Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/16—Purine radicals
  • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
  • C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/16—Purine radicals
  • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
  • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
  • A61K31/7068—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines having oxo groups directly attached to the pyrimidine ring, e.g. cytidine, cytidylic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
  • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
  • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/02—Antineoplastic agents specific for leukemia
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/06—Pyrimidine radicals
  • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids

Definitions

• the present invention provides compounds useful in the treatment of cancer, e.g. leukaemia.
• the compounds comprise a salt of a diphosphate phosphoramidate.
• the present invention also provides formulations of said compounds and uses of said
• Nucleoside based drugs have become a powerful tool in the treatment of human disease.
• nucleoside drugs such as gemcitabine, clofarabine, cytarabine, fludarabine is widespread.
• nucleoside drugs can be improved by administration as a drug of the ProTide class.
• Drugs of the ProTide class are prodrugs of monophosphorylated nucleosides and have been shown to be particularly potent therapeutic agents in the fields of both antivirals and oncology. These compounds appear to avoid many of the resistance mechanisms which limit the utility of the parent nucleosides (see, for example,‘Application of ProTide Technology to Gemcitabine: A Successful Approach to Overcome the Key Cancer Resistance Mechanisms Leads to a New Agent (NUC-1031) in Clinical Development’ Slusarczyk et al; J. Med. Chem.] 2014, 57, 1531-1542; McGuigan et al.
• An exemplary ProTide is NUC-1031 , a ProTide of gemcitabine:
• Drugs of the ProTide class can also be poorly soluble in aqueous solvents and this can make administration challenging.
• R 1 is independently at each occurrence selected from: C 1 -C 24 -alkyl, C 3 -C 24 -alkenyl, C 3 -C 24 - alkynyl, C 0 -C 4 -alkylene-C 3 -C 8 -cycloalkyl and C 0 -C 4 -alkylene-aryl;
• R 2 and R 3 are each independently at each occurrence selected from H, CrC 6 -alkyl and Cr C 3 -alkylene-R 7 ; or R 2 and R 3 together with the atom to which they are attached form a 3- to 6-membered cycloalkyl or heterocycloalkyl group;
• R 4 is independently at each occurrence H or CrC 4 -alkyl
• R 4 a group selected from R 2 and R 3 and the atoms to which they are attached may form a 3- to 6-membered heterocycloalkyl group;
• R 5 is independently at each occurrence selected from aryl, 5-, 6-, 9- or 10-membered heteroaryl, C 3 -C 8 -cycloalkyl, 3- to 7-membered heterocycloalkyl, CrC 3 -alkylene-R 5a and CrC 8 -alkyl; said aryl being optionally fused to C 6 -C 8 -cycloalkyl;
• R 5a is independently at each occurrence selected from aryl, 5-, 6-, 9- or 10-membered heteroaryl, C 3 -C 8 -cycloalkyl, 3- to 7-membered heterocycloalkyl, said aryl being optionally fused to C 6 -C 8 -cycloalkyl;
• R 6 is independently selected from:
• R 8 is independently selected from H and
• Z 1 and Z 2 are each independently selected from O and S;
• Y is independently selected from H, F, Cl and OMe
• X is independently at each occurrence a pharmaceutically acceptable cation
• any aryl group is either phenyl or naphthyl
• R 1 , R 2 , R 3 , R 4 , R 5 or R 7 is an alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, that alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl group is optionally substituted with from 1 to 4 substituents selected from: halo, nitro, cyano, NR a R a ,
• R a is independently at each occurrence selected from: H and C 1 -C 4 -alkyl; and R b is independently at each occurrence selected from: H, C 1 -C 4 -alkyl and C(0)-C 1 -C 4 -alkyl.
• phosphoramidated diphosphates of formula (I) have good activities against a range of cancer cell lines, including solid tumour and
• diphosphates of formula (I) are more active than the corresponding ProTide.
• Diphosphate prodrugs reported in the literature such as for example nucleoside pyrophosphate diesters based on glicerides (K. Y. Hostetler et al. J. Biol. Chem. 1990, 265, 6112-6117; G. M. T. can Wijk, K. Y.Hostetler et al. Biochim. Biophys. Acta Lipids Lipid Metab. 1991 , 1084, 307-310) are known to be unstable due to cleavage of a bond of the P- O-P group, thus releasing the corresponding nucleoside monophosphate instead of desired diphosphate.
• the inventors have found, however, that deprotonation of the a-phosphate group of diphosphate phosphoramidates provides high stability.
• the ionic nature of the compounds of the invention also means that they are more soluble in water than the corresponding ProTide.
• a number of enzymatic processes are needed to convert a ProTide into the corresponding monophosphate nucleotide, including ester cleavage by carboxylesterases and cleavage of the resultant amino acid from the monophosphate nucleoside
• diphosphate phosphoramidates of the invention are larger molecules, having a different shape and they are ionic, casting doubt over whether they will interact with the relevant enzymes in the same way for conversion to the diphosphate nucleotide. Indeed, in preliminary data, the inventors have shown that the diphosphate phosphoramidates of formula (I) are stable with respect to carboxypeptidases, for example, carboxypeptidase Y, the enzymes that carry out the ester cleavage step in the conversion of drugs of the ProTide class to monophosphates. Nevertheless, the diphosphate phosphoramidates of formula (I) are stable with respect to carboxypeptidases, for example, carboxypeptidase Y, the enzymes that carry out the ester cleavage step in the conversion of drugs of the ProTide class to monophosphates. Nevertheless, the diphosphate phosphoramidates of formula (I) are stable with respect to carboxypeptidases, for example, carboxypeptidase Y, the enzymes
• diphosphate phosphoramidates of formula (I) are still active in cells, suggesting that an alternative mechanism of action is taking place compared to drugs of the ProTide class.
