EP1951307A2 - Combination of a cdk-inhibitor and a hdac-inhibitor - Google Patents

Combination of a cdk-inhibitor and a hdac-inhibitor

Info

Publication number
EP1951307A2
EP1951307A2 EP06808516A EP06808516A EP1951307A2 EP 1951307 A2 EP1951307 A2 EP 1951307A2 EP 06808516 A EP06808516 A EP 06808516A EP 06808516 A EP06808516 A EP 06808516A EP 1951307 A2 EP1951307 A2 EP 1951307A2
Authority
EP
European Patent Office
Prior art keywords
thiazol
pyrimidin
dimethyl
methyl
phenylamino
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP06808516A
Other languages
German (de)
French (fr)
Inventor
Simon Green
Sheelagh Frame
Ian Fleming
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cyclacel Ltd
Original Assignee
Cyclacel Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB0523040.4A external-priority patent/GB0523040D0/en
Priority claimed from GB0523160A external-priority patent/GB0523160D0/en
Application filed by Cyclacel Ltd filed Critical Cyclacel Ltd
Publication of EP1951307A2 publication Critical patent/EP1951307A2/en
Pending legal-status Critical Current

Links

Classifications

    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4188—1,3-Diazoles condensed with other heterocyclic ring systems, e.g. biotin, sorbinil
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a pharmaceutical combination suitable for the treatment of proliferative disorders.
  • the present invention relates to combinations for the treatment of cancer, preferably non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • CDKs Cyclin-dependent kinases
  • CDKs are serine/threonine kinases that play a crucial regulatory role in the cell cycle. CDKs regulate cell cycle progression by phosphorylation of various proteins involved in DNA replication and cell division, including transcription factors and tumour suppressor proteins (Senderowicz, AM. Small-molecule cyclin-dependent kinase modulators, Oncogene, 2003; 22: 6609- 6620). Certain CDKs also play a role in the regulation of RNA synthesis by their involvement in the phosphorylation of the carboxy terminal domain (CTD) of the largest subunit of RNA polymerase II (pol II). It is not surprising, therefore, that CDKs have become attractive therapeutic targets.
  • CCD carboxy terminal domain
  • Cdc2 (also known as cdkl) is a catalytic sub-unit of a family of cyclin dependent kinases that are involved in cell cycle regulation.
  • kinases comprise at least two sub-units, namely a catalytic sub-unit (of which cdc2 is the prototype) and a regulatory sub-unit (cyclin).
  • the cdks are regulated by transitory association with a member of the cyclin family: cyclin A (cdc2, CDK2), ⁇ : cyclin B1-B3 (cdc2), cyclin C (CDK8), cyclin D1-D3 (CDK2-CDK4- CDK5-CDK6), cyclin E (CDK2), cyclin H (CDK7).
  • CDK activity is regulated by post-translatory modification, by transitory associations with other proteins and by modifications of their intra-cellular localization.
  • the CDK regulators comprise activators (cyclins, CDK7/cyclin H, cdc25 phosphateses), the p9.sup.CKS and pi 5. sup. CDK-BP sub-units, and the inhibiting proteins (pl6.sup.INK4A, pl5.sup.INK4B, p21.sup.Cipl, pl8, p27.sup.Kipl).
  • Roscovitine has been demonstrated to be a potent inhibitor of cyclin dependent kinase enzymes, particularly CDK2.
  • CDK inhibitors are understood to block passage of cells from the Gl /S and the G2/M phase of the cell cycle.
  • the pure R-enantiomer of roscovitine, seliciclib (R-Roscovitine; CYC202) has recently emerged as a potent inducer of apoptosis in a variety of tumour cells (McClue SJ, Blake D, Clarke R, et al,
  • Roscovitine has also been shown to be an inhibitor of retinoblastoma phosphorylation and therefore implicated as acting more potently on Rb positive tumors.
  • active pharmaceutical agents can often be administered in combination in order to optimise the treatment regime.
  • a CDK inhibitor in combination with a second chemotherapeutic agent is described in WO 03/077999, WO 03/082337, WO 2004/041262, WO 2004/041267, WO 2004/041268, WO 2004/041308, WO 2004/110455 and WO 2005/053699 (all to Cyclacel Limited).
