the purpose of diluting the include aqueous and non-aqueous sterile injection solutions agent in the pharmaceutical preparation. The diluent may be which may contain anti-oxidants, buffers, bacteriostats and one or more of Saline, water, polyethylene glycol, propylene solutes which render the formulation isotonic with the blood US 2015/0307441 A1 Oct. 29, 2015

of the intended recipient; and aqueous and non-aqueous ster intramuscular), topical, ocular, nasal, pulmonar, buccal, oral, ille Suspensions which may include Suspending agents and parenteral, vaginal and rectal. Also administration from thickening agents. The formulations may be presented in implants is possible. unit-dose or multi-dose containers, for example sealed I0123. Alternatively, the pharmaceutical compositions ampoules and vials, and may be stored in a freeze-dried may be administered intranasally or by inhalation (for (lyophilised) condition requiring only the addition of the example, in the form of an aerosol spray presentation from a sterile liquid carrier, for example water for injections, imme pressurised container, pump, spray or nebuliser with the use diately prior to use. Extemporaneous injection solutions and of a suitable propellant. Such as dichlorodifluoromethane, Suspensions may be prepared from sterile powders, granules trichlorofluoro-methane, dichlorotetrafluoro-ethane, a and tablets of the kind previously described. hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 0117. In a further embodiment, the pharmaceutical com 134A3 or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA3), positions of the invention may be in the form of a liposome, in carbon dioxide or other Suitable gas). In the case of a pres which the agent is combined, in addition to other pharmaceu Surised aerosol, the dosage unit may be determined by pro tically acceptable carriers, with amphipathic agents such as viding a valve to deliver a metered amount. The pressurised lipids, which exist in aggregated forms as micelles, insoluble container, pump, spray or nebuliser may contain a solution or monolayers and liquid crystals. Suitable lipids for liposomal Suspension of the active polypeptide, e.g. using a mixture of formulation include, without limitation, monoglycerides, ethanol and the propellant as the solvent, which may addi diglycerides, sulfatides, lysolecithin, phospholipids, Saponin, tionally contain a lubricant, e.g. Sorbitan trioleate. Capsules bile acids, and the like. Suitable lipids also include the lipids and cartridges (made, for example, from gelatin) for use in an above modified by poly(ethylene glycol) in the polar head inhaler or insufflator may be formulated to contain a powder group for prolonging bloodstream circulation time. Prepara mix of a compound of the invention and a suitable powder tion of Such liposomal formulations is can be found in for base Such as lactose or starch. example U.S. Pat. No. 4,235,871, the disclosures of which are 0.124. The pharmaceutical compositions will be adminis incorporated herein by reference. tered to a patient in a pharmaceutically effective dose. A therapeutically effective amount, or 'effective amount, or 0118. The pharmaceutical compositions of the invention therapeutically effective’, as used herein, refers to that may also be in the form of biodegradable microspheres. Ali amount which provides a therapeutic effect for a given con phatic polyesters, such as poly(lactic acid) (PLA), poly(gly dition and administration regimen. This is a predetermined colic acid) (PGA), copolymers of PLA and PGA (PLGA) or quantity of active material calculated to produce a desired poly(carprolactone) (PCL), and polyanhydrides have been therapeutic effect in association with the required additive widely used as biodegradable polymers in the production of and diluent, i.e. a carrier or administration vehicle. Further, it microspheres. Preparations of Such microspheres can be is intended to mean an amount Sufficient to reduce and most found in U.S. Pat. No. 5,851,451 and in EP O 213 303, the preferably prevent, a clinically significant deficit in the activ disclosures of which are incorporated herein by reference. ity, function and response of the host. Alternatively, a thera 0119. In a further embodiment, the pharmaceutical com peutically effective amount is Sufficient to cause an improve positions of the invention are provided in the form of polymer ment in a clinically significant condition in a host. As is gels, where polymers such as starch, cellulose ethers, cellu appreciated by those skilled in the art, the amount of a com lose carboxymethylcellulose, hydroxypropylmethyl cellu pound may vary depending on its specific activity. Suitable lose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, dosage amounts may contain a predetermined quantity of alginates, carageenans, hyaluronic acid and derivatives active composition calculated to produce the desired thera thereof, polyacrylic acid, polyvinyl imidazole, polysulpho peutic effect in association with the required diluent. In the nate, polyethylenglycol/polyethylene oxide, polyethyleneox methods and use for manufacture of compositions of the ide/polypropylene oxide copolymers, polyvinylalcohol/ invention, a therapeutically effective amount of the active polyvinylacetate of different degree of hydrolysis, and component is provided. A therapeutically effective amount polyvinylpyrrolidone are used for thickening of the Solution can be determined by the ordinary skilled medical or veteri containing the agent. The polymers may also comprise gela nary worker based on patient characteristics, such as age, tin or collagen. weight, sex, condition, complications, other diseases, etc., as 0120 Alternatively, the compounds may simply be dis is well known in the art. The administration of the pharma Solved in saline, water, polyethylene glycol, propylene gly ceutically effective dose can be carried out both by single col, ethanol or oils (such as safflower oil, corn oil, peanut oil, administration in the form of an individual dose unit or else cottonseed oil or sesame oil), tragacanth gum, and/or various several Smaller dose units and also by multiple administra buffers. tions of subdivided doses at specific intervals. Alternatively, the dose may be provided as a continuous infusion over a 0121. It will be appreciated that the pharmaceutical com prolonged period. positions of the invention may include ions and a defined pH 0.125. It will be appreciated that the compositions of the for potentiation of action of the active agent. Additionally, the invention may be formulated in unit dosage form, i.e. in the compositions may be subjected to conventional pharmaceu form of discrete portions containing a unit dose or a multiple tical operations such as sterilisation and/or may contain con or sub-unit of a unit dose. ventional adjuvants such as preservatives, stabilisers, wetting 0.126 Whilst the dosage of the compound used will vary agents, emulsifiers, buffers, fillers, etc. according to the activity of the particular compound and the 0122 The pharmaceutical compositions according to the condition being treated, it may be stated by way of guidance invention may be administered via any suitable route known that a dosage selected in the range from 0.1 to 20 mg/kg per to those skilled in the art. Thus, possible routes of adminis body weight per day, particularly in the range from 0.1 to 5 tration include parenteral (intravenous, Subcutaneous, and mg/kg of body weight per day, will often be suitable although US 2015/0307441 A1 Oct. 29, 2015 higher doses than this, for example in the range from 0.1 to 50 gastric cancer, gestational tumours, head and neck cancer, mg/kg of body weight per day (or possibly even as high as kidney (renal cell) cancer, laryngeal cancer, leukaemias (Such described in U.S. Pat. No. 4,197,249) may be considered in as ALL, AML, CLL, CML and hairy cell leukaemias), liver view of the lower level of toxic side effects obtained with the cancer, lung cancer, lymphomas (such as Hodkin's lym compounds. This dosage regime may be continued for how phoma and non-Hodkin's lymphoma), melanoma, mesothe ever many days is appropriate to the patient in question, the lioma, mouth cancer, myeloma, nasal and sinus cancers, daily dosages being divided into several separate administra nasopharyngeal cancer, oesophageal cancer, ovarian cancer, tions if desired. Thus, for example, in the case of conditions pancreatic cancer, penile cancer, prostate cancer, stomach Such as advanced breast cancer, non-Hodgkin’s lymphyoma cancer, testicular cancer, thyroid cancer, uterine cancer, vagi and hepatoma, treatment for one day followed by a repeated nal cancer, Vulvar cancer and womb cancer. dose after an interval. Such as 21 days, may be appropriate 0.139. In one embodiment, the compound is for use in the whilst for the treatment of acute non-lymphocytic leukaemia, treatment of a solid tumour, Such as various forms of sarcoma treatment over 5 consecutive days may be more Suitable. and carcinoma. 0127. A fourth aspect of the invention provides a com 0140. The compounds of the invention may be of particu pound according to the first aspect of the invention for use in lar use in the treatment of a tumour that is naturally hypoxic, medicine (clinical and/or veterinary). at least in part (for example, having a median oxygen level of 0128. A fifth aspect of the invention provides a compound below 3%, e.g. lower than 2.5%, 2%, 1.5%, 1% or 0.5%). An according to the first aspect of the invention for use as a example of Such tumours are pancreatic cancer and prostate cytotoxin, or a hypoxia activated prodrug thereof. cancer, both typically exhibiting low oxygen levels and a 0129. In one embodiment, the compound is for use in vivo propensity for malignant progression. as a cytotoxin, or a hypoxia activated prodrug thereof. 0.141. The hypoxia-activated cytotoxicity of the prodrug 0130 By “hypoxia activated prodrug thereof we include compounds of the invention allows the cytotoxicity to be that the compound is preferentially cytotoxic under, or fol targeted to the tumour cells, reducing the risk of damage to lowing exposure to, hypoxic conditions (i.e. exhibits greater healthy cells. cytotoxicity under, or following exposure to, hypoxic condi 0142. It is believed that hypoxia may play a role in facili tions). For example, N-oxide compounds of the invention, tating the malignant progression of certain cancers (for such as those of formulae V, VI, IX and X, are relatively example, see Rudolfsson & Bergh, 2009, Exp. Opin. Ther. non-cytotoxic under normoxic conditions but are readily Tar: 13:219-225). By exerting a cytotoxic effect preferentially reduced under hypoxic conditions to generate cytotoxic com within the regions of tumour hypoxia, the compounds of the pounds, such as those of formulae III, IV, VII and VIII. invention may be able to target cancer cells that are otherwise 0131. In this context, “hypoxia” may be regarded as an resistant to treatment, e.g. by radiotherapy or conventional oxygenation level of 4% or lower (or s23 mmHg) when chemotherapeutic agents. Eradication of Such resistant cells measured directly by electrode methods. For example, the may, in turn, lead to a reduction in metastasis. level of oxygenation may be lower than 3.0%, 2.5%, 2%, 0143. Thus, in one embodiment, the compounds are for 1.5%, 1% or 0.5 or 0.1%. use in the treatment or prevention of metastases (which may 0.132. It will be appreciated by persons skilled in the art arise from the aetiology of the cancer or as a consequence of that the hypoxia-induced activation of a compounds cyto treatment). toxic activity may be determined either in vitro or in vivo. 0144. It will be appreciated by persons skilled in the art 0.133 For example, cytotoxicity may be determined in that the compounds of the invention may be used on their own vitro at various oxygenation levels measured by direct elec or in combination with other cancer treatments (such as radio trode methods. therapeutic modalities, e.g. radioisotopes and external beam 0134. Alternatively, the level of oxygenation in a tissue radiation, and chemotherapeutic agents; see below). may be measured indirectly, for example using histological 0145. In one embodiment, the compounds are for use as a sections probed with an enzyme detection assay or by gene monotherapy (i.e. without any other cancer treatments). expression analysis. However, it will be appreciated that the cancer patient may 0135 For confirmation of hypoxia-activated cytotoxicity also be receiving different types of beneficial medication in vivo, oxygenation levels in living tissue may be determined (such as a painkiller, sedative, antidepressant, antibiotic, etc). using both the Helzel and OxyLite systems (for example, see 0146) However, the compounds of the invention may alter Wen et al., 2008, Radiat. Res. 169:67-75). natively be for use in combination with one or more addi 0136. The results of blood flow and perfusion analyses tional cancer treatments. For example, the compounds may be may also infer the existence of hypoxia in a given tissues. The used in combination with one, two, three, four, five or more application of agents that modify blood flow or compromise additional cancer treatments. blood vessel formation would also on first principles be 0147 By “in combination' we include that the compound expected to reduce oxygenation in affected tissues. is administered to a subject who is receiving one or more 0.137 In particular, the invention provides a compound additional cancer treatments in the same course of therapy. according to the first aspect of the invention for use in the Thus, the term covers not only the concomitant administra treatment of cancer in mammals (most notably in humans). tion of the compound with one or more additional cancer 0138 For example, the compound may be for use in the treatments (either as bolus doses or infusions) but also the treatment of a cancer selected from the group consisting of temporally separate administration of these cancer treat bladder cancer, breast cancer, bone cancer (primary and sec ments. For example, the compound may be administered ondary, such as osteosarcoma and Ewings sarcoma), brain within a treatment schedule/cycle as defined by the patients cancer (including glioblastoma multiforme and astrocy oncologist to include one or more additional cancer treat toma), cervical cancer, choriocarcinoma, colon and rectal ments, administered either before, concomitantly with or cancer, endometrial cancer, eye cancer, gallbladder cancer, after the compound; for example within ten weeks, nine US 2015/0307441 A1 Oct. 29, 2015