• the inventors have also shown that where there is an alkyloxy group on the phosphoramidate phosphorous (OR 5 in Formula I) the compounds of the invention are active. In drugs of the ProTide class, this is not typically the case and only compounds having aryloxy groups at this position provide good activity. This is understood to be because the aryloxy group acts as a leaving group during the activation of drugs of the ProTide class. Again, this indicates that the diphosphate phosphoramidates of the invention are activated via a different mechanism to drugs of the ProTide class.
• the compound of formula (I) is a compound of formula (II):
• Diphosphate phosphoramidates of clofarabine such as those of formula (II) were more active than the corresponding ProTide against a number of cancer cell lines, including the haematological cell lines.
• the compound of formula (I) is a compound of formula (III):
• the compound of formula (I) is a compound of formula (IV):
• the compound of formula (I) is a compound of formula (V):
• the compound of formula (I) is a compound of formula (VI):
• the compound of formula (I) is a compound of formula (VII):
• R 1 , R 2 , R 3 , R 5 and X are the same at both
• R 1 may be independently at each occurrence selected from: C 1 -C 24 -alkyl, C 3 -C 24 - alkenyl, C 3 -C 24 -alkynyl, C 0 -C 4 -alkylene-C 3 -C 6 -cycloalkyl and C 0 -C 4 -alkylene-aryl.
• R 1 may be independently at each occurrence selected from: C 1 -C 24 -alkyl, C0-C 4 - alkylene-Cs-C 8 -cycloalkyl and CH 2 -aryl.
• R 1 may be independently at each occurrence selected from: CrCio-alkyl, C 4 - C 6 -cycloalkyl and benzyl.
• R 1 may be independently at each occurrence selected from: CrCe-alkyl, C 6 -cycloalkyl and benzyl.
• R 1 may be C 1 -C 8 alkyl.
• R 1 may be selected such that it comprises three or more carbon atoms.
• R 1 may be selected such that it comprises five or more carbon atoms.
• R 1 may therefore be selected such that it includes six or more carbon atoms.
• R 1 is preferably selected such that it comprises only carbon and hydrogen atoms.
• R 1 may be selected from C 5 -Cycycloalkyl, C 5 - C 8 -alkyl and benzyl, optionally wherein said groups are unsubstituted.
• R 1 may be unsubstituted benzyl.
• R 1 may be neopentyl.
• R 1 may be ethyl.
• R 3 is H. It may be that R 3 is C 1 -C 4 -alkyl. It may be that R 3 is methyl. It may be that R 2 is selected from C 1 -C 6 -alkyl and C 1 -C 3 -alkylene-R 7 . It may be that R 2 is Cr C 4 -alkyl. It may be that R 2 is selected from methyl and isopropyl. R 2 may be methyl. R 2 may be H.
• R 4 is preferably H.
• R 4 a group selected from R 2 and R 3 , and the atoms to which they are attached form a 3- to 6-membered heterocycloalkyl group. It may be that R 4 , a group selected from R 2 and R 3 , and the atoms to which they are attached do not form a 3- to 6- membered heterocycloalkyl group.
• R 2 and R 3 are each independently at each occurrence selected from H, C 1 -C 6 -alkyl and C 1 -C 3 -alkylene-R 7 ; or R 2 and R 3 together with the atom to which they are attached form a 3- to 6-membered heterocycloalkyl group; and R 4 is independently at each occurrence H or C 1 -C 4 -alkyl.
• R 5 is substituted or unsubstituted phenyl, which may be optionally fused to a C 6 -C 8 -cycloalkyl ring, e.g. a cyclohexane ring. It may be that R 5 is substituted or unsubstituted phenyl. It may be that R 5 is substituted or unsubstituted naphthyl (e.g. 1- naphthyl). Preferably, R 5 is selected from unsubstituted phenyl or unsubstituted naphthyl (e.g. 1-naphthyl). Thus, R 5 may be unsubstituted phenyl. Alternatively, R 5 may be unsubstituted naphthyl (e.g. 1 -naphthyl).
• R 6 may be
• R 6 may be
• R 6 may be
• R 6 may be
• Y may be H.
• Y may be F.
• R 8 maybeH.
• R 8 may be Z 2 may be S.
• Z 2 may be
• R 8 is it may be that R 1 , R 2 , R 3 , R 5 , X and Z 2
• R 6 may be
• R 6 may be
• R 6 may be
• the compound of formula (I) may be selected from:
• X + may be a metal cation or it may be an ammonium cation.
• X + may be a metal cation, e.g. a cation of an alkali or alkali earth metal.
• X + may be an ammonium cation, e.g. a trialkylammonium cation or an ammonium cation of a nitrogen heterocycle.
• Illustrative cations include those derived from aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine, triethylamine and zinc cations.
• X + may be a triethylamine cation.
• Compounds of the invention comprise a chiral centre at the phosphorus atom (b- phosphorus) that is bonded to OR 5 .
• the compound may be present as a mixture of phosphate diastereoisomers, as the (S)-epimer at the phosphorus atom in substantially diastereomerically pure form or as the ( )-epimer at the phosphorus atom in substantially diastereomerically pure form.
• ‘Substantially diastereomerically pure’ is defined for the purposes of this invention as a diastereomeric purity of greater than about 90%. If present as a substantially diastereoisomerically pure form, the compound may have a
• the compound may be present as a mixture of phosphate diastereoisomers.
• the ( R )- and/or (S)-epimers of the compound can be obtained in substantially diastereomerically pure form by chromatography, e.g. HPLC optionally using a chiral column.
• the (R)- and/or (S)-epimers of the compound can be obtained in substantially diastereomerically pure form by crystallisation from an appropriate solvent or solvent system.
• a compound of formula (I), or a pharmaceutically acceptable salt thereof for use in a method of treatment.
• a compound of formula (I), or a pharmaceutically acceptable salt thereof for use in the prophylaxis or treatment of cancer.
• a compound of formula (I), or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the prophylaxis or treatment of cancer.
• a method of prophylaxis or treatment of cancer comprising administration to a patient in need of such treatment an effective dose of a compound of formula (I), or a pharmaceutically acceptable salt thereof.