  • the present invention seeks to provide a new combination of known pharmaceutical agents that is particularly suitable for the treatment of proliferative disorders, especially cancer. More specifically, a preferred aspect of the invention centres on combinations useful in the treatment of non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • a first aspect of the present invention relates to a combination comprising a histone deacetylase inhibitor and a compound of formula I or II, or a pharmaceutically acceptable salt thereof,
  • R 1 and R 2 are methyl, ethyl or isopropyl, and the other is H;
  • R 3 and R 4 are each independently H, branched or unbranched C 1 -C 6 alkyl, or aryl, and wherein at least one of R 3 and R 4 is other than H;
  • R 5 is a branched or unbranched Cj -C 5 alkyl group or a Ci-C 6 cycloalkyl group, each of which may be optionally substituted with one or more OH groups;
  • R 6 , R 7 , R 8 and R 9 are each independently H, halogen, NO 2 , OH, OMe, CN,
  • R 10 and R 14 are each independently H, C(OR' ) or a hydrocarbyl group optionally substituted by one or more R 15 groups;
  • R 11 , R 12 , and R 13 are each independently H, alkyl or alkenyl, each of which may be optionally substituted with one or more R 1 groups;
  • R 15 and R 16 are each independently halogen, NO 2 , CN, (CH 2 ) m OR a ,
  • R 8 , R h , R 1 and R j are each independently selected from alkyl, aryl, aralkyl and heteroaryl, each of which may be optionally substituted with one or more substituents selected from halogen, OH, NO 2 , NH 2 CF 3 and COOH; m, p, q and r are each independently O, 1, 2 or 3; n and s are each independently 1, 2, or 3; and
  • R a'n and R a "F are each independently H or alkyl.
  • a second aspect relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a combination according to the invention and a pharmaceutically acceptable carrier, diluent or excipient.
  • a third aspect relates to the use of a combination according to the invention in the preparation of a medicament for treating a proliferative disorder.
  • a fourth aspect relates to a pharmaceutical product comprising a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, and a HDAC inhibitor, as a combined preparation for simultaneous, sequential or separate use in therapy.
  • a fifth aspect relates to a method of treating a proliferative disorder, said method comprising simultaneously, sequentially or separately administering a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, and a HDAC inhibitor.
  • a sixth aspect relates to the use of a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment of a proliferative disorder, wherein said treatment comprises simultaneously, sequentially or separately administering a HDAC inhibitor.
  • a seventh aspect relates to the use of a HDAC inhibitor in the preparation of a medicament for the treatment of a proliferative disorder, wherein said treatment comprises simultaneously, sequentially or separately administering a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof.
  • An eighth aspect relates to the use of (i) a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, and (ii) a HDAC inhibitor, in the preparation of a medicament for treating a proliferative disorder.
  • a ninth aspect relates to the use of a HDAC inhibitor, in the preparation of a medicament for treating a proliferative disorder, wherein said medicament is for use in combination therapy with a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof.
  • a tenth aspect relates to the use of a HDAC inhibitor, in the preparation of a medicament for treating a proliferative disorder, wherein said medicament is for use in pretreatment therapy with a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof.
  • An eleventh aspect relates to the use of a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating a proliferative disorder, wherein said medicament is for use in combination therapy with a HDAC inhibitor.
  • a twelfth aspect relates to the use of a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating a proliferative disorder, wherein said medicament is for use in pretreatment therapy with a HDAC inhibitor.
  • a thirteenth aspect relates to the use of a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, and a HDAC inhibitor, in the preparation of a medicament for the treatment of non small cell lung cancer (NSCLC).
  • NSCLC non small cell lung cancer
  • the invention relates to a combination which comprises a histone deacetylase (HDAC) inhibitor and a compound of formula I, or a pharmaceutically acceptable salt thereof.
  • HDAC histone deacetylase
  • the invention provides a combination comprising a 2,6,9-substituted purine derivative of formula I, which exhibits improved resistance to metabolic deactivation, and a HDAC inhibitor.
  • one of R 1 and R 2 is ethyl or isopropyl, and the other is H.
  • R s is isopropyl or cyclopentyl.
  • R 6 , R 7 , R 8 and R 9 are all H.
  • R 1 or R 2 is ethyl and the other is H.
  • R 3 and R 4 are each independently H 5 methyl, ethyl, propyl, butyl or phenyl.
  • R 3 and R 4 are each independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, t-butyl or phenyl.
  • R 3 and R 4 are each independently H, methyl, ethyl, propyl or butyl.
  • R 3 and R 4 are each independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl or t-butyl.
  • R 3 and R 4 are each independently H, methyl, ethyl, isopropyl or t-butyl.
  • said compound of formula I is selected from the following:
  • said compound of formula I is selected from the following: (26'3J?)-3- ⁇ 9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino ⁇ -pentan-2- ol; (2i?3jS)-3- ⁇ 9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purm-2-ylamino ⁇ -pentan-2- ol;
  • said compound of formula I is selected from the following: (3R)-3- ⁇ 9-isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino ⁇ -2-methyl- pentan-2-ol; (3 S)-3 - ⁇ 9-isopropy l-6-[(pyridin-3 -ylmethy l)-amino] -9H-purin-2-ylamino ⁇ -2-methyl- pentan-2-ol;
  • said compound of formula I is selected from the following:
  • the present invention provides a combination comprising a compound of formula II and a HDAC inhibitor.