weeks, eight weeks, seven weeks, six weeks, five weeks, four tro-L-arginine (L-NNA), L-nitroarginine methyl ester weeks, three weeks, two week, ten days, one week, five days, (L-NAME), LG-nitro-L-arginine (L-NO-Arg) and 7-Nitro four days, three days, two days, one day, 12 hours, 10 hours, Indazole (7-NI). 8 hours, 6 hours, 4 hours, 3 hours, 2 hours, 1 hour, 45 minutes, 0164. By “vasoconstricting agents' we include alpha 1 30 minutes, 20 minutes, 10 minutes or five minutes. Each adrenoceptor agonists (e.g. methoxamine, phenylephrine, treatment cycle may be repeated on several occasions, nor oxymetazoline, tetrahydralazine, Xylometazoline), alpha 2 mally up to 6 cycles, but could be more or less than this adrenoceptor agonists (e.g. clonidine, guanabenz, guanfa number depending on the nature of the cancer and its response cine, C.-methyldopa) and vasopressinanalogues (e.g. arginine tO treatment. vasopressin and triglycyl lysine vasopressin). 0148. It will be appreciated by persons skilled in the art 0.165. By “vasodilating (vascular steal) agents’ we that the one or more additional cancer treatments may be include alpha-adrenoceptor antagonists (alpha-blockers), chemotherapeutic agents or radiotherapeutic modalities. angiotensin converting enzyme (ACE) inhibitors, angio 0149. In one embodiment, however, the one or more addi tensin receptor blockers (ARBs), beta2-adrenoceptor ago tional cancer treatments comprise or consist of one or more nists (B2-agonists), calcium-channel blockers (CCBs), cen chemotherapeutic and/or radiotherapeutic modality. trally acting sympatholytics, direct acting vasodilators, 0150. Given the hypoxia-activated cytotoxicity of the pro endothelin receptor antagonists, ganglionic blockers, nitrodi drug compounds of the invention, it is advantageous to lators, phosphodiesterase inhibitors, potassium-channel administer them as part of a combination treatment with one openers and renin inhibitors. or more chemotherapeutic agents and/or radiotherapeutic 0166 By “radiotherapy modalities' we include conven modalities capable of decreasing (at least, transiently) tumour tional external beam radiation therapy (2DXRT), stereotactic oxygenation levels in vivo. For example, the one or more radiosurgery (SRS), stereotactic body radiation therapy chemotherapeutic agents and/or radiotherapeutic modalities (SBRT) and particle therapy such as proton therapy; brachy may be capable of lowering the median oxygen level of the therapy such as SAVITM, MammoSiteTM, ConturaTM, Prox tumour to below 3%, for example below 2.5%,2%, 1.5%, 1%, celanTM, TheraSeedTM and I-SeedTM; radioisotope therapy 0.5%, 0.4%, 0.3%, 0.2% or below 0.1%. such as metaiodobenzylguanidine (MIBG), iodine-131, hor 0151. It will be appreciated by skilled persons that a reduc mone-bound lutetium-177 and yttrium-90 (peptide receptor tion in tumour oxygenation levels may be achieved by a radionuclide therapy). number of different means, for example by the disruption of 0167. In one preferred embodiment, the one or more can established tumour vasculature, prevention of angiogenesis cer treatments is/are non-steroidal anti-androgens, such as (new blood vessel formation) and/or vasoconstriction. flutamide, nilutamide, bicalutamide, finasteride, dutasteride, 0152 Suitable cancer treatments may be selected from the bexlosteride, izonsteride, turosteride, epristeride and abi group consisting of anti-androgens (steroidal and non-steroi raterOne. dal), Vascular disrupting agents, anti-angiogenic agents, anti 0168 Thus, in one embodiment, a compound according to VEGFR agents, IL8 inhibitors, NO synthase inhibitors, vaso the first aspect of the invention is used in combination with constricting agents, vasodilating agents and radiotherapy. bicalutamide in the treatment of cancer, e.g. the prevention or 0153. By “steroidal anti-androgens' we include cyproter reduction of metastasis. One acetate. 0169. Thus, in one embodiment, a compound according to the first aspect of the invention is used in combination with 0154 By “anti-angiogenic agents' we include: cancer chemotherapeutic agents and/or radiotherapeutic 0155 (a) anti-VEGF antibodies or antibody fragments modalities and/or methods to reduce or increase the air being Such as bevacizumab, axitinib, paZopanib and ranibi breathed by the patients e.g. carbogen (with or without nico Zumab, pegaptainib sodium, tryptophanyl-tRNA syn tinamide). thetase, AdPEDF, EYLEA, AG-013958, JSM6427, 0170 A related, sixth aspect of the invention provides the TG100801, ATG3, rapamycin, endostatin: use of a compound of the first aspect of the invention in the 0156 (b) drugs that block signalling within the cell such preparation of a medicament for treating cancer. as lapatinib, Sunitinib, Sorafenib, axitinib, paZopanib (0171 Preferred embodiments of the sixth aspect of the and AZ2171; invention are described above in relation to the fifth aspect of 0157 (c) tetrahydrocannabinol (THC) and cannabidiol; the invention. 0158 (d) thiazolidinediones such as rosiglitazone, 0172 A seventh aspect of the invention provides a method pioglitaZone and troglitaZone of treating cancer in a patient comprising administering to the 0159 (e) erlotinib, imatinib, gefitinib, dasatinib, nilo patient a therapeutically effective amount of a compound of tinib, lapatinib, and the first aspect of the invention. 0160 (f) drugs that affect signals between cells, such as 0173. In one embodiment, the patient is mammalian (e.g. thalidomide and lenalidomide. human). 0161. By “vascular disrupting agents' we include small 0.174 Preferred embodiments of the seventh aspect of the molecules (such as taxanes, taxol, paclitaxel combretastatins, invention are described above in relation to the fifth aspect of CA4P. Oxia.503, aurostatins, dolostatins, colchine, azacolchi the invention. cinol, ZD6126I, MMP-activated colchicines, ICT2588, 0.175. An eighth aspect of the invention provides the use of DMXAA, TZT1027 and AVE8062) and biologicals (such as a compound of the first aspect of the invention as a marker of ADEPT GDEPT and antibody drug-conjugates that target the oxygenation level of cells. In particular, Such compounds the tumour vasculature). may be used as a cellular hypoxic marker, either in vitro or in (0162 By “IL8 inhibitors' we include repertaxin. vivo. (0163. By “NO synthase inhibitors' we include N-me (0176) In one embodiment, the cells are mammalian (e.g. thyl-1-arginine hydrochloride (546C88: 1-NMMA), NG-ni human). US 2015/0307441 A1 Oct. 29, 2015