• embodiments of the invention comprise a cancer selected from but not restricted to the group consisting of: pancreatic cancer, bladder cancer, other urothelial cancers (e.g.
• cancers of ureter and renal pelvis include gastrointestinal cancers (also known as cancer of the digestive tract, including: oesophageal cancer, gastric cancer, stomach cancer, bowel cancer, small intestine cancer, colon cancer, colorectal cancer, appendix mucinous, goblet cell carcinoid, liver cancer, biliary cancer, gallbladder cancer, anal cancer and rectal cancer), lung cancer, renal (or kidney) cancer, biliary cancer, prostate cancer, cholangiocarcinoma, neuroendocrine cancer, sarcoma, lymphoma, thymic cancer, glioblastoma multiforme, a cancer of an unknown primary origin, mesothelioma, adrenal cancer, testicular cancer, cancer of the central nervous system, basal cell carcinoma, Bowens disease, other skin cancers (such as malignant melanoma, merckel cell tumour and rare appendage tumours), ocular surface squamous neoplasia, germ cell
• the cancer is a leukaemia or a lymphoma.
• the cancer may be a leukaemia.
• leukaemia There are four main types of leukaemia depending on whether they are chronic or acute, and myeloid or lymphoid in origin. These are: acute myeloblastic leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myelogenous leukaemia (CML) and chronic lymphocytic leukaemia (CLL).
• AML acute myeloblastic leukaemia
• ALL acute lymphoblastic leukaemia
• CML chronic myelogenous leukaemia
• CLL chronic lymphocytic leukaemia
• BAL biphenotypic acute leukaemia
• the leukaemia is selected from the group consisting of: acute myeloblastic leukaemia (AML), acute lymphoblastic leukaemia (ALL), chronic myelogenous leukaemia (CML), chronic lymphocytic leukaemia (CLL) and biphenotypic acute leukaemia (BAL; which is a mix of AML and ALL).
• AML acute myeloblastic leukaemia
• ALL acute lymphoblastic leukaemia
• CML chronic myelogenous leukaemia
• CLL chronic lymphocytic leukaemia
• BAL biphenotypic acute leukaemia
• Chronic lymphocytic leukaemia includes the subtypes: B-cell CLL (B-CLL), B- cell prolymphocytic leukaemia (PLL) and T-cell chronic prolymphocytic leukaemia (T-PLL) and several subtypes that differ at the genetic level.
• Acute lymphoblastic leukaemia includes the subtypes: precursor B-cell ALL (B-ALL), precursor T-cell ALL (T-ALL), Burkitt-type ALL and Philadelphia chromosome positive (BCR-ABL fusion) ALL.
• Acute myeloblastic leukaemia includes the subtypes: myeloid leukaemia, monocytic leukaemia and acute promyelocytic leukaemia (APL).
• Chronic myelogenous leukaemia includes the subtypes: chronic granulocytic leukaemia (CGL) (CGL), Juvenile CML (JCML), chronic neutrophilic leukaemia (CNL), chronic myelomonocytic leukaemia (CMML) and atypical CML (aCML).
• any of the above sub-types of CLL, ALL, AML and CML are suitable for treatment with the compounds of the invention or pharmaceutical compositions containing them
• erythroleukemia arising from red blood cell precursors
• lymphoma that has gone into the blood.
• the cancer may be a lymphoma, e.g. a solid lymphoma.
• lymphoma There are two main types of lymphoma: Hodgkin’s lymphoma and non-Hodgkin’s lymphoma. Within each of these there are various subtypes.
• the lymphoma is selected from the group consisting of: Hodgkin’s lymphoma and non-Hodgkin’s lymphoma.
• the lymphoma is selected from the group consisting of: Burkitt’s lymphoma (BL), mantle cell lymphoma (MCL), follicular lymphoma (FL), small lymphocytic lymphoma (SLL) indolent B-cell non-Hodgkin’s lymphoma, histiocytic lymphoma (aka immunoblastic lymphoma; IBL) and diffuse large B-cell lymphoma (DLBCL), including activated-cell like diffuse large B-cell lymphoma (DLBCL-ABC) and germinal center B-cell like diffuse large B-cell lymphoma (DLBCL-GCB).
• BL Burkitt’s lymphoma
• MCL mantle cell lymphoma
• FL follicular lymphoma
• SLL small lymphoc
• the cancer is a gastrointestinal cancer and can be selected from the group consisting of: oesophageal cancer, gastric cancer, stomach cancer, bowel cancer, small intestine cancer, colon cancer, colorectal cancer, appendix mucinous, goblet cell carcinoid, liver cancer, biliary cancer, gallbladder cancer, anal cancer and rectal cancer.
• the cancer is of gynaecological origin and can be selected from the group consisting of: a cancer of the uterus, cancer of the fallopian tube, cancer of the endometrium, cancer of the ovary, cancer of the peritoneum and cancer of the cervix).
• the ovarian cancer may be epithelial ovarian cancer.
• the peritoneal cancer may be primary peritoneal cancer.
• the cancer may be selected from, but not restricted to pancreatic cancer, lung cancer, bladder cancer, breast cancer, biliary cancer, lymphoma, a leukaemia, a gastrointestinal cancer and a gynaecological cancer.
• the compounds of the invention have been found to retain activity even under hypoxic conditions. Cancers particularly associated with hypoxia are pancreatic and renal (or kidney) cancers.
• the cancer may be relapsed.
• the cancer may be metastatic.
• the cancer may be previously untreated.
• the cancer may be refractory cancer that has previously been treated but has proven unresponsive to prior treatment.
• the cancer patient may be intolerant of a previous therapy, for example, may develop side effects that make the patient intolerant to further treatment with the agent being administered.
• composition comprising a compound of formula (I), or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient.
• the pharmaceutical composition may be for use in the prophylaxis or treatment of cancer, e.g. a cancer or group of cancers mentioned above.