  • One embodiment of the present invention relates to a combination comprising a compound of formula Ha, or a pharmaceutically acceptable salt thereof,
  • R 10 and R 14 are each independently H or a hydrocarbyl group optionally substituted by one or more R 15 groups;
  • R 11 , R 12 , and R 13 are each independently H, alkyl or alkenyl, each of which may be optionally substituted with one or more R groups;
  • R 15 and R 16 are each independently halogen, NO 2 , CN, (CH 2 ) m OR a , where m is 0, 1, 2 or 3, O(CH 2 ) n OR b , where n is 1, 2, or 3, NR c R d , CF 3 , COOR e , CONR f R g , COR h , SO 3 H 5 SO 2 R 1 , SO 2 NR j R k , heterocycloalkyl or heteroaryl, wherein said heterocycloalkyl and heteroaryl may be optionally substituted by one or more substituents selected from R m and COR n ; and
  • R a"n are each independently H or alkyl.
  • hydrocarbyl refers to a group comprising at least C and H. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain heteroatoms. Suitable heteroatoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen, oxygen, phosphorus and silicon.
  • the hydrocarbyl group is an aryl, heteroaryl, alkyl, cycloalkyl, aralkyl or alkenyl group.
  • alkyl includes both saturated straight chain and branched alkyl groups which may be substituted (mono- or poly-) or unsubstituted.
  • the alkyl group is a Ci -20 alkyl group, more preferably a Ci. 15 , more preferably still a Ci -J2 alkyl group, more preferably still, a Cj -6 alkyl group, more preferably a C1. 3 alkyl group.
  • Particularly preferred alkyl groups include, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl and hexyl.
  • Suitable substituents include, for example, one or more R 5 groups.
  • cycloalkyl refers to a cyclic alkyl group which may be substituted (mono- or poly-) or unsubstituted.
  • the cycloalkyl group is a C3-12 cycloalkyl group.
  • Suitable substituents include, for example, one or more R 15 groups.
  • heterocycloalkyl refers to a cycloalkyl group containing one or more heteroatoms selected from O, N and S.
  • heterocycloalkyl include 1- (1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, pyrrolidinyl, dihydrofuranyl, tetrahydropyranyl, pyranyl, thiopyranyl, aziridinyl, oxiranyl, methylenedioxyl, chromenyl, isoxazolidinyl, l,3-oxazolidin-3-yl, isothiazo
  • heterocycloalkyl a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule.
  • connection of said heterocycloalkyl rings is through a carbon or a sp 3 hybridized nitrogen heteroatom.
  • Preferred heterocycloalkyl groups include piperazine, morpholine, piperidine and pyrrolidine.
  • alkenyl refers to a group containing one or more carbon- carbon double bonds, which may be branched or unbranched, substituted (mono- or poly-) or unsubstituted.
  • the alkenyl group is a C 2-20 alkenyl group, more preferably a C 2-15 alkenyl group, more preferably still a C 2-I2 alkenyl group, or preferably a C 2-6 alkenyl group, more preferably a C 2 - 3 alkenyl group.
  • Suitable substituents include, for example, one or more R 15 groups as defined above.
  • aryl refers to a Q ⁇ -n aromatic group which may be substituted (mono- or poly-) or unsubstituted. Typical examples include phenyl and naphthyl etc. Suitable substituents include, for example, one or more R 15 groups.
  • heteroaryl refers to a C 4-12 aromatic, substituted (mono- or poly-) or unsubstituted group, which comprises one or more heteroatoms.
  • Preferred heteroaryl groups include pyrrole, pyrazole, pyrimidine, pyrazine, pyridine, quinoline, triazole, tetrazole, thiophene and furan.
  • suitable substituents include, for example, one or more R 15 groups.
  • R 8 , R h , R 1 and R* are each independently selected from alkyl, phenyl, benzyl and pyridyl, each of which may be optionally substituted with one or more substituents selected from halogen, OH, NO 2 , NH 2 CF 3 and COOH;
  • R a"n and R a "f are each independently H, methyl, ethyl or isopropyl.
  • R 10 and R 14 are each independently H or a C 1-20 hydrocarbyl group optionally comprising up to six heteroatoms selected from from N, O, and S, and which is optionally substituted by one, two or three R 15 groups;
  • R 14 is aryl or heteroaryl, each of which may be optionally substituted by one or more R 15 groups.
  • R 14 is H, CO(R J ), aryl or heteroaryl, wherein said aryl or heteroaryl groups may be optionally substituted by one or more R 15 groups.