0177 Exposure of the N-oxide forms of the compounds of DESCRIPTION OF THE DRAWINGS the invention (such as those of formulae V and VI) to hypoxic 0189 Preferred, non-limiting examples which embody cells causes their reduction to the corresponding amine form certain aspects of the invention will now be described, with (such as those of formulae III and IV), which can be readily reference to the following figures: detected by known means. (0190 FIG. 1: The metabolites AQ4 and OCT1001 have 0.178 The presence of the reduced compound (such as similar cell cyle arresting actions, under normal oxygenation those of formulae III and IV) can be used to detect hypoxic conditions, indicating that selective deuteration has not modi cells in vitro or in vivo. The innate fluorescence properties fied intrinsic biological activity. retained by the reduced compound(s) and the intracellular (0191) See Example B persistence of the reduced compound(s) are advantageous for 0.192 FIG. 2: Similar hypoxia-enhanced cytotoxicity for the discrimination, quantification and localisation of cells AQ4N and OCT1002 that have been exposed to, or continue to be exposed to (0193 See Example B hypoxic conditions. (0194 FIG. 3: Exemplification of that the bioactivity of 0179 For example, when acting as a cellular marker for AQ4N and OCT1002 is dependent upon the degree of hypoxia, the reduced compound (such as those of formulae III hypoxia and IV) maybe detected using method(s) that identify chemi (0195 See Example B cal composition or physical properties that include but are not 0.196 FIG. 4: Hypoxia-dependent growth inhibition by limited to mass spectrometry, infrared spectroscopy, colorim AQ4N and OCT1002 arises from a similar mechanism of cell etry, Raman spectroscopy, nuclear magnetic resonance or cycle arrest and is dependent on the degree of hypoxia positron emission tomography. Affinity capture methods (0197) See Example B would exploit the high affinity binding potential of the (0198 FIG.5 (A & B): Exemplification of shared bioactiv reduced compound to DNA or synthetic polynucleotide ity of AQ4N and OCT1002 under hypoxic conditions for Sequences. functional p53 (DoHH2) and mutant p53 (SU-DHL-4) 0180 Optical properties of the reduced compound(s) may human B cell lymphoma cells be used to detect compound in biological samples and include (0199. See Example B but are not limited to flow cytometry and microscopy utilising 0200 FIG. 6: Intracellular accumulation of the OCT1001 the innate fluorescent properties of the reduced compound. far-red fluorescent chromophore under hypoxia is responsive Secondary methods of detection of reduced compound to OCT1002 pro-drug dose and oxygenation level include but are not limited to a combination with other 0201 See Example B molecular reporter compounds with the reduced compound 0202 FIG. 7: Deuteration does not affect the intrinsic participating in resonant energy transferreactions as eitheran capacity of the metabolite (AQ4 or OCT1001) to accumulate acceptor or donor. Other secondary methods of detection of within a cell reduced compound include but are not limited to methods (0203. See Example B using antibody based methods for molecular detection. 0204 FIG. 8: Accumulation of converted pro-drug 0181. In one embodiment, the compounds of the invention OCT1001 correlates with growth arrest are used to identify hypoxic tumour cells in Vivo, which may (0205 See Example then be visualised in situ or excised Surgically. (0206 FIG.9 (A & B): Demonstration of intracellular fluo 0182. In a further embodiment, a compound of the first rescence following exposure to OCT1002 under hypoxic con aspect of the invention is used as a cellular hypoxic marker in ditions and that prodrug deuteration reduces intracellular combination with a non-deuterated form of a compound of accumulation but increases persistence of the metabolite. the first aspect of the invention. 0207. See Example B 0183 By “in combination' in this context this includes 0208 FIG.10: Effect of bicalutamide on the oxygenation that the compounds may be applied to the cells (e.g. admin of 22RV1 prostate tumours grown as Xenografts istered to a patient) either concomitantly or sequentially (for (0209. See Example C example, within 24 hours, 12 hours, 6 hours, 4 hours, 3 hours, 0210 FIG. 11: Effect of bicalutamide on blood vessels in 2 hours, 1 hour, 30 minutes, 30 minutes, 10 minutes or less). 22RV 1 tumour xenografts 0184 Thus, in a preferred embodiment, a compound of 0211 See Example C formulae IX or X is used as a cellular hypoxic marker (in vivo 0212 FIG.12: Effect of bicalutamide only or AQ4N single or in vitro) in combination with a compound as disclosed in dose or OCT1002 single dose on 22RV1 xenografts in mice U.S. Pat. No. 5,132,327 (for example, AQ4N). 0213 See Example C 0185. A related, ninth aspect of the invention provides a kit 0214 FIG. 13: Combined effect of AQ4N single dose or of parts for use in detecting the oxygenation level of cells OCT1002 single dose on 22Rv1 xenografts in mice treated comprising a compound according to the first aspect of the daily with bicalutamide invention. 0215 See Example C 0186 Optionally, the kit further comprises a non-deuter 0216 FIG. 14: Effect of OCT1002 on LNCaP xenografts ated form of a compound according to the first aspect of the in mice treated with/without bicalutamide invention (such as a compound as disclosed in U.S. Pat. No. 0217 See Example C 5,132,327, for example AQ4N). 0218 FIG. 15: OCT1002 is reduced in hypoxic LNCaP 0187 Preferably, the compound(s) is/are provided in a tumour cells in vivo sterile, pyrogen-free form. 0219. See Example C 0188 It will be appreciated that the kits of the invention 0220 FIG. 16: OCT1002 reduces the metastatic spread of may further comprise one or more regents, control samples LNCaP tumours to the lungs and/or instructions. 0221 See Example C US 2015/0307441 A1 Oct. 29, 2015

DETAILED DESCRIPTION OF THE -continued EMBODIMENTS DC n N Examples s. CD3 HCI Example A Synthesis of Alkylaminoalkylaminoanthraquinones 0227. A suspension of deuterated-do-dimethylamine and their N-oxides hydrochloride (18.4g, 210 mmol) and 2-bromoacetonitrile (14.63 ml, 210 mmol) in anhydrous THF (250 mL) in a round (a) Preparation of bottom flask was cooled to -10°C. with vigorous stifling and 1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione treated portion-wise with potassium carbonate (58.1 g, 420 0222 mmol). After addition of the base, the reaction was fitted with a reflux condensor and balloons and allowed to warm slowly to 5° C. over 2 hours. TLC (1:1 EtOAc/Iso-Hexanes) indi OH F O cated the presence of product. The mixture was stirred at room temperature over a weekend. AlCl3 O -e- 0228. The residue was diluted with DCM (250 mL) and Sulfolane filtered, washing with copious amounts of DCM. The mother O liquors were degassed with N for 1 hour, then reduced in OH F volume by half on the rotavap. Then a 4M dioxane solution of OH O F hydrogen chloride (52.5 ml, 210 mmol) was added, precipi tating a white solid and the mixture allowed to stand for 10 minutes before being filtered, washing with DCM to afford deuterated-do-dimethylacetonitrile (21.73 g, 172 mmol. 82% yield). OH O F 0229 H NMR (400 MHz, d-DMSO)8:4.47 (2H, s) was consistent with the desired material. 0223. A mixture of 4,7-difluoroisobenzofuran-1,3-dione (8.50 g., 46.2 mmol), hydroquinone (5.64 g, 51.3 mmol), (c) Preparation of aluminium trichloride (36.9 g, 277 mmol) and sulfolane (10 deuterated-do-N,N-dimethylethylenediamine mL) was stirred together for 16 hours at 165°C. The reaction was effectively a melt as the mixture does not become a 0230 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. p f 0224. The mixture was poured carefully into ice and 2M DC1 N LiAlH4 —-Ether DC1 N N1)-Nil, HCl added (50 mL). The mixture was stirred, then filtered, n N washing the resultant slurry with further 2M HC1. The solid HCI 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 flaskat 60°C. until 0231. To a stirred suspension of deuturated-d6-dimethy constant weight afforded 1,4-difluoro-5,8-dihydroxyan lacetonitrile (21.72 g, 172 mmol) in Et2O (200 mL) at 0°C. thracene-9,10-dione (9.82 g, 35.6 mmol. 77% yield). was added d/w a 1M ether solution of lithium aluminium 0225 H NMR (DMSO-d) was clean and consistent with hydride (515 ml, 515 mmol) via dropping funnel over 1.5 the desired material. hours. After the addition, the cooling bath was removed. After a further 1.5 hr, the reaction was quenched at 15°C. (no higher (b) Preparation of 1,4-bis-(2-(deuterated-d6-dim than 18°C.) with sodium sulfate decahydrate (0.5 eq rel. to ethylamino)ethylamino)-5,8-dihydroxy-anthracene LiAlH4, 80 g) cautiously (delayed reaction) over 1.5 hours. 9,10-dione The mixture was left to stir for 1 hour and subsequently 0226 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-do-N,N-dimethylethyl CD3 enediamine (15.89 g, 160 mmol. 93% yield was clean and K2CO3 consistent with the desired material but contained ~0.25 eq HCI DC1 N NH B1NŠ Tetrahydrofuran ether). HCI 0232 H NMR (400 MHz, CDC1) 8: 2.76 (2H, t), 2.33 (2H, t) US 2015/0307441 A1 Oct. 29, 2015