• the compound of formula (I) may be as described in the following numbered paragraphs:
• R 1 is independently at each occurrence selected from: C 1 -C 24 -alkyl, C 3 -C 24 -alkenyl, C 3 -C 24 - alkynyl, C 0 -C 4 -alkylene-C 3 -C 6 -cycloalkyl or C 0 -C 4 -alkylene-aryl;
• R 2 and R 3 are each independently at each occurrence selected from H, C 1 -C 6 -alkyl and Cr C 3 -alkylene-R 7 ; or R 2 and R 3 together with the atom to which they are attached form a 3- to 6-membered cycloalkyl or heterocycloalkyl group;
• R 4 is independently at each occurrence H or CrC 4 -alkyl
• R 5 is independently at each occurrence selected from aryl and 5-, 6-, 9- or 10-membered heteroaryl;
• R 6 is independently selected from:
• R 8 is independently selected from H and
• Y is independently selected from H, F, Cl and OMe
• X is independently at each occurrence a pharmaceutically acceptable cation
• any aryl group is either phenyl or naphthyl
• R 1 , R 2 , R 3 , R 4 , R 5 or R 7 is an alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl, that alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl group is optionally substituted with from 1 to 4 substituents selected from: halo, nitro, cyano, NR a R a ,
• R a is independently at each occurrence selected from: H and C 1 -C 4 -alkyl; and R b is independently at each occurrence selected from: H, and C 1 -C 4 -alkyl and C(0)-C 1 -C 4 -alkyl.
• R 1 is selected from C5-C7- cycloalkyl, CrC 8 -alkyl and benzyl.
• a compound of paragraph 14 wherein R 1 is benzyl. 16. A compound of paragraph 14, wherein R 1 is CrCe-alkyl, e.g. ethyl.
• the cancer is a leukaemia selected from the group consisting of acute lymphoblastic leukaemia, acute myelogenous leukaemia, myelodysplastic syndromes, acute promyelocytic leukaemia, acute lymphocytic leukaemia, chronic myelogenous leukaemia, chronic lymphocytic leukaemia, monoblastic leukaemia and hairy cell leukaemia.
• a leukaemia selected from the group consisting of acute lymphoblastic leukaemia, acute myelogenous leukaemia, myelodysplastic syndromes, acute promyelocytic leukaemia, acute lymphocytic leukaemia, chronic myelogenous leukaemia, chronic lymphocytic leukaemia, monoblastic leukaemia and hairy cell leukaemia.
• a pharmaceutical composition comprising compounds of any one of claims 1 to 22 and at least one pharmaceutically acceptable excipient.
• Figure 1 shows sigmoidal dose-response curves for (A) clofarabine, (B) Example 1 and (C) Example 2. Clofarabine and Example 1 showed the highest potency. All assays were carried out using KG 1a cells and data are presented as mean ( ⁇ SD) of three independent experiments.
• Figure 2 shows analysis of the LSC targeting capacity of clofarabine Example 1 and Example 2. Under normoxic conditions, all of the compounds showed stem cell targeting at concentrations above 1x10 7 M. All data are the mean ( ⁇ SD) of three independent experiments.
• Figure 3 shows comparison of the fraction of KG1a cells expressing an LSC phenotype under normoxic and hypoxic conditions.
• Figure 4 shows comparison of the mean LD50 values for (A) clofarabine, (B) Example 1 , and (C) Example 2 under normoxic and hypoxic conditions. Clofarabine showed a significant loss in potency under hypoxic conditions. In contrast, the compounds of the invention maintained their potency under hypoxic conditions. All data are the mean ( ⁇ SD) of three independent experiments.
• Figure 5 shows comparison of the DNA damage, as measured by gH2A.C phosphorylation assay, induced in purified LSCs and bulk tumour cells following 2-hour exposure to (A) clofarabine, (B) Example 1 and (C) Example 2 under hypoxic conditions (5% O2). All data are the mean ( ⁇ SD) of three independent experiments.
• Saline is intended to refer to an aqueous solution of sodium chloride.
• Saline solutions of the present invention will typically be sterile and will typically be at a concentration suitable for use in parenteral administration. Suitable concentrations are up to 2 w/v% or up to 1 w/v%. To optimise osmolarity different concentrations of saline can be used in the formulations of the invention, e.g. 0.9% or 0.45%.
• formulations of the present invention can be used in the treatment of the human body. They may be used in the treatment of the animal body.
• Suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, malic, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benzenesulphonic, salicylic, sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and va
• Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts. Hemisalts of acids and bases may also be formed, for example, hemisulfate, hemioxalate and hemicalcium salts. [0076] For the above-mentioned formulations of the invention the dosage administered will, of course, vary with the compound employed, the precise mode of administration, the treatment desired and the disorder indicated.
• Dosage levels, dose frequency, and treatment durations of compounds of the invention are expected to differ depending on the formulation and clinical indication, age, and co-morbid medical conditions of the patient.
• the size of the dose for therapeutic purposes of compounds of the invention will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well known principles of medicine.
• a pharmaceutical formulation typically takes the form of a composition in which active compounds, or pharmaceutically acceptable salts thereof, are in association with a pharmaceutically acceptable adjuvant, diluent or carrier.
• a pharmaceutically acceptable adjuvant, diluent or carrier in the formulations of the invention is the polar aprotic solvent.
• the formulations may be suitable for topical application (e.g. to the skin or bladder), for oral administration or for parenteral (e.g. intravenous administration).
• topical application e.g. to the skin or bladder
• parenteral e.g. intravenous administration
• Any solvents used in pharmaceutical formulations of the invention should be pharmaceutical grade, by which it is meant that they have an impurity profile which renders them suitable for administration (e.g. intravenous administration) to humans.
• the formulations of the invention may comprise the active compound admixed with an adjuvant or a carrier, for example, lactose, saccharose, sorbitol, mannitol; a starch, for example, potato starch, corn starch or amylopectin; a cellulose derivative; a binder, for example, gelatine or polyvinylpyrrolidone; and/or a lubricant, for example, magnesium stearate, calcium stearate, polyethylene glycol, a wax, paraffin, and the like, and then compressed into tablets.