  • R 14 is H, COMe, phenyl or pyridyl, wherein said phenyl or pyridyl groups may be optionally substituted by one or more R 15 groups.
  • R 14 is phenyl or pyridinyl, each of which may be optionally substituted by one or more R 15 groups.
  • R 10 is H or alkyl. More preferably, R 10 is H, methyl, ethyl or 3-methylbutyl.
  • R 11 , R 12 , and R 13 are each independently H, C 1 -C 6 alkyl or C 2 -C 6 alkenyl, each of which may be optionally substituted with one, two or three R 16 groups.
  • R is C 1-6 alkyl. More preferably still, R 11 is methyl.
  • R 12 and R 13 are both H.
  • R 15 and R 16 are each independently F, Cl, Br, I, NO 2 , CN 3 OH, OMe, OEt, CH 2 OH, O(CH 2 ) 2 OMe, NH 2 , NHMe, NMe 2 , CF 3 , COOH, CONH 2 , CONHMe, CONMe 2 , COMe, SO 3 H, SO 2 Me, SO 2 NH 2 , SO 2 NHMe, SO 2 NMe 2 , morpholine, piperidine, piperazine, N-acetylpiperazine, N-methylpiperazine, triazole, or tetrazole.
  • R 12 and R 13 are both H and R 11 is Me.
  • the compound of the invention is of formula III, or a pharmaceutically acceptable salt thereof,
  • R 10 is as defined above in claim 1 or claim 12;
  • X is C; or X is N and R 17 is absent;
  • R , 1 1 7 ', ⁇ R> 1 l 8 iS ⁇ R» 1 i 9 y and R >2 z 0 u are each independently H or as defined for R 15 and R , 16
  • R 10 is H or alkyl
  • R 17 is H, NO 2 , OR", halogen, CF 3 , CN, COR q , alkyl, NR r R s , 0(CHa) 1 OR 1 ;
  • R 18 is H, OR U , halogen, alkyl, NR V R W , or a heterocycloalkyl optionally substituted with one or more substituents selected from R m and C0R n ;
  • t is O, 1, 2 or 3;
  • R 19 is H, alkyl or NR x R y ; and R p"y are each independently H or alkyl.
  • R 10 is H, Me, Et or 3-methylbutyl.
  • R 17 is H, NO 2 , OH, Me, I, CF 3 , CN, CH 2 OH, CO 2 H, CO 2 Me OrNH 2 ;
  • R 18 is H, F, OH, I, Cl, Br, OMe, NMe 2 , morpholine, Me, N-methylpiperazine, N- acetylpiperazine or piperazine; and R 19 is H, Me or NMe 2 .
  • R 17 is selected from H, NO 2 , halogen, CN, CF 3 , SO 3 H, (CH 2 ) m OR a , COOR e , (CH 2 ) p NR c R d , (CH 2 ) r NR b' SO 2 R h' , (CH 2 ) q NR a' C0R gl , SO 2 NR j R k , C0NR f R g , SO 2 NR e' (CH 2 ) s OR c> , S0 2 NR d R' and heterocycloalkyl optionally substituted by one or more COR" or sulfonyl groups.
  • R 17 is selected from H, NO 2 , OH, Me, I, CN, CH 2 OH, CF 3 , CO 2 H, CO 2 Me, NH 2 , Cl, 4-acetylpiperazin-l-yl, OMe, SO 3 H, CH 2 NHSO 2 Me, CH 2 NHCOPh, CH 2 NHSO 2 CF 3 , SO 2 NH 2 , CONH 1 Pr, SO 2 NHEt, SO 2 NH(CH 2 ) 2 OMe, SO 2 NH 1 Pr, SO 2 NH(CH 2 ) 2 OH, NHMe, SO 2 NH-benzyl and morpholin-4-sulfonyl.
  • R 18 is selected from H, NO 2 , SO 3 H, halogen, (CH 2 ) m OR a , (CH 2 ) p NR c R d , (CH 2 ) q NR a' COR s> , SO 2 NR e' (CH 2 ) s OR c> , SO 2 NR d> R' ' and heterocycloalkyl optionally substituted by one or more COR", R m or aralkyl groups.
  • R 18 is selected from H, F, OH, Cl, Br, OMe, NMe 2 , morpholin-4-yl, 4-methylpiperazin-l-yl, Me, 4-acetyl-piperazin-l-yl, I, CH 2 NHCOMe, NO 2 , SO 3 H, SO 2 NH(CH 2 ) 2 OMe, 4-benzylpiperazin-l-yl, SO 2 NH(CH 2 ) 2 OH, SO 2 NH-benzyl, CH 2 NH 2 , CH 2 NHCO-(pyrid-2-yl) and piperazin-1-yl.