(d) Preparation of 1,4-bis-(2-(deuterated-d6-dim was treated with deuterated-do-N,N-dimethylethylenedi ethylamino)ethylamino)-5,8-dihydroxy-anthracene amine, (16.57 ml, 142 mmol) as a steady stream. The mixture 9,10-dione (“OCT1001) was warmed to 40° C. and allowed to stir for 24 hours under a flow of nitrogen. The reaction was taken offheat and cooled 0233 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 OH O F CD3 was filtered washing with a 10% ammonium hydroxide solu N tion (130 mL). The solid was air-dried for 30 minutes, then HN 1N1 n CD transferred to a tared flask and dried under vacuum at 60° C. He Pyridine until constant weight (-2 h). 0235. The bulk material was purified by flash chromatog raphy (Biotage, 120 g) loading in DCM (through cottonwool OH O F plug) eluting with 6 then 10% MeOH (containing 1% NH)/ DCM to give 1,4-bis-(2-(deuterated-d6-dimethylamino) ethylamino)-5,8-dihydroxyanthracene-9,10-dione (2.01 g, 4.73 mmol, 26.7% yield). 0236. 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%. 0237 H NMR (CDC1) was clean and consistent with the desired material 'H NMR (400 MHz, CDC1) 8: 13.51 (2H, s), 10.40 (2H, brt), 7.17 (2H, s), 7.11 (2H, s), 3.47 (4H, q), 2.66 (4H, t). CD (e) Preparation of 1,4-bis-(2-(deuterated-d6-dim ethylamino-N-oxide)ethylamino)-5,8-di-hydroxyan 0234. A solution of 1,4-difluoro-5,8-dihydroxyan thracene-9,10-dione (“OCT1002) thracene-9,10-dione, (4.9 g, 17.74 mmol) in pyridine (35 mL) 0238

N1,N1 N NCD 6H2O

H Methanol DCM HCI Ethyl Acetate/Ethanol

CD3 oH o HN1N1, NCD,-o-

OH O HN CD3 N-1 | Yo US 2015/0307441 A1 Oct. 29, 2015

0239 A suspension containing magnesium monoperoX dione (AQ4N) and 1,4-bis-2-(deuterated-d6-dim yphthalate, MMPP (3.10 g. 6.27 mmol) in methanol (8 mL) ethyl-amino-N-oxide)ethylamino)-5,8-di-hydroxy was added dropwise to a stirred solution of 281-041 (1.90 g, anthracene-9,10-dione (“OCT1002) both exhibit 4.48 mmol), AQ4 in methanol (8 mL) and DCM (30 mL) pronounced cytostatic activity under conditions of cooled to -11° C. After the addition was complete, the reac hypoxia (1% oxygen). tion solution was allowed to warm to 0°C. and stirred over 0248. As a control it is shown that hypoxia does not night in the dark (warmed to room temperature during this modify the cytostatic action of a direct acting DNA time). Pre-cooled EtOAc (30 mL) and EtOH (6 mL) were topoisomerase inhibitor (VP-16), achieving similar added the reaction mixture at 0°C. This mixture was allowed levels of prolonged cytostatic action. to stir for 30 minutes then a 4M solution of hydrogen chloride 0249 (c) Exemplification of that the bioactivity of (4.48 ml, 17.90 mmol) in dioxane was added dropwise at AQ4N and OCT1002 is dependent upon the degree of approximately -10 to -15°C. The resulting slurry was then hypoxia stirred for 10 minutes then filtered, washing with EtOH/Water 0250 A549 human lung cancer cells were cultured (9:1, 100 mL), MeOH/EtOAc (1:1, 100 mL) and EtOAc (60 using conventional methods for adherent cells and mL) and dried under vacuum (on rotavap) at 40°C. for 2 hours exposed for 4 days to varying concentrations of either (constant weight) to afford 1,4-bis-(2-(deuterated-d6-dim AQ4N and OCT1002 agents under standard cell cul ethylamino-N-oxide)ethyl-amino)-5,8-di-hydroxyan ture conditions of 5% carbon dioxide in air (nor thracene-9,10-dione (2.15g, 3.99 mmol. 89% yield) as a dark moxia) at 37 degC, or under conditions of reduced blue powder. oxygen (1% and 3%). 0240. The product was analysed by LCMS (standard 4 0251 Data are plotted as relative population dou min. method, agilent), m/z. 458.2 (M--H)" (ES), at 3.07 min, blings determined by cell detachment and Coulter 98.3% purity (a 254 nm. "H NMR (400 MHz, DO) 8: 6.73 Counterparticle counting of cell densities at the start (2H, brs), 6.43 (2H, brs), 3.76 (4H, brs), 3.58 (4H, brs). and end of the exposure period. 0241 H NMR (DO) was consistent with the desired 0252 FIG. 3 shows that for the compounds tested, material. namely 1,4-bis-(2-(dimethylamino-N-oxide)ethyl amino)-5,8-di-hydroxy-anthracene-9,10-dione Example B (AQ4N) and 1,4-bis-(2-(deuterated-d6-dimethyl amino-N-oxide)ethylamino)-5,8-di-hydroxyan In vitro properties of 1,4-bis-(2-(deuterated-d6 thracene-9,10-dione (“OCT1002), growth inhibition dimethylamino-N-oxide)ethylamino)-5,8-di-hy is dependent upon the degree of hypoxia and drug droxyanthracene-9,10-dione and its active metabolite concentration, with the two agents showing similar 0242 (a) The metabolites AQ4 and OCT1001 have responses. similar cell cyle arresting actions, under normal oxygen 0253 (d) Hypoxic sensitisation by AQ4N and ation conditions, indicating that selective deuteration OCT10O2 has not modified intrinsic biological activity. 0254 A549 human lung cancer cells were used in 0243 A549 human lung cancer cells were cultured this experiment; culture conditions were as described using conventional methods for adherent cells and in (c) above. exposed for 4 days to 0, 1, 3 or 10 nM agents under 0255 Cell cycle analysis was performed as described standard cell culture conditions of 5% carbon dioxide in (a) above. in air at 37 degC. Harvested cells were permeabilised 0256 FIG. 4 shows that the compounds tested, and stained with the DNA fluorescent dye ethidium namely 1,4-bis-(2-(dimethylamino-N-oxide)ethyl bromide and cell cycle distributions determined by amino)-5,8-di-hydroxy-anthracene-9,10-dione conventional flow cytometry. (AQ4N) and 1,4-bis-(2-(deuterated-d6-dimethyl 0244 FIG. 1 (flow cytometry) shows similar amino-N-oxide)ethylamino)-5,8-di-hydroxy-an increases in the G2 peaks of the DNA content distri thracene-9,10-dione (“OCT1002), generate similar butions between 3-10 nM (indicating cell cycle arrest) cell cycle arrest (determined by flow cytometry) for cells exposed to exogenous metabolites 1,4-bis within the bioactive drug dose range. 2-(dimethylamino)ethylamino)-5,8-dihydroxy-an 0257 The degree of late cell cycle arrest is increased thracene-9,10-dione (AQ4) and 1,4-bis-(2-(deu as oxygenation levels are reduced. terated-d6-dimethylamino)-ethylamino)-5.8- 0258 (e) Exemplification of shared bioactivity of dihydroxy-anthracene-9,10-dione (“OCT1001). AQ4N and OCT1002 under hypoxic conditions for p53 0245 (b) Similar hypoxia-enhanced cytotoxicity for functional and mutant p53 human B cell lymphoma cell AQ4N and OCT1002 lines 0246 Human T cell leukemia cells (Jurkat) were cul 0259 Human B cell lymphoma cells were cultured tured using conventional methods for Suspension cul using conventional methods for Suspension cultures tures in air or under 1% oxygen conditions for 4 days in air, 1% or 3% oxygenation conditions for 4 days in in the presence of a range of concentrations of either the presence of a range of concentrations of either AQ4N or OCT1002. The relative cell number was AQ4N or OCT1002. The relative cell numbers were determined using a conventional Coulter Counterpar determined using a conventional Coulter Counterpar ticle counting method. ticle counting method. 0247 FIG. 2 shows that the compounds tested 0260 FIG. 5(A) shows that the compounds tested are require hypoxic conditions for the inhibition of cell equally and selectively cytotoxic in hypoxic condi proliferation. Thus, 1,4-bis-2-(dimethylamino-N- tions against DoHH2 human B cell lymphoma cells oxide)ethylamino)-5,8-di-hydroxyanthracene-9,10 (bcl2 overexpressing, p53 wt) grown in Suspension US 2015/0307441 A1 Oct. 29, 2015 14

and exposed to prodrugs for 4 days under 21% 0265 Table 1 shows a comparison of HPLC analysis (circles), 3% (triangles) or 1% 0 (squares). Thus, of metabolite generation following exposure of 1,4-bis-(2-(dimethylamino-N-oxide)ethylamino)- human A549 cells to AQ4N and OCT1002 under 5,8-di-hydroxyanthracene-9,10-dione (AQ4N) and varying degrees of hypoxia and concentration (data 1,4-bis-(2-(deuterated-d6-dimethyl-amino-N-ox- derived from two determinations) where 21% is taken ide)ethylamino)-5,8-di-hydroxy-anthracene-9,10- to represent normal oxygenation conditions. dione (“OCT1002) both exhibit pronounced cyto static activity under conditions of hypoxia (1% 10266 Data show the consistent reduction 1. the gen oxygen), with the growth inhibition being sensitive to eration of OCT1001 compared with AQ4 in cells the degree of hypoxia. exposed to the conditions indicated and washed prior 0261 Likewise, FIG. 5(B) shows that the prodrugs to assay for the presence of prodrug or their metabo AQ4N and OCT1002 are equally selectively cyto- lites. Data also shows that the molecular forms toxic in hypoxic conditions against SU-DHL-4 present in cells experiencing hypoxia are the metabo human B cell lymphoma cells (bcl2 overexpressing: lites and not parent prodrugs. TABLE 1.