• an adjuvant or a carrier for example, lactose, saccharose, sorbitol, mannitol
• a starch for example, potato starch, corn starch or amylopectin
• a cellulose derivative for example, gelatine or polyvinylpyrrolidone
• a lubricant for example, magnesium stearate, calcium stearate, polyethylene glycol
• the cores may be coated with a concentrated sugar solution which may contain, for example, gum arabic, gelatine, talcum and titanium dioxide.
• a concentrated sugar solution which may contain, for example, gum arabic, gelatine, talcum and titanium dioxide.
• the tablet may be coated with a suitable polymer dissolved in a readily volatile organic solvent.
• the active compounds may be admixed with, for example, a vegetable oil or polyethylene glycol.
• Hard gelatine capsules may contain granules of the compound using either the above-mentioned excipients for tablets.
• liquid or semisolid formulations of the active compounds may be filled into hard gelatine capsules.
• Liquid preparations for oral application may be in the form of syrups or suspensions, for example, solutions containing the compound of the invention, the balance being sugar and a mixture of ethanol, water, glycerol and propylene glycol.
• such liquid preparations may contain colouring agents, flavouring agents, sweetening agents (such as saccharine), preservative agents and/or carboxymethylcellulose as a thickening agent or other excipients known to those skilled in art.
• the formulations may be for parenteral (e.g. intravenous) administration.
• parenteral (e.g. intravenous) administration the active compounds may be administered as a sterile aqueous or oily solution.
• the active compounds are administered as a sterile aqueous solution.
• the aqueous solution may further comprise at least one surfactant and/or organic solvent.
• organic solvents include dimethylacetamide, ethanol, ethyleneglycol, /V-methyl-pyrrolidinone, dimethylsulfoxide, dimethylformamide and isopropanol.
• surfactants include polyethoxylated fatty acids and fatty acid esters and mixtures thereof.
• Suitable surfactants include polyethoxylated castor oil (e.g. that sold under the trade name Kolliphor ® ELP); or polyethoxylated stearic acid (e.g. that sold under the trade names Solutol ® or Kolliphor ® HS15); or polyethoxylated (e.g. polyoxyethylene (20)) sorbitan monooleate, (e.g. that are sold under the trade names Polysorbate 80 or Tween ® 80).
• polyethoxylated castor oil e.g. that sold under the trade name Kolliphor ® ELP
• polyethoxylated stearic acid e.g. that sold under the trade names Solutol ® or Kolliphor ® HS15
• polyethoxylated e.g. polyoxyethylene (20)
• sorbitan monooleate e.g. that are sold under the trade names Polysorbate 80 or Tween ® 80.
• the pharmaceutical composition of the invention will preferably comprise from 0.05 to 99 %w (per cent by weight) compound of the invention, more preferably from 0.05 to 80 %w compound of the invention, still more preferably from 0.10 to 70 %w compound of the invention, and even more preferably from 0.10 to 50 %w compound of the invention, all percentages by weight being based on total composition.
• Cyclodextrins have been shown to find wide application in drug delivery (Rasheed et al, Sci. Pharm., 2008, 76, 567-598). Cyclodextrins are a family of cyclic oligosaccharides. They act as a‘molecular cage’ which encapsulates drug molecules and alters properties of those drug molecules such as solubility. Cyclodextrins comprise (a-1 ,4)-linked a-D- glucopyranose units. Cyclodextrins may contains 6, 7 or 8 glucopyranose units (designated a-, b- and g-cyclodextrins respectively).
• Cyclodextrins used in pharmaceutical formulations are often b-cyclodextrins.
• the pendant hydroxyl groups can be alkylated with a C1-C6 substituted or unsubstituted alkyl group.
• Examples of cyclodextrins are a-cyclodextrin, b- cyclodextrin, g-cyclodextrin, 2-hydroxypropyl ⁇ -cyclodextrin (HR-b-CD), sulfobutylether b- cyclodextrin sodium salt, partially methylated b-cyclodextrin.
• the formulations of the invention may also comprise at least one cyclodextrin.
• C m -C n refers to a group with m to n carbon atoms.
• alkyl refers to a linear or branched hydrocarbon group.
• An alkyl group is monovalent.
• CrC 6 -alkyl may refer to methyl, ethyl, n-propyl, /so-propyl, 17- butyl, sec-butyl, tert- butyl, n-pentyl and n-hexyl.
• the alkyl groups are preferably
• alkylene refers to a linear hydrocarbon chain.
• An alkylene group is divalent.
• Ci.alkylene may refer to a CH2 group.
• C2-alkylene may refer to - CH2CH2- group.
• the alkylene groups are preferably unsubstituted.
• haloalkyl refers to a hydrocarbon chain substituted with at least one halogen atom independently chosen at each occurrence from: fluorine, chlorine, bromine and iodine.
• the halogen atom may be present at any position on the hydrocarbon chain.
• CrC 4 -haloalkyl may refer to chloromethyl, fluoromethyl, trifluoromethyl, chloroethyl e.g. 1 -chloromethyl and 2-chloroethyl, trichloroethyl e.g. 1 ,2,2-trichloroethyl, 2,2,2- trichloroethyl, fluoroethyl e.g.
• a halo alkyl group may be a fluoroalkyl group, i.e. a hydrocarbon chain substituted with at least one fluorine atom.
• alkenyl refers to a branched or linear hydrocarbon chain containing at least one carbon-carbon double bond.
• the double bond(s) may be present as the E or Z isomer.
• the double bond may be at any possible position of the hydrocarbon chain.
• “C2-C 4 -alkenyl” may refer to ethenyl, allyl and butenyl.
• the alkenyl groups are preferably unsubstituted.
• alkynyl refers to a branched or linear hydrocarbon chain containing at least one carbon-carbon triple bond.
• the triple bond may be at any possible position of the hydrocarbon chain.