  • R 19 is selected from H, R f and (CH 2 ) p NR 0 R d .
  • R 19 is selected from H, Me and NMe 2 .
  • R 20 is selected from H 5 R f , CF 3 , halogen and (CH 2 ) q NR a COR g> .
  • R 20 is selected from H, NHCOMe, CF 3 , Br and Me.
  • X is N and R 1 is absent.
  • X is C.
  • the compound of formula II is selected from the following:
  • the compound of formula II is selected from the following:
  • said compound of formula II is 3,4-dimethyl-5 ⁇ [2-(4-piperazin-l-yl-phenylamino)pyrimidin-4-yl]-3H-thiazol-2-one.
  • the presently claimed combinations comprise a compound of formula I or formula II and a histone deacetylase (HDAC) inhibitor.
  • HDAC histone deacetylase
  • Histones are small positively charged proteins that are rich in basic amino acids (positively charged at physiological pH). There are five main types of histones namely, Hl, H2A, H2B, H3, and H4 which exhibit a high degree of structural similarity. Histones are not found in eubacteria (e.g., E. coli), although the DNA of these bacteria is associated with other proteins that presumably function like histones to package the DNA within the bacterial cell. Archaebacteria, however, do contain histones that package their DNAs in structures similar to eukaryotic chromatin (G. M. Cooper, "The Cell - A Molecular Approach", 2 nd Edition, Chapter II). The majority of histones are synthesized during the S phase of the cell cycle, and newly synthesized histones quickly enter the nucleus to become associated with DNA. Within minutes of its synthesis, new DNA becomes associated with histones in nucleosomal structures.
  • eubacteria e.g., E. coli
  • the amino-terminal tail domains of histones may be enzymatically modified by post- translational addition of methyl (to lysine and arginine groups), acetyl (to lysine groups), or phosphate groups (to serine groups) (Spencer et al, Gene, 1999, 240(1), 1). This results in a reduction of the net positive charge of the histone which, consequently, may weaken the binding of the histone to DNA.
  • HDACs histone deacetylators
  • HDACs therefore, are believed to be associated with a number of different diseases which include proliferative disorders such as leukemia (Lin et al, Nature, 1998, 391, 811), melanomas/squamous cell carcinomas (Gillenwater et al, Int. J. Cancer, 1998, 75217; Saunders et al, Cancer Res., 1999, 59, 399), breast cancer, prostrate cancer, bladder cancer (Gelmetti et al, MoI. Cell Biol., 1998, 18, 7185; Wang et al, PNAS, 1998, 951, 10860) and colon cancer (C. A. Hassig, et al, 1997, Chem. Biol., 4, 783; S. Y. Archer et al, PNAS, 1998, 95(12), 6791).
  • proliferative disorders such as leukemia (Lin et al, Nature, 1998, 391, 811), melanomas/squamous cell carcinomas (Gillenwater e
  • US 2005/0004007 discloses a method for promoting apoptosis in cancer cells which involves administering a cyclin dependent kinase inhibitor and an agent which induces cellular differentiation.
  • agent which induces cellular differentiation namely, histone deacetylase inhibitors, protein kinase C, retinoids and vitamin D3.
  • combinations comprising a compound of formula I or formula II and a HDAC inhibitor are not specifically disclosed, nor is the use of this combination in the treatment of solid tumours, such as NSCLC.
  • the exemplification of US 2005/0004007 is limited to combinations of flavopiridol with selected HDAC inhibitors tested on leukemia cell lines. Accordingly, to date, there has been no disclosure of the specific combinations claimed in the present application, let alone any suggestion that they would be therapeutically useful in the treatment of lung cancers such as NSCLC.
  • the HDAC inhibitor is sodium butyrate.
  • the HDAC inhibitor is a prodrug of sodium butyrate.
  • the prodrug is pivaloyloxymethyl butyrate.
  • Pivaloyloxymethyl butyrate (Pivanex ® ) is an acyloxyalkyl ester prodrug of butyric acid and has been shown to induce the instrinsic pathway of apoptosis in leukemia and neuroblastoma cells (S. Mei et al, International Journal of Oncology, 2004, 25, 1509).
  • the HDAC inhibitor is trichostatin A (TSA).
  • Trichostatin A is a specific and reversible inhibitor of HDAC. At nanomolar concentrations, TSA causes a marked accumulation of highly acetylated histones in vivo and strongly inhibits the activity of the partially purified histone deacetylase in vitro (M. Yoshida et al, J. Biol. Chem., 1990, 265(28), 17174).
  • TSA arrests cell cycle progression in Gl and inhibits the activity of the HDl deacetylase with an IC 50 of 70 nM (Y. Hoshikawa et al, Exp. Cell Res., 1994, 214, 189).