Dose of Humidified Relative prodrug oxygenation pmoles metabolite generated prodrug (OCT1002 conditions perior cells reduction to or AQ4N) pO2 mm range range metabolite nMx days % O2 Hg AQ4 OCT1001 AQ4 OCT1001 OCT1001/AQ4 30 190 7.1 9.25 5.64 1.46 120 O.61 30 3% 21.4 O.78 O49 0.05 OO6 O.62 30 21%. 142.2 <0.10 O.1O O.O3 O.O2 1.02 100 190 7.1 >42.9S 16.17 6.59 8.16 <0.38 100 3% 21.4 5.58 1.93 113 O16 O.35 100 21%. 142.2 O.23 O.11 O.O8 O.O3 O.SO “No 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,

p53 mutant) grown in Suspension and exposed to pro 0267 (g) Intracellular accumulation of the OCT1001 drugs for 4 days under 21% (circles), 3% (triangles) or far-red fluorescent chromophore under hypoxia is 1% O (squares). Again, the growth inhibition is sen responsive to OCT1002 prodrug dose and oxygenation sitive to the degree of hypoxia. level 0262 (f) Reciprocity between an imposed pC) level 0268 Adherent A549 cells were cultured by conven and the degree of end-product generation tional methods and exposed to 0, 30 or 100 nM 0263 OCT1002 and AQ4N show reciprocity OCT1002 for 4 days in air, 1% or 3% oxygenation between an imposed pC) level and the degree of end levels. Detached cells were analysed far red fluores product generation in the biologically relevant range cence intensity using conventional flow cytometry of hypoxia with low or undetectable levels of conver and 633 nm wavelength excitation (1x10" cells analy sion under normoxia (and undetectable levels of sed). AQ4N or OCT1002 showing that the metabolites are 0269 FIG. 6 shows mean fluorescence intensity the primary persistent anthraquinone forms) increases in a linear function of pro-drug dose and is 0264. Relative to AQ4N, the deuterated variant dependent upon oxygenation levels. This provides a OCT1002 shows a reduction in overall capacity for convenient fluorometric, single live cell analytical reduction/accumulation (HPLC analysis) within method for analyzing cell population experience of moribund cells, under protracted exposure conditions prevailing pC) levels. showing a reduction of redundant targeting in a 0270 (h) Deuteration does not affect the intrinsic human lung cancer cell line. In this case redundant capacity of the active metabolite (OCT1001) to accumu targeting of a prodrug refers to the over-generation of late the cytotoxic form beyond that required for cell inac 0271 A549 human lung cancer cells were used in tivation since conversion of the prodrug can continue this experiment, as described in (g) above. even when cell cycle arrest has occurred. The conse quences of over-generation will be increased delete 0272 Under normoxia conditions, similar levels of rious effects of the converted form when released accumulation of OCT1001 and AQ4 were observed from the initial target cell. This undesirable bystander within cells (see FIG. 7). Thus, the overlaid histo effect on nearby tissue not initially subject to hypoxic grams for the population distribution of fluorescence conditions will comprise non-target normal and in cells exposed to AQ4 or OCT1001 under normoxia tumour cells. Damage to normal cells is clearly unde shows similar cellular accumulation potential. sirable. Suboptimal exposure of non-target tumour 0273 (i) Accumulation of converted pro-drug cells through a bystander effect may compromise OCT1001 correlates with growth arrest (increasingly their responses to other agent(s) delivered in combi moribund cells) nation or generate selective conditions for the devel 0274 A549 human lung cancer cells were used in opment of drug resistance. this experiment, as described in (g) above, with the US 2015/0307441 A1 Oct. 29, 2015 15

exception that light side scatter (488 nm wavelength) 0285 (c) Reciprocity between pC) level and end-prod was collected versus fluorescence intensity (>695 nm. uct generation in the biologically relevant range of wavelength). hypoxia; (0275 FIG. 8 shows collected flow cytometry data for 0286 (d) The ability of cellular fluorescence to reportin A549 cells exposed to 0, 30 and 100 nM OCT1002 situ generation of of metabolite providing for the sens under 21%, 3% and 1% oxygen over 4 days. ing and reporting of hypoxic environments; 0276 Plotting all data points reveals that increasing 0287 (e) A distinct molecular/atomic signature pro light side scatter parameter (reflecting the expansion vided by site-specific deuteration that can be used to of cell size and complexity associated with growth trace prodrug conversion and metabolism by physico arrest) correlates with the increase in fluorescence chemical methods; and intensity (indicating co-accumulation of OCT1001). 0288 (f) Prodrug deuteration results in reduced accu mulation of the reduced form under hypoxia but 0277 (i) Demonstration of intracellular fluorescence increased persistence/retention of the reduced form following exposure to OCT1002 under hypoxic condi upon removal of exernal drug and re-oxygenation. This tions and that prodrug deuteration reduces intracellular property demonstrated in moribund cells confirms both accumulation but increases persistence of the metabo reduced redundant targeting of the deuterated form and lite. convenient signal persistence for hypoxia sensing appli 0278 A549 cells were cultured using conventional cations. methods and allowed to attach to the glass Substrate in chamber slides and exposed to OCT1002 under Example C hypoxia. Fluorescence imaging of live cells used con ventional confocal fluorescence microscopy using Effect of OCT1002 on Tumour Growth and red-line laser excitation. Metastasis. In Vivo 0279 FIG. 9a shows that the far red fluorescence 0289 Given the hypoxia-activated cytotoxicity of the pro detected in cells is intracellular (background fluores drug compounds of the invention, it may be advantageous to cence not detectable in control cultures) with evi administer them as part of a combination treatment with one dence of regions of cytoplasmic accumulation. The or more chemotherapeutic agents and/or radiotherapeutic data exemplify the single cell hypoxia sensing prop modalities capable of decreasing (at least, transiently) tumour erties of the deuterated pro-drug at the single-cell oxygenation levels in vivo. Bicalutamide (marketed as Caso level. dex, CoSudex, Calutide, Kalumid) is an oral non-steroidal 0280 Given the confirmation of intracellular fluores anti-androgen used in the treatment of prostate cancer includ cence associated with conversion of OCT1002 to ing as monotherapy for the treatment of earlier stages of the OCT1001 under hypoxia, A549 human lung cancer disease. 22RV1 is a human prostate carcinoma epithelial cell cells were further used to assess differential accumu line (Sramkoski RM, Pretlow TG 2nd, Giaconia JM, Pretlow lation or retention of the metabolites using flow T P. Schwartz S, Sy MS, Marengo SR, Rhim J S. Zhang D, cytometry as described in (g) above. Following expo Jacobberger J W A new human prostate carcinoma cell line, sure to AQ4N or OCT1002 under 1% oxygen, cells 22Rv1. In Vitro Cell Dev Biol Anim. 1999 July-August: were detached for analysis, or washed and incubated 35(7):403-9). The cell line expresses prostate specific antigen for 24h in drug free medium and held under normal (PSA). Growth is weakly stimulated by dihydroxytestoster oxygenation conditions prior to detachment and one and lysates are immunoreactive with androgen receptor analysis by flow cytometry antibody by Western blot analysis. (0281 Flow cytometry data in FIG. 9b shows the 0290 (i) Effect of Bicalutamide on the Oxygenation of reduced cellular accumulation (after 4 day exposure) 22Rv1 Prostate Tumours Grown as Xenografts but also reduced loss (after 24h post exposure recov 0291 Male SCID mice (>8 weeks) bearing 22Rv1 pros ery) of intracellular fluorescence attributable to the tate tumours of 100-150 mm were treated daily for 28 metabolite OCT1001, compared with the fluores days by oral gavage with either vehicle (0.1% DMSO in cence attributable to the metabolite AQ4, following corn oil) or bicalutamide (2 mg/kg/day in vehicle). exposure of A549 cells to pro-drugs OCT1001 and 0292 Before commencement of treatment (day 0) pCO2 AQ4 in A549 under hypoxia. Thus, deuteration (mmHg) was measured using an Oxylite oxygen elec changes the in situ intracellular compartment loading/ trode probe; this was repeated on the days indicated. retention of hypoxia converted forms of OCT1002. Conclusions Day of Mean p0 + SD Significance Significance Treatment Treatment (mmHg) (to vehicle) (to day O) 0282. The above studies demonstrate the in vitro proper Vehicle only O 15.277 11.254 ties of an exemplary deuterated compound of the invention 7 14741 - 4.290 (the N-oxide prodrug, OCT1002, and its active metabolite, 14 3.16S 3.275 21 2.660 - 1889 OCT1001). 28 3.546 1.563 0283 (a) Evidence of primary biological activity fol Bicalutamide O 15.277 11.254 S lowing reduction of the prodrug in hypoxia that elicits (2 mg/kg/ 7 1996 - 1989