• “C2-C 6 -alkynyl” may refer to ethynyl, propynyl, butynyl.
• the alkynyl groups are preferably unsubstituted.
• cycloalkyl refers to a saturated hydrocarbon ring system containing 3, 4, 5 or 6 carbon atoms.
• “3- to 6- membered cycloalkyl” may refer to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl.
• the cycloalkyl groups are preferably unsubstituted.
• heterocycloalkyl may refer to a saturated or partially saturated monocyclic group comprising 1 or 2 heteroatoms independently selected from O, S and N in the ring system (in other words 1 or 2 of the atoms forming the ring system are selected from O, S and N).
• heterocycloalkyl groups include; piperidine, piperazine, morpholine, thiomorpholine, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, tetrahydropyran, dihydropyran, dioxane, azepine.
• the heterocycloalkyl groups are preferably unsubstituted or substituted.
• the present invention also includes formulations of all pharmaceutically acceptable isotopically-labelled forms of compound wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number of the predominant isotope usually found in nature.
• isotopes suitable for inclusion in the compounds of the invention include isotopes of hydrogen, such as 2 H and 3 H, carbon, such as 11 C, 13 C and 14 C, chlorine, such as 36 CI, fluorine, such as 18 F, iodine, such as 123 l and 125 l, nitrogen, such as 13 N and 15 N, oxygen, such as 15 0, 17 0 and 18 0, phosphorus, such as 32 P, and sulphur, such as 35 S.
• Radioactive isotopes tritium, i.e. 3 H, and carbon-14, i.e. 14 C, and 18 F are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.
• Isotopically-labelled compounds can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described using an appropriate isotopically-labelled reagent in place of the non-labelled reagent previously employed.
• the method of treatment or the formulation for use in the treatment of cancer may involve, in addition to the formulations of the invention, conventional surgery or radiotherapy or chemotherapy.
• Such chemotherapy may include the administration of one or more other active agents.
• a further active agent is administered as part of a method of treatment of the invention
• such combination treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment.
• Such combination products employ the compounds of this invention within a therapeutically effective dosage range described hereinbefore and the one or more other pharmaceutically- active agent(s) within its approved dosage range.
• the pharmaceutical formulations of the invention may comprise another active agent.
• the one or more other active agents may be one or more of the following categories of anti-tumour agents:
• antiproliferative/antineoplastic drugs and combinations thereof such as alkylating agents (for example cyclophosphamide, nitrogen mustard, bendamustin, melphalan, chlorambucil, busulphan, temozolamide and nitrosoureas); antimetabolites (for example gemcitabine and antifolates such as fluoropyrimidines like 5-fluorouracil and tegafur, raltitrexed, methotrexate, pemetrexed, cytosine arabinoside, and hydroxyurea); antibiotics (for example anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic agents (for example vinca alkaloids like vincristine, vinblastine, vindesine and vinorelbine and taxoids like taxol and tax
• epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan, mitoxantrone and camptothecin);
• cytostatic agents such as antiestrogens (for example tamoxifen, fulvestrant, toremifene, raloxifene, droloxifene and iodoxyfene), antiandrogens (for example
• bicalutamide, flutamide, nilutamide and cyproterone acetate), LHRH antagonists or LHRH agonists for example goserelin, leuprorelin and buserelin
• progestogens for example megestrol acetate
• aromatase inhibitors for example as anastrozole, letrozole, vorazole and exemestane
• inhibitors of 5a-reductase such as finasteride
• anti-invasion agents for example dasatinib and bosutinib (SKI-606), and
• metalloproteinase inhibitors inhibitors of urokinase plasminogen activator receptor function or antibodies to Heparanase;
• inhibitors of growth factor function include growth factor antibodies and growth factor receptor antibodies, for example the anti-erbB2 antibody trastuzumab [HerceptinTM], the anti-EGFR antibody panitumumab, the anti-erbB1 antibody cetuximab, tyrosine kinase inhibitors, for example inhibitors of the epidermal growth factor family (for example EGFR family tyrosine kinase inhibitors such as gefitinib, erlotinib and 6- acrylamido-/ ⁇ /-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)-quinazolin-4-amine (Cl 1033), erbB2 tyrosine kinase inhibitors such as lapatinib); inhibitors of the hepatocyte growth factor family; inhibitors of the insulin growth factor family; modulators of protein regulators of cell apoptosis (for example Bcl-2 inhibitors
• antiangiogenic agents such as those which inhibit the effects of vascular endothelial growth factor, [for example the anti-vascular endothelial cell growth factor antibody bevacizumab (AvastinTM); thalidomide; lenalidomide; and for example, a VEGF receptor tyrosine kinase inhibitor such as vandetanib, vatalanib, sunitinib, axitinib and pazopanib;
• vascular endothelial growth factor for example the anti-vascular endothelial cell growth factor antibody bevacizumab (AvastinTM); thalidomide; lenalidomide; and for example, a VEGF receptor tyrosine kinase inhibitor such as vandetanib, vatalanib, sunitinib, axitinib and pazopanib;
• immunotherapy approaches including for example adoptive ceil transfer, such as CAR T-cel! therapy; antibody therapy such as alemtuzumab, rituximab, ibritumomab tiuxetan (Zevalin®) and ofatumumab; interferons such as interferon a; interleukins such as IL-2 (aldesleukin); interleukin inhibitors for example IRAK4 inhibitors; cancer vaccines including prophylactic and treatment vaccines such as HPV vaccines, for example Gardasil, Cervarix, Oncophage and Sipuleucel-T (Provenge); and toll-like receptor modulators for example TLR- 7 or TLR-9 agonists;
• adoptive ceil transfer such as CAR T-cel! therapy
• antibody therapy such as alemtuzumab, rituximab, ibritumomab tiuxetan (Zevalin®) and ofatumumab
• cytotoxic agents for example fludarabine (fludara), cladribine, pentostatin (NipentTM);
• steroids such as corticosteroids, including glucocorticoids and mineralocorticoids, for example aclometasone, aclometasone dipropionate, aldosterone, amcinonide,
• beclomethasone beclomethasone dipropionate, betamethasone, betamethasone
• betamethasone sodium phosphate betamethasone valerate
• budesonide clobetasone
• clobetasone butyrate clobetasol propionate
• cloprednol cortisone, cortisone acetate, cortivazol, deoxycortone, desonide, desoximetasone, dexamethasone,
• hydrocortisone valerate icomethasone
• icomethasone enbutate meprednisone
• methylprednisolone mometasone paramethasone, mometasone furoate monohydrate, prednicarbate, prednisolone, prednisone, tixocortol, tixocortol pivalate, triamcinolone, triamcinolone acetonide, triamcinolone alcohol and their respective pharmaceutically acceptable derivatives.