  • TSA can also concomitantly modify the expression of genes.
  • Mishra et al demonstrated that TSA significantly downregulated CDl 54 and IL-IO and up- regulated IFN- ⁇ gene expression in systemic lupus erythematosis (SLE) T cells.
  • SLE is an autoimmune disease characterised by dysregulated production of antibodies which leads to irreversible, immune complex-mediated end-organ failure ( N. Mishra et al, PNAS, 2001, 98(5), 2628).
  • the HDAC inhibitor is suberoylanilide hyroxamic acid (SAHA).
  • SAHA Suberoylanilide hyroxamic acid
  • WO 2005/097747 discloses the use of prodrugs of hydroxamic based HDAC inhibitors, such as SAHA, in the treatment of neoplasms, thioredoxin (TRX)-mediated diseases and in the prevention and/or treatment of CNS diseases.
  • WO 2005/039498 and WO 2005/018578 disclose further methods for the treatment of neoplasms, wherein WO2005/039498 relates to leukemia and WO 2005/018578 relates to mesothelioma or lymphoma.
  • the HDAC inhibitor is sodium valproate (otherwise known as sodium 2-propylpentanoate).
  • Sodium valproate is the sodium salt of valproic acid and is a NICE-approved anticonvulsant drug used in the treatment of epilepsy. More recently, studies have investigated the use of sodium valproate for the treatment of advanced solid tumour malignancies and cancer-related neuropathic pain. Combination studies involving valproic acid and UCN-Ol have also been undertaken. In this regard, although valproic acid itself has only a weak anticancer effect, studies have shown that it becomes highly effective against cancer cells when used in combination.
  • Another aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, and a HDAC inhibitor.
  • Another aspect relates to a pharmaceutical product comprising the combination of the present invention for use in the treatment of a proliferative disorder, wherein the disorder is preferably cancer, and more preferably, NSCLC.
  • a further aspect of the present invention relates to a pharmaceutical product comprising a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, and a HDAC inhibitor as a combined preparation for simultaneous, sequential or separate use in therapy.
  • the HDAC inhibitor is selected from sodium butyrate, or a prodrug thereof, suberoylanilide hydroxamic acid (SAHA), sodium valproate and trichostatin (TSA).
  • SAHA suberoylanilide hydroxamic acid
  • TSA sodium valproate
  • TSA trichostatin
  • Yet another aspect relates to a method of treating a proliferative disorder, said method comprising simultaneously, sequentially or separately administering a combination of the present invention.
  • “simultaneously” is used to mean that the two agents are administered concurrently, whereas the term “in combination” is used to mean that they are administered, if not simultaneously, then “sequentially” with a timeframe that they are able to act therapeutically within the same time frame.
  • administration “sequentially” may permit one agent to be administered within 5 minutes, 10 minutes or a matter of hours after the other provided that they are both concurrently present in therapeutic amounts.
  • the time delay between administration of the components will vary depending on the exact nature of the components, the interaction therebetween and their respective half-lives.
  • the HDAC inhibitor is administered sequentially or separately prior to the compound of formula I or formula II, or a pharmaceutically acceptable salt thereof.
  • the compound of formula I or formula II, or a pharmaceutically acceptable salt thereof is administered sequentially or separately prior to the HDAC inhibitor.
  • the compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, and the HDAC inhibitor are administered simultaneously.
  • the HDAC inhibitor is sodium butyrate, or a prodrug thereof
  • the compound of formula I or formula II is administered simultaneously with, or separately or sequentially with, the HDAC inhibitor, irrespective of the order of administration. More preferably, the sodium butyrate and compound of formula I or formula II are administered separately or sequentially.
  • the HDAC inhibitor is sodium butyrate and the compound of formula I is compound [I].
  • the sodium butyrate and compound [1] are administered separately or sequentially, irrespective of the order of administration.
  • the HDAC inhibitor is sodium butyrate and the compound of formula II is compound [2].
  • the sodium butyrate and compound [2] are administered separately or sequentially, irrespective of the order of administration, or simultaneously.
  • the HDAC inhibitor is sodium valproate
  • the compound of formula I or formula II is administered simultaneously with, or separately or sequentially with, the HDAC inhibitor, irrespective of the order of administration. More preferably, the sodium valproate and compound of formula I or formula II are administered separately or sequentially. Even more preferably, the sodium valproate is administered separately or sequentially prior to the compound of formula I or II.
  • the HDAC inhibitor is sodium valproate
  • the compound of formula I is compound [I].
  • sodium valproate and compound [1] are administered separately or sequentially, irrespective of the order of administration.
  • the HDAC inhibitor is sodium valproate
  • the compound of formula II is compound [2].