0293 Table 3 shows mean pC2 values +SD. Also shown Conclusions are statistical comparisons of the bicalutamide group com 0308 Vehicle has no effect on blood vessels for at least pared to control and to day 0 values; ns not significant. 7 days. By day 14 there is a slight pruning of vessels 0294 22RV1 cells grow as a solid tumour on the backs which is clearly seen by day 21. This vessel loss, of SCID mice. although not as dramatic as seen in the bicalutamide 0295 Tumour oxygenation was measured over 28 days treated tumours (at days 7 and 14; Ming et al., 2007), in vehicle and bicalutamide (2 mg/kg/day) treated mice may be due to vascular collapse and necrosis seen at this (see Table 3 above). time in this fast growing vehicle-treated tumour. The 0296 Bicalutamide caused a drop in tumour oxygen oxygen levels drop somewhat earlier, i.e. Sometime ation (as shown in FIG. 10); from ~15.3 mmHg (2% between days 7 and 14 (see FIG. 10). oxygen) to 2.0 mmHg (0.3% oxygen) at day 7 and to 0.5 0309. In bicalutamide-treated 22RV 1 tumours there is a mmHg (0.1% oxygen) at day 14. This drop persists for marked early loss of tumour vasculature (by day 7). The approximately 2 weeks before recovering to almost nor data provide evidence that bicalutamide causes a pro mal somewhere beyond 21 and 28 (at which time it is not found drop in tumour oxygenation through an anti-vas significantly different from the starting level of oxygen cular effect; this may be director alternatively it could be ation). due to inhibition of the production of pro-angiogenic 0297. The faster-growing, vehicle-treated, controls factors by the tumour cells. showed no significant drop in oxygen levels up to day 7. 0310. By day 21, the small vessels have returned which However, during the subsequent week (probably related is consistent with the increased level of oxygenation to tumour size) the median oxygen levels drop to about seen in FIG. 10. 3 mmHg (0.4% oxygen) and do indicate recovery. 0311 (iii) Effect of Bicalutamide Only or AQ4N Single Dose or OCT1002 Single Dose on 22RV1 Xenografts in Conclusion Mice. 0312 Male SCID mice (>8 weeks) bearing 22Rv1 0298 Hypoxia exists in the 22RV1 solid tumour model. xenograft tumours of 100-150 mm3 were treated for 28 The addition of bicalutamide alters the patterns of oxygen days. Treatment included Vehicle (0.1% DMSO in corn levels indicated by the tumour. Hypoxia is clearly relevant to oil) or bicalutamide (2 mg/kg/day in vehicle) both the 22Rv1 model and the response of such a model to mono administered daily via oral gavage. Alternatively, at day therapy (+bicalutamide); and the potential role of OCT1002 7 of the experimental period AQ4N or OCT1002 (50 in a combination treatment. mg/kg in sterile PBS) was administered intraperito 0299 (ii) Effect of Bicalutamide on Blood Vessels in neally as a single dose. 22Rv1 Tumour Xenografts 0313 Tumour volumes were measured using callipers every other day. 0300 Dorsal skin folds were secured using window 0314 Data analysis to determine the time dependent chambers onto the backs of male SCID mice (>8 weeks). effect of treatment(s) on tumour volume was performed. 22RV 1 tumour fragments were implanted and allowed to Tumour Volume was normalised to day 6 (ie pre-produg vascularise for 7 days before commencement of treat addition). Time series and regression analysis was ment. undertaken. 0301 Animals were treated daily via oral gavage with 0315 Tumour growth is normalised to day 6, so that either vehicle (0.1% DMSO in corn oil) or bicalutamide overall tumour growth, and patterns can be compared (2 mg/kg in vehicle). FIG. 12 (A and B). 0302 Anaesthetised mice were injected i.v. with FITC 0316 Despite the lack of sensitivity to bicalutamide in labelled dextran immediately prior to imaging with a vitro, the 22RV 1 tumours show a small but significant confocal microscope. slowing of growth. Classical cross-sectional comparison 0303. Each image is representative of a minimum of 5 of growth delay showed that mice treated with vehicle animals per treatment group. required 14.0+0.3 days to reach four times the volume at 0304 22RV 1 tumours were grown in window cham the start of treatment. Bicalutamide treatment (2 mg/kg/ bers/dorsal skin flaps on the backs of SCID mice. day) increased this to 18.5+0.8 days; thus this was a Tumour fragments were imaged (see FIG. 11) before growth delay of 4.5 days. treatment began (A) vehicle and (E) bicalutamide pre 0317 Graphical regression fits indicate that 22RV1 treatment groups and then after 7, 14 and 21 days of tumours treated with bicalutamide only show a delay in treatment, (B-D) vehicle only (F-H) bicalutamide (10x growth (during days 10-20), despite continuing daily exposure to bicalutamide; the tumours exhibit an overall magnification). exponential growth pattern (R-09915) to day 24. 0305 Within 7 days tumour fragments showed the 0318. Addition of AQ4N given as a single dose (50 development of extensive Small vessels indicated as day mg/kg) on day 7, a different growth pattern was 0 of the experimental period (see FIG. 11). observed compared to that of the bicalutamide treatment 0306 In vehicle-treated tumours vessel density showed alone, regression fitting showed a non-linear polynomial a slight change by day 14 and by day 21 the Small vessel growth pattern (R=0.9948). numbers were reduced. 0319. Addition of OCT1002 given as a single dose (50 0307. In bicalutamide-treated tumours, loss of small mg/kg) on day 7; tumours treated with this single dose vessels was seen at days 7 and 14 with some recovery by were capable of maintaining a polynomial (x) growth day 21. This is consistent with oxygen electrode data i.e., rate pattern, this was also a non-linear pattern (R-0. fall and then recovery of oxygenation. 9978). US 2015/0307441 A1 Oct. 29, 2015 17

0320 OCT1002 treated tumours showed an overall istered daily via oral gavage. OCT1002 (50 mg/kg in reduced rate of growth over the remaining period of the sterile PBS) was administered intraperitoneally as a experiment (beyond day 22) compared to the bicaluta single dose at day 7. mide only and AQ4N only treated tumours. Culmulative growth over the entire period (progressive area under the 0337 Tumour volumes were measured using callipers curve), indicates this difference (FIG. 12B). every other day. 0321 (iv) Combined Effect of AQ4N Single Dose or 0338 Growth curves are the mean of >5 animals in OCT1002 Single Dose on 22Rv1 Xenografts in Mice Treated bicalutamide and vehicle treatment groups; bicaluta Daily with Bicalutamide mide+OCT1002 group (n=5 until day 14; then n=3) and 0322 Male SCID mice (>8 weeks) bearing 22Rv1 xenograft tumours of 100-150 mm were treated for 28 vehicle+OCT1002 (n=5 until day 13; n=1)+s.e. days. Vehicle (0.1% DMSO in corn oil) and bicaluta 0339 Table 6 below shows the growth delays calculated mide (2 mg/kg/day in vehicle) treatments were admin for the time to reach twice the treatment size. istered daily via oral gavage. 0323 AQ4N or OCT1002 (50 mg/kg in sterile PBS) 0340 Bicalutamide causes a 5.1 day delay in LNCaP was administered intraperitoneally as a single dose at tumour growth compared to vehicle. day 7. (0341 When OCT1002 (50 mg/kg single dose on day 7) 0324 Tumour volumes were measured using callipers was given in combination with vehicle (daily adminis every other day. tration) there was no appreciable effect on tumour 0325 Animals were culled once the tumour burden growth (Table 6 below). reached 2800 mm. 0326 Tumour growth is normalised to day 6, so that 0342 Bicalutamide (daily for 28 days) initially slows overall tumour growth, and patterns can be compared tumour growth until day 12-14. Tumour growth then (FIG. 13 (A and B). recovers and the tumours are the same size as the 0327 Bicalutamide treatment alone (2 mg/kg/day) is vehicle-treated tumours by day 28 (Table 6 below). discussed above; it exhibits a overall exponential growth (0343 Tumours treated with a single dose of OCT1002 pattern (R=0.9915) to day 24. reduced the growth rate in combination with bicaluta 0328 Bicalutamide treatment was combined with an mide and this was significantly different from control at AQ4N single dose (50 mg/kg) given on day 7, a modified all times between days 14 and days 28 at the termination growth pattern was observed compared to that of the of the experiment (FIG. 15). bicalutamide treatment alone, regression fitting showed a non-linear polynomial growth pattern (R=0.9982), with divegence of growth to bicalutamide alone appar Conclusions ent at beyond day 20. 0329 Bicalutamide treatment treatment was combined 0344 Administration of OCT1002 at day 7 had no sig with an OCT1002 single dose (50 mg/kg) given on day nificant effect on LNCaP tumour growth. 7; a different modified growth pattern was observed 0345. This shows that the better-oxygenated tumours (i.e. regression fitting showed a linear tumour growth as compared to bicalutamide-treated tumours) there is low response (R-0.9955), with divegence of growth to toxicity of OCT1002 and that this better-oxygenated fraction bicalutamide alone apparent at beyond day 14. of cells is predominant in contributing to growth in vehicle treated control tumours. Conclusions (0346 Combination of a single dose of OCT1002 with 0330. The combined treatment indicates two critical bicalutamide blocked the increase in growth rate features. observed in the bicalutamide-treated group. OCT1002 is 0331 (i) the first is an earlier effective tumour growth very effective at blocking tumour growth from 12 days inhibition of OCT1002 on the bicalutamide treated onwards where, for bicalutamide alone, there is a delay tumours compared to AQ4N: and then recovery. 0332 (ii) the second indicates a sustained tumour growth inhibition (indicated by a maintained linear 0347 The initial slowing and then recovery after day 14 response); that reflects a persistance OCT1002 and of LNCaP tumour growth, during daily treatment with tumour growth inhibition. bicalutamide, is consistent with the drop and then recov 0333. Thus with OCT1002 administered at the time ery of tumour oxygenation and blood vessels (Ming et when hypoxia/low oxygen levels were achieved; an al., 2012, Supra.). early and sustained effect was obtained. The combina tion of OCT1002 with bicalutamide was more effective TABLE 6 at inhibiting tumour growth as compared to AQ4N with Time to 2x start volume Growth bicalutamide. Treatment (days) Delay (days) 0334 (v) Effect of OCT1002 on LNCaPXenografts in Vehicle Only 11.2 1.88 Mice Treated with/without Bicalutamide Bicalutamide 16.21.94 53.82 0335 Male SCID mice (>8 weeks) bearing LNCaP OCT1002 only 13 - 0.89 1.8 2.77 xenograft tumours of 100-150 mm were treated for 28 OCT1002 -- 2S.S 3.22 14.35.1 days. Bicalutamide 0336 Vehicle (0.1% DMSO in corn oil) and bicaluta mide (2 mg/kg/day in vehicle) treatments were admin US 2015/0307441 A1 Oct. 29, 2015