• a combination of steroids may be used, for example a combination of two or more steroids mentioned in this paragraph;
• (x) targeted therapies for example PI3Kd inhibitors, for example idelalisib and perifosine; or checkpoint inhibitor compounds including anti-PD-1 , anti- PD-L1 and anti-CTLA4 molecules, such as nivolumab, pembrolizumab, pidilizumab, atezolizumab, durvalumab and avelumab.
• PI3Kd inhibitors for example idelalisib and perifosine
• checkpoint inhibitor compounds including anti-PD-1 , anti- PD-L1 and anti-CTLA4 molecules, such as nivolumab, pembrolizumab, pidilizumab, atezolizumab, durvalumab and avelumab.
• the one or more other active agents may also be antibiotic.
• nucleoside 5'- monophosphate triethylammonium salt B was prepared by negative ion mass spectrometry.
• a 0.5 M solution of TEAB was prepared by bubbling C0 2 through a 0.5 M triethylamine (TEA) solution in water at 0-4°C for 30-45 min (pH of approximately 7.4-7.6, unless stated otherwise)
• nucleoside 5'-monophosphate triethylammonium salt in the first step was accomplished following modified reference: El-Tayeb A. et al. J. Med. Chem. 2006, 49(24), 7076-7087.
• Example 1 2-Chloro-9-(2'-Deoxy-2'-fluoro-fi-D-arabinofuranosyl)adenine-5'-0- [phenyl(benzoxy-L-alaninyl)] diphosphate triethylammonium salt.
• Example 2 2-Chloro-9-(2'-Deoxy-2'-fluoro-fi-D-arabinofuranosyl)adenine-5'-0- [phenyl(ethoxy-L-alaninyl)] diphosphate triethylammonium salt.
• Example 4 5-Fluoro-2'- deoxyuridine-5’-0-[1 -naphthyl-(benzoxy-L-a/a/?//?y/)7 diphosphate triethylammonium salt.
• Example 5 8-Chloroadenosine-5'-0-[1-naphthyl-(benzoxy-L- alaninyl)]diphosphate triethylammonium salt.
• Examples 6 and 7 3'-Deoxyadenosine-5'-0-[phenyl(benzoxy-L- alaninyl)]diphosphate triethylammonium salt (6) and 3'-Deoxyadenosine-5',2'-bis-0- [phenyl(benzoxy-L-alaninyl)]-diphosphate triethylammonium salt (7).
• Example 8 2'-Deoxy-2',2'-difluoro-D-cytidine-5'-0-[phenyl(benzoxy-L-alaninyl)] diphosphate triethylammonium salt.
• Example 9 2-Chloro-9-(2’-Deoxy-2’-fluoro-fi-D-arabinofuranosyl)adenine-5’-O- [phenyl(neopentoxy-L-alaninyl)] diphosphate triethylammonium salt.
• Example 10 2-Chloro-9-(2 '-Deoxy-2’-fluoro-fi-D-arabinofuranosyl)adenine-5’-O- [phenyl(benzoxy-dimethylglycinyl)] diphosphate triethylammonium salt.
• Example 11 2-Chloro-9-(2 '-Deoxy-2’-fluoro-fi-D-arabinofuranosyl)adenine-5’-0-[ 1 - naphthyl(benzoxy-dimethylglycinyl)] diphosphate triethylammonium salt.
• Example 12 2-Chloro-9-(2 '-Deoxy-2 '-fluoro-fi-D-arabinofuranosyl)adenine-5’-O- [phenyl(cyclohexoxy-L-valinyl)] diphosphate triethylammonium salt.
• Example 13 2-Chloro-9-(2 '-Deoxy-2 '-fluoro-fi-D-arabinofuranosyl)adenine-5’-0-[ 1 - naphthyl(neopentoxy-L-alaninyl)] diphosphate triethylammonium salt.
• Example 16 2-Chloro-9-(2 '-Deoxy-2’-fluoro-fi-D-arabinofuranosyl)adenine-5’-0-[ 1 - phenyl(methoxy-L-alaninyl)] diphosphate triethylammonium salt.
• Example 18 2-Chloro-9-(2 '-Deoxy-2 '-fluoro-fi-D-arabinofuranosyl)adenine-5’-0-[ 1 - phenyl(hexoxy-L-alaninyl)] diphosphate triethylammonium salt.
• Example 21 2-Chloro-9-(2 '-Deoxy-2 '-fluoro-fi-D-arabinofuranosyl)adenine-5’-0-[ 1 - phenyl(benzoxy-L-leucinyl)] diphosphate triethylammonium salt.
• Example 22 2-Chloro-9-(2 '-Deoxy-2 '-fluoro-fi-D-arabinofuranosyl)adenine-5’-0-[ 1 - phenyl(benzoxy-L-phenylalaninyl)] diphosphate triethylammonium salt.
• Example 23 2-Chloro-9-(2 '-Deoxy-2 '-fluoro-fi-D-arabinofuranosyl)adenine-5’-0-[ 1 - phenyl(benzoxy-L-prolinyl)] diphosphate triethylammonium salt.