  • sodium valproate and compound [2] are administered separately or sequentially, irrespective of the order of administration.
  • the HDAC inhibitor is sodium valproate
  • the compound of formula I is compound [3].
  • the sodium valproate is administered separately or sequentially prior to compound [3], i.e. the subject is pretreated with sodium valproate.
  • the HDAC inhibitor is sodium valproate
  • the compound of formula I is compound [4].
  • the sodium valproate is administered separately or sequentially prior to compound [4], i.e. the subject is pretreated with sodium valproate.
  • the compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, and the HDAC inhibitor are each administered in a therapeutically effective amount with respect to the individual components. In another preferred embodiment, the compound of formula I or formula II, or pharmaceutically acceptable salt thereof, and the HDAC inhibitor are each administered in a sub-therapeutic amount with respect to the individual components.
  • sub-therapeutic amount means an amount that is lower than that typically required to produce a therapeutic effect with respect to treatment with the compound of formula I or formula II alone or the HDAC inhibitor alone.
  • a further aspect relates to the use of the combination of the present invention in the preparation of a medicament for treating a proliferative disorder.
  • preparation of a medicament includes the use of one or more of the above described components directly as the medicament or in any stage of the manufacture of such a medicament.
  • Another aspect relates to the use of a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment of a proliferative disorder, wherein said treatment comprises simultaneously, sequentially or separately administering a HDAC inhibitor to a subject.
  • Yet another aspect relates to the use of a HDAC inhibitor, in the preparation of a medicament for the treatment of a proliferative disorder, wherein said medicament is for use in combination therapy with the compound of formula I or formula II, or a pharmaceutically acceptable salt thereof.
  • the therapy can be pretreatment therapy.
  • a further aspect relates to the use of the compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment of a proliferative disorder, wherein said medicament is for use in combination therapy with a HDAC inhibitor.
  • the therapy can be pretreatment therapy.
  • the term “combination therapy” refers to therapy in which the HDAC inhibitor and a compound of formula I or formula II are administered, if not simultaneously, then sequentially within a time frame that they both are available to act therapeutically within the same time frame.
  • pretreatment therapy means a regimen in which one agent is administered prior to, either separately or sequentially, the second agent.
  • the second agent is administered at least 2 hours after the administration of the first agent. More preferably, the second agent is administered at least 4 hours, or more preferably at least 6 or 8 hours, after the administration of the first agent. Even more preferably, the second agent is administered at least 12 hours, or more preferably at least 18 or 24 hours, after the administration of the first agent.
  • the compound of formula I or formula II and the HDAC inhibitor interact in a synergistic manner.
  • the term “synergistic” means that the compound of formula I or formula II and the HDAC inhibitor produce a greater effect when used in combination than would be expected from adding the individual effects of the two components.
  • a synergistic interaction may allow for lower doses of each component to be administered to a patient, thereby decreasing the toxicity of chemotherapy, whilst producing and/or maintaining the same therapeutic effect.
  • each component can be administered in a sub-therapeutic amount.
  • the CDK inhibitor and the HDAC inhibitor interact in a manner so as to alleviate or eliminate adverse side effects associated with use of the individual components in monotherapy, or associated with their use in known combinations.
  • the HDAC inhibitor is selected from sodium butyrate, or a prodrug thereof, suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA).
  • SAHA suberoylanilide hydroxamic acid
  • TSA trichostatin A
  • Another aspect of the invention relates to a combination comprising:
  • a HDAC inhibitor (i) a HDAC inhibitor; and (ii) (a) a 2,6,9-substituted purine derivative or a pharmaceutically acceptable salt thereof; or (b) a 2-substituted-4-heteroaryl-pyrimidine derivative, or a pharmaceutically acceptable salt thereof.
  • proliferative disorder is used herein in a broad sense to include any disorder that requires control of the cell cycle, for example cardiovascular disorders such as restenosis and cardiomyopathy, auto-immune disorders such as glomerulonephritis and rheumatoid arthritis, dermatological disorders such as psoriasis, anti-inflammatory, anti-fungal, antiparasitic disorders such as malaria, emphysema and alopecia.
  • cardiovascular disorders such as restenosis and cardiomyopathy
  • auto-immune disorders such as glomerulonephritis and rheumatoid arthritis
  • dermatological disorders such as psoriasis, anti-inflammatory, anti-fungal, antiparasitic disorders such as malaria, emphysema and alopecia.
  • the compounds of the present invention may induce apoptosis or maintain stasis within the desired cells as required.
  • the proliferative disorder is cancer.
  • the proliferative disorder is lung cancer, more preferably, non-small cell lung cancer (NSCLC).
  • NSCLC non-small cell lung cancer
  • Lung cancers may be divided into two broad categories namely, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The distinction between these two types of cancer is based on the appearance of the tumour cells when viewed under a microscope.