(0348 (vi) OCT1002 Prodrug is Converted to Metabolites 0366 By day 21, tumour blood vessels show some in Hypoxic LNCaPTumour Cells In Vivo recovery and OCT1001 levels are lower although still above background. Methods 0367 The persistence of the reduced product, 0349. A dorsal skin flap (window chamber) was OCT1001, for >7 days shows that the half-life of the attached to the dorsum of male SCID mice and a 1 mm converted compound is long. LNCaP-Luc tumour fragment inserted; this was left to 0368. However it may be less than AQ4 since by day 21 vascularise for 7 days. the OCT1001 signal is very much decreased. 0350 Mice were then treated orally for 21 days with 0369. This may be due to the different cellular binding either vehicle (0.1% DMSO in corn oil) or bicalutamide properties of OCT1001 as compared to AQ4 and poten (2 mg/kg/day). tially will provide a rationale for less cumulative sys 0351. Seven days after induction of (a) vehicle or (b) temic toxicity which might be caused through persis bicalutamide mice were dosed intraperitoneally with tence of Small amount of reduced compound in OCT1002 (50 mg/kg). marginally hypoxic peripheral tissues. This should not 0352. Two hours after injection of OCT1002 mice were affect the primary efficacy of OCT1002/OCT1001 at the injected intravenously with FITC-dextran. predominant site of metabolism (i.e. the hypoxic cells in 0353 Images were captured using a confocal laser tumours) since large amounts are seen throughout the Scanning microscopy to show blood vessels (green) and hypoxic tumour fragment which persists for greater than OCT1001 (blue) patterns in the tumour. (Magnification 7 days. 10x with 3x digital Zoom) (pixel resolution). 0370 (vii) OCT1002 Reduces the Metastatic Spread of 0354) Images were also acquired at day 0 (i.e. 7 days LNCaPTumours to the Lungs after tumour fragment implantation), 14 and 21. 0355 Only FITC-dextran was administered on days 0, Methods 14 and 21. (c) Full panel of images 0, 7, 14 and 21 days. 0356 Control mice were treated orally for 21 days with 0371 Male SCID mice (>8 weeks) bearing LNCaP-luc vehicle (0.1% DMSO in corn oil): vascularisation was xenograft tumours of 100-150 mm were treated for 28 maintained throughout. By 7 days the tumour fragment days (the luciferase-expressing cells had similar charac was vascularised (day 0 of experiment shown in FIG. teristics to parental LNCaP cells; Ming et al., 2012, 15C green). Supra.). 0357. In mice treated with vehicle+OCT1002 at day 7: 0372 Vehicle (0.1% DMSO in corn oil) and bicaluta the converted compound OCT1001 (blue) is in a few mide (2 mg/kg/day in vehicle) treatments were admin areas where vascularisation is poor (FIG. 15A). istered daily via oral gavage. 0358 Mice treated with bicalutamide (2 mg/kg/day in 0373 OCT1002 (50 mg/kg in sterile PBS) was admin vehicle): vascularisation was reduced at days 7. On day istered intraperitoneally as a single dose at day 7. 7, two hours after intraperitoneal injection of a single dose of OCT1002 (50 mg/kg) large quantities of con 0374. On day 28 of treatment, animals were injected i.p. verted compound (OCT1001; blue) can be seen across with a solution of D-luciferin (150 mg/kg in PBS) 15 the whole tumour fragment (FIG. 15B). mins prior to imaging. 0359 Mice treated with bicalutamide (2 mg/kg/day in 0375 Animals were then killed and a range of tissues vehicle): vascularisation was reduced at days 7 and 14. were removed for the detection of bioluminescence this recovered by day 21 (Ming et al., 2012, Supra.). using the IVIS imaging system (Xenogen, USA). 0360 Tumours were re-examined at days 14 and 21. 0376 Images were taken for 5 minutes and quantifica 0361. OCT1001 (blue) is still localised to the tumour at tion of bioluminescence was achieved by drawing a day 14, by day 21 the amount of compound was consid region of interest around the area and measuring total erably lower (FIG. 15C). flux in photons/second (ph/sec). 0377. A range of tissues were excised, however only the Conclusions lungs and tumour showed measurable bioluminescence. 0362 OCT1002, administered intraperitoneally, dis The meants.e of bioluminescence in the lungs is shown tributed widely throughout the tumour fragments loca in FIG. 16; bicalutamide and vehicle treatment groups lised in the skin fold on the backs of the mice. (n=10); bicalutamide--OCT1002 group (n=3). and vehicle+OCT1002 (n=1). * Bicalutamide vs bicaluta 0363 Distribution was extensive even when the vascu mide+OCT1002 (p=0.024). Mice treated with vehicle lature was significantly decreased (i.e. by the bicaluta showed some metastatic spread to the lung. OCT1002, mide treatment at days 7 and 14). single dose day 7, had no effect on this spread. 0364 OCT1001 was found predominantly where the oxygen levels are low (i.e. areas of poor vascularisation); 0378 Bicalutamide appeared to increase the extent of Small areas were seen in the control also (indicating that metastatic spread although the result did not reach sig hypoxia can occur in untreated tumours but to a lesser nificance. eXtent. 0379 Combination of OCT1002 with bicalutamide 0365 Extensive localisation of OCT1001 was still showed that OCT1002 significantly reduces the meta observed at day 14 of bicalutamide treatment showing static spread to the lungs caused by bicalutamide. (P=0. that the compound remains for at least 7 days. 024) US 2015/0307441 A1 Oct. 29, 2015

Conclusions of —CH, —CHCH. —CH2CHCH, —CH2CH2OH. - CHCHCH-OH, CH(CH)CH-OH, 0380 OCT1002 given as a single dose at day 7 was able - CHCHOHCH-OH and deuterated forms thereof. to reduce significantly the metastatic spread to the lungs 10. The compound according to claim 1, wherein one or caused by bicalutamide treatment. two of X, X, X and X are independently selected from the 1. A compound of Formula I group consisting of NH-(CH), N(O)(CH), —NH (CH), N(O)(CH)CHs, NH-(CH), N(O)(CHs), O - NH-(CH), N(O)(CHCH-OH), NH-(CH), N X4 X (O)(CHCHCH-OH), -NH-(CH), N(O)CH(CH) sa OH, -NH-(CH), N(O)(CHCHOHCH-OH), and deu terated forms thereof. 11. The compound according to claim 1, wherein one or S. two of X, X, X and X are independently selected from the X3 X2 group consisting of —NH-(CH2): NCCH), —NH O (CH2), N(CH)CHs. —NH-(CH2): NCCHs), —NH-(CH), N(CHCH-OH), NH-(CH), N wherein X1,X2, X and X are each independently selected (CHCHCH-OH), - NH-(CH), NCH(CH)OH, from the group consisting of hydrogen, hydroxy, halo NH-(CH), N(CHCHOHCH-OH), and deuterated geno, amino, Calkoxy, Cs alkanoyloxy, —NH-A- forms thereof. NHR, NH-A-NR'R'' and NH-A-N(O) R'R" 12. The compound according to claim 1, which contains wherein A is an alkylene group with a chain length of at one group —NH-A-N(O)RR" and one group—NH-A-NHR, least two carbon atoms (between NH and NHR or the —NH-A-NHR group being selected from NH-(CH2) N(O)R'R"), NHCH, -NH (CH), NHCHs, NH-(CH) wherein R, R and R" are each independently selected NHCHCH-OH, -NH. (CH), NHCHCHCH-OH, from C alkyl groups and Ca hydroxyalkyl and NH-(CH), NHCH(CH)CH-OH, -NH-(CH)— C-dihydroxyalkyl groups in which the carbonatom NHCHCHOHCH-OH and deuterated forms thereof. attached to the nitrogenatom does not carry a hydroxy 13. The compound according to claim 1, which contains group and no carbon atom is Substituted by two one group—NH-A-NR'R'' and one group —NH-A-NHR, the hydroxy groups, or wherein R and R" together are a —NH-A-NHR group being selected from NH-(CH)— C. alkylene group which with the nitrogen atom to NHCH, -NH-(CH), NHCHs, NH-(CH)— which R" and R" are attached forms a heterocyclic NHCHCH-OH, -NH. (CH), NHCHCHCH-OH, group having 3 to 7 atoms in the ring, NH-(CH), NHCH(CH)CH-OH, -NH-(CH)— wherein at least one of X, X, X and X is selected from NHCHCHOHCH-OH and deuterated forms thereof. the group consisting of deuterated forms of —NH-A- 14. The compound according to claim 1, selected from the NHR, NH-A-NR'R'' and NH-A-N(O) R'R". group consisting of: 2. The compound according to claim 1 wherein the com (a) X = NH-A-N(O)RR", X= H and XX= OH: pound is of Formula II: (b) X = NH-A-N(O)RR", X= OH, X= OH and X = H: (c) X = NH-A-N(O)RR" and X=X=X= OH: X4 O X (d) X=X = NH-A-N(O)RR" and XX = OH: (e) X=X = NH-A-N(O)RR" and XX= OH: (f) X=X= NH-A-N(O)RR" and X=X= OH: (g) X = NH-A-NR'R'', X= H and XX OH: (h) X = NH-A-NR'R", X= OH at position 4, X = - OH and X, H: (i) X = NH-A-NR'R" and XXX= OH: X3 O X2 (i) X=X NH-A-NR'R" and X=X = OH: (k) X=X = NH-A-NR'R" and XX= OH: 3. The compound according to claim 1 wherein X, X2, X (1) X=X= NH-A-NR'R" and X=X= OH: and X are each separately selected from the group consisting and deuterated forms thereof. of hydrogen, hydroxy, NH-A-NHR, NH-A-NR'R", 15. The compound according to claim 1, selected from the —NH-A-N(O)RR" and deuterated forms thereof. group consisting of: 4. (canceled) (a) X = NH-A-N(O)R'R", X = NH-A-NHR, and 5. The compound according to claim3, wherein X and X XX= -OH: are both hydroxy and X and X are both NH-A-N(O)R'R" (b) X = NH-A-N(O)RR", X= OH, X= NH-A- or deuterated forms thereof. NHR and X= OH: 6. The compound according to claim3, wherein X and X (c) X = NH-A-N(O)RR", X=X= OH and X = - are both hydroxy and X and X are both NH-A-NR'R' or NH-A-NHR: deuterated forms thereof. (d) X = NH-A-NR'R", X = NH-A-NHR, and 7. (canceled) XX = -OH: 8. The compound according to claim 1 wherein A is ethyl (e) X = NH-A-NR'R", X= OH, X= NH-A-NHR CC. and X = OH: 9. The compound according to claim 1, wherein R, R and (f) X = NH-A-NR'R", XX= OH and X= NH-A- R" are each independently selected from the group consisting NHR; US 2015/0307441 A1 Oct. 29, 2015 20