• Example 24 2-Chloro-9-(2 '-Deoxy-2 '-fluoro-fi-D-arabinofuranosyl)adenine-5’-0-[ 1 - phenyl(methoxy-L-metioninyl)] diphosphate triethylammonium salt.
• Example 25 2-Chloro-9-(2 '-Deoxy-2’-fluoro-fi-D-arabinofuranosyl)adenine-5’-0-[ 1 - phenyl(ethoxy-L-isoleucinyl)] diphosphate triethylammonium salt.
• Example 26 2-Chloro-9-(2 '-Deoxy-2’-fluoro-fi-D-arabinofuranosyl)adenine-5’-0-[ 1 - naphthyl(benzoxy-L-alaninyl)] phosphorothiol-phosphate triethylammonium salt.
• A represents an ECso of no more than 0.2 pm
• B represents an ECso of greater than 0.2 pm but no more than 2 pm
• C represents an ECso of greater than 2 pm but no more than 5 pm
• D represents an ECso of greater than 5 pm but no more than 10 pm
• E represents an ECso of greater than 10 pm.
• A represents an ECso of no more than 0.2 pm
• B represents an ECso of greater than 0.2 pm but no more than 1 pm
• C represents an ECso of greater than 1 pm but no more than 5 pm
• D represents an ECso of greater than 5 pm but no more than 10 pm
• E represents an ECso of greater than 10 pm.
• Letters in bold represent an ECso less than that of the exemplary ProTide A.
• the assay was performed according to the published procedure (Kuhnz, W.; Gieschen, H. Drug Metab. Dispos. 1998, 26, 1120-1127).
• Example 29 Evaluation of clofarabine and selected clofarabine diphosphate phosphoramidates in a KG1a cell line model of acute myeloid leukaemia (AML) Summary
• Clofarabine and Examples 1 and 2 were selected for analysis to determine whether: (1) the compounds of the invention possessed increased potency when compared to the parental compound and (2) the compounds of the invention preferentially targeted leukaemic stem cells (LSCs).
• LSCs leukaemic stem cells
• the acute myeloid leukaemia cell line, KG1a was employed as it manifests a minor stem cell-like compartment with a distinct immunophenotype (LinYCD347CD38YCD123 + ).
• the compounds were evaluated over an extended dose range.
• the effects of the compounds on the stem cell compartment were evaluated over the entire dose range.
• the mean clofarabine LD50 (the concentration of drug required to kill 50% of the cells) was 1.69 x 10 8 M.
• Example 1 showed a similar mean LD50 value (1.37 x 10 8 M), whereas Example 2 showed significantly increased mean LD50 values (4.38 x 10 8 M and 7.10 x 10 8 M respectively).
• AML acute myeloid leukaemia
• KG1a cells were cultured for 48h in the presence of a wide range of concentrations of each nucleoside analogue and their respective diphosphate phosphramidate. Cells were then harvested and labelled with a cocktail of anti-lineage antibodies (PE-cy7), anti-CD34 (FITC), anti-CD38 (PE) and anti-CD123 (PERCP cy5). The sub-population expressing a leukaemic stem cell (LSC) phenotype were subsequently identified and were expressed as a percentage of all viable cells left in the culture. The percentages of stem cells remaining were then plotted on a dose-response graph and the effects of the diphosphate
• LSC leukaemic stem cell
• phosphoramidate were compared with the parental nucleosides.
• tumour cells were purified by high speed cell sorting using a FACS Melody cell sorter (Becton Dickenson) and were placed back into hypoxic cell culture conditions prior to the addition of clofarabine or clofarabine diphosphate phosphoramidates.
• FACS Melody cell sorter Becton Dickenson
• the gH2A.C Phosphorylation Assay Kit (Flow cytometry) is a cell-based assay formatted for flow cytometric detection of levels of phosphorylated Histone gH2A.C (Merck, UK).
• LSC and‘bulk tumour’ cells were cultured in 96-well plates in the presence of clofarabine or clofarabine diphosphate phosphoramidates. After 2h of exposure to drug, the cells were harvested by centrifugation and then fixed and permeabilised in preparation for staining and detection.
• Histone gH2A.C phosphorylated at serine 139 is detected by the addition of the anti-phospho-Histone gH2A.C conjugated to APC. Cells were then run on a flow cytometer to quantitate the number of cells staining positive for phosphorylated Histone gH2A.C.
• Clofarabine and the compounds of the invention showed potency in the nanomolar range; clofarabine and Example 1 had similar LD50 values with Example 1 being slightly more potent.
• the fraction of KG1 a cells expressing an LSC phenotype is modulated by hypoxia KG1a cells were grown under normoxic and hypoxic conditions and the LSC phenotype was monitored over time. Under normoxic conditions, the LSC phenotype was stably maintained in approximately 3.5% of the cells in culture. In contrast, under hypoxic conditions, the fraction of the cells in culture expressing the LSC phenotype increased in a time-dependent manner ( Figure 3A). When cells were then transferred back into normoxic culture conditions, the fraction of cells expressing the LSC phenotype returned to -3.5%, again in a time- dependent manner (Figure 3B).
• clofarabine showed a significant reduction in potency when used on cells cultured under hypoxic conditions. This reduction in potency was not observed when Examples 1 and 2 were used under the same hypoxic conditions.
• Clofarabine induces significantly less DNA damage in LSCs than bulk tumour cells under hypoxic conditions
• Example 1 showed a trend towards increased selectivity against LinYCD347CD38YCD123 + LSCs.
• the LSC phenotype was shown to be inducible under hypoxic conditions and this was reversed when the cells were reintroduced to normoxic culture conditions.
• the dynamics of the changes in LSC fraction suggests a plasticity in the phenotype rather than a selective expansion/contraction in a fixed LSC sub-population.
• Clofarabine showed a significant reduction in potency when used on cells cultured under hypoxic conditions. This reduction in potency was not observed when
• Examples 1 and 2 were used under the same hypoxic conditions. Many cancers exist in a hypoxic state in the human body.

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