and deuterated forms thereof. wherein Y are each independently selected from the group 16. The compound according to claim 14, selected from the consisting of hydrogen, hydroxy, halogeno, amino, Ca group consisting of alkoxy and C2-s alkanoxy (a) X=X = NH-A-N(O)R'R" and XX= OH: (b) X=X = NH-A-N(O)RR" and XX OH: or a prodrug thereof. (c) X=X = NH-A-NR'R" and X=X= OH; and 19. The compound according to claim 18 wherein the pro (d) X=X = NH-A-NR'R" and XX OH drug is a compound of Formula V or VI: wherein both - NH-A-N(O)RR" are NH-(CH)N(O)(CH), or —NH (CH)N(O)(CHCH-OH), or deuterated Formula V forms thereof and CD3 both NH-A-NR'R" are - NH-(CH)N(CH), or —NH-(CH)N(CHCH-OH), or deuterated forms - O thereof. Y o HN1)-1 17. The compound according to claim 15, selected from the CD group consisting of (a) X = NH-A-N(O)R'R", X = NH-A-NHR and XX OH: (b) X = NH-A-N(O)R'R", X= OH, X= NH-A- CD NHR and X= OH: (c) X = NH-A-NR'R", X = NH-A-NHR and Y O HNS-1a - O XX OH; and (d) X = NH-A-NR'R", X= OH, X= NH-A-NHR CD3 and X = OH, wherein - NH-A-N(O)RR" is NH-(CH)N(O)(CH), or —NH-(CH2)N(O)(CHCH-OH) or a deuterated Formula VI form thereof NH-A-NHR is NH-(CH),NHCH, or NH(CH.) p NHCHCH-OH or a deuterated form thereof O and NH-A-NR'R" is NH-(CH)N(CH), or -NH Y o HN1\1 (CH)N(CH2CH2OH) or a deuterated form thereof. CD3 18. The compound of claim 1, wherein the compound is of Formula III or IV:

CD 3 Formula III NH O Y lCD3 o-r- Y o HN1N1 NCD, CD

wherein Y are each independently selected from the group consisting of hydrogen, hydroxy, halogeno, amino, Ca alkoxy and C2-s alkanoxy. Y O HN 20. The compound according to claim 1, wherein the com n1N – CD3 pound is of Formula VII or VIII: CD3 Formula IV Formula VII CD p l N Y o HN1N1, NCD, oH o HN1 N1 NCD,

OH O HN N-- CD CD US 2015/0307441 A1 Oct. 29, 2015

-continued with a monoperoxyphthalate to under conditions suitable for Formula VIII the production of an N-oxide derivative of the alkylamino alkylaminoanthraquinone. p 27. The process according to claim 25 comprising reacting 1,4-difluoro-5,8-dihydroxyanthracene-9,10-dione, 281-005 OH O HN 1n-N CD3 with deuterated N,N-dimethylethylene-diamine under con ditions suitable for the production of 1,4-bis-2-(deuterated d6-dimethylamino)ethylamino)-5,8-dihydroxyanthracene 9,10-dione. 28. The process according to claim 27 further comprising the step of reacting the 1,4-bis-(2-(deuterated-d6-dimethy lamino)ethylamino)-5,8-dihydroxyanthracene-9,10-dione CD3 n1n-N" O OH with magnesium monoperoxyphthalate under conditions suitable for the production of 1,4-bis-(2-(deuterated-d6 CD3 dimethylamino-N-oxide)ethylamino)-5,8-dihydroxy-an thracene-9,10-dione. 29. (canceled) or a prodrug thereof. 30. (canceled) 21. The compound according to claim 20 wherein the pro 31. (canceled) drug is a compound of Formula IX or X: 32. (canceled) 33. (canceled) Formula IX 34. (canceled) CD3 35. (canceled) 36. (canceled) oH Q HN1N1 O 37. (canceled) CD3 38. (canceled) 39. (canceled) 40. (canceled) 41. (canceled) 42. (canceled) OH O HN 43. (canceled) n1n P- O 44. (canceled) 45. (canceled) CD3 46. (canceled) Formula X 47. (canceled) CD3 48. (canceled) 49. (canceled) oH Q HN1N1 O 50. (canceled) CD 51. (canceled) 52. (canceled) 53. (canceled) 54. A method of treating cancer in a patient comprising administering to the patient a therapeutically effective amount of a compound according to claim 1. f NH O OH 55. (canceled) 56. The method according to claim 54 for treating or pre o-r-CD3 venting the formation of metastases. 57. (canceled) 58. The method according to claim 54 further comprising 22. The compound according to claim 1, wherein the com administering to the patient one or more additional cancer pound is in the form of a halide salt, for example a chloride treatmentS. salt. 59. The method according to claim 58 wherein the one or 23. The pharmaceutical composition comprising a com more cancer treatmentsis/are selected from the group consist pound according to claim 1 together with pharmaceutically ing of anti-androgens (steroidal and non-steroidal), Vascular acceptable buffer, diluent, carrier, adjuvant or excipient. disrupting agents, anti-angiogenic agents, anti-VEGFR agents, IL8 inhibitors, NO synthase inhibitors, vasoconstrict 24. (canceled) ing agents, vasodilating agents, and radiotherapeutic modali 25. A process for making a compound according to claim 1, ties. comprising reacting an anthracene-9,10-dione with a deuter 60. The method according to claim 59 wherein the one or ated alkylenediamine under conditions suitable for the pro more chemotherapeutic and/or radiotherapeutic agents is/are duction of an alkylaminoalkyl-aminoanthraquinone. anti-androgens. 26. The process according to claim 25 further comprising 61. The method according to claim 60 wherein the non the step of reacting the alkylaminoalkylaminoanthraquinone steroidal anti-androgens comprise or consist of flutamide, US 2015/0307441 A1 Oct. 29, 2015 22 nilutamide, bicalutamide, finasteride, dutasteride, bexlos 78. (canceled) teride, iZonsteride, turosteride, epristeride and abiraterone. 79. (canceled) 62. The method according to claim 61 wherein the non 80. (canceled) steroidal anti-androgens comprise or consist of bicalutamide. 81. The method according to claim 54 wherein the cancer 63. Use of a compound according to claim 1, or a non is selected from the group consisting of bladder cancer, breast deuterated form thereof, as a marker of the oxygenation level cancer, bone cancer (primary and secondary, Such as osteosa of cells in vitro or in vivo. rcoma and Ewings sarcoma), brain cancer (including glio 64. (canceled) blastoma multiforme and astrocytoma), cervical cancer, cho 65. The use according to claim 63 wherein the compound is riocarcinoma, colon and rectal cancer, endometrial cancer, a cellular hypoxic marker. eye cancer, gallbladder cancer, gastric cancer, gestational 66. The use according to claim 63 in vitro. tumours, head and neck cancer, kidney (renal cell) cancer, 67. The use according to claim 63 in vivo. laryngeal cancer, leukaemias (Such as ALL, AML, CLL, 68. The use according to claim 67 wherein cells identified CML and hairy cell leukaemias), liver cancer, lung cancer, as being hypoxic are excised Surgically after their detection. lymphomas (such as Hodkin's lymphoma and non-Hodkins 69. The use of a compound according to claim 63 wherein lymphoma), melanoma, mesothelioma, mouth cancer, the compound is used in combination with a non-deuterated myeloma, nasal and sinus cancers, nasopharyngeal cancer, form of a compound as defined in claim 1. oesophageal cancer, ovarian cancer, pancreatic cancer, penile 70. The use of a compound according to claim 63 wherein cancer, prostate cancer, stomach cancer, testicular cancer, the compound(s) is/are detected using a method selected from thyroid cancer, uterine cancer, vaginal cancer, Vulvar cancer the group consisting of mass spectrometry, nuclear magnetic and womb cancer. resonance, infrared spectroscopy, colorimetrically, Raman 82. The method according to claim 54 for use in the treat spectroscopy, nuclear magnetic resonance, affinity capture ment of Solid tumours. methods, immunohistochemistry, flow cytometry, micros 83. The method according to claim 82 wherein the tumour, copy and antibody-based detection methods. or part thereof, is naturally hypoxic. 71. A kit of parts for use in detecting the oxygenation level of cells comprising a compound according to claim 1. 84. The method according to claim 58 wherein the one or 72. The kit according to claim 7170 further comprising a more cancer treatments is/are capable of decreasing tumour non-deuterated form of a compound according to claim 1. oxygenation in vivo. 73. (canceled) 85. The method according to claim 84 wherein the one or 74. (canceled) more cancer treatments is/are capable of lowering the median 75. (canceled) oxygen level of the tumour to below 3%, for example below 76. (canceled) 2.5%, 2%, 1.5%, 1%, 0.5%, 0.4%, 0.3%, 0.2% or below 0.1%. 77. (canceled) k k k k k