  • SCLC small cell lung cancer
  • NSCLC non-small cell lung cancer
  • SCLC accounts for 20% of lung cancers diagnosed and it is characterised by small cells which are mostly filled with the nucleus (hence the name). It is sometimes also referred to as "oat cell” cancer. SCLC is the most aggressive type of cancer, which metastasizes rapidly to other parts of the body. Diagnosis with SCLC often occurs only after the cancer has spread throughout the body. In general, SCLC is almost always caused as a result of smoking. NSCLC can be subdivided into a group of related lung cancers which include epidermoid or squamous cell carcinoma, adenocarcinoma and large cell carcinoma.
  • Squamous cell lung cancer accounts for approximately 30% of all lung cancer cases and develops from reserve cells (which have the role of replacing damaged epithelium cells) in the lining of the lungs and bronchi. As a result, the cancer often initially develops in the centre of the chest. Squamous cell lung cancers are frequently slow growing and can take several years to progress from a confined tumour into invasive cancer. In 10-20% of cases, the cancer cavitates within the lungs. On metastasis, it often spreads to the bone, liver, adrenal glands, small intestine and brain.
  • Adenocarcinoma is the most common form of lung cancer making up 30-40% of all lung cancer cases. Adenocarcinoma develops in the outer part of the lung and develops from mucus-producing cells. The course of this cancer varies widely but often progresses slowly and the patient will present with few or no symptoms. In some cases, however, it can be extremely aggressive and rapidly fatal. In 50% of cases when it metastasises, it spreads only to the brain. Other locations to which adrenocarcinoma spreads include the liver, the adrenal glands, and bone.
  • the incidence of large cell carcinoma occurs less frequently than that of either adenocarcinoma or squamous cell carcinoma and accounts for 10-20% of lung cancer cases.
  • the cancer is composed of large-sized cells that are anaplastic in nature and often arise in the bronchi. Large cell carcinoma develops on the periphery of the lungs and can spread to the plura.
  • NSCLC drugs and regimens include camptosar (irinotecan; CPT-I l), camptothecin, carboplatin (paraplatin), cisplatin (platinol), epirubicin, gemcitabine, navelbine (vinorelbine), oxaliplatin, taxol (paclitaxel) and taxotere (docetaxol) (NSCLC Treatment - Chemotherapy, Lung Cancer Online).
  • Cisplatin is acknowledged to have certain disadvantages in that significant non-hematological toxicity (ototoxicity and nephroxicity) occurs in patients, along with emesis (P. Zatloukal et al, Lung Cancer, 2002, 38, S33).
  • the pharmaceutical product of the invention is in the form of a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent or excipient.
  • the compounds of the present invention can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy.
  • a pharmaceutical carrier excipient or diluent
  • the pharmaceutical compositions may be for human or animal usage in human and veterinary medicine.
  • suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like.
  • suitable diluents include ethanol, glycerol and water.
  • compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
  • Suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.
  • Suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
  • Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition.
  • preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • Antioxidants and suspending agents may be also used.
  • the agents of the present invention can be present as salts or esters, in particular pharmaceutically acceptable salts or esters.
  • compositions of the agents of the invention include suitable acid addition or base salts thereof.
  • suitable pharmaceutical salts may be found in Berge et al, J Pharm Sci, 66, 1-19 (1977). Salts are formed, for example with strong inorganic acids such as mineral acids, e.g.
  • sulphuric acid, phosphoric acid or hydrohalic acids with strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (Cl-C4)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid.
  • Esters are formed either using organic acids or alcohols/hydroxides, depending on the functional group being esterified.
  • Organic acids include carboxylic acids, such as alkanecarboxylic acids of 1 to 12 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acid, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (Cl-C4)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluen
  • Suitable hydroxides include inorganic hydroxides, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide.
  • Alcohols include alkanealcohols of 1-12 carbon atoms which may be unsubstituted or substituted, e.g. by a halogen).
  • the invention also includes where appropriate all enantiomers and tautomers of the agents.
  • the man skilled in the art will recognise compounds that possess optical properties (one or more chiral carbon atoms) or tautomeric characteristics.
  • the corresponding enantiomers and/or tautomers may be isolated/prepared by methods known in the art. STEREO AND GEOMETRIC ISOMERS
  • agents of the invention may exist as stereoisomers and/or geometric isomers — e.g. they may possess one or more asymmetric and/or geometric centres and so may exist in two or more stereoisomeric and/or geometric forms.
  • the present invention contemplates the use of ail the individual stereoisomers and geometric isomers of those inhibitor agents, and mixtures thereof.
  • the terms used in the claims encompass these forms, provided said forms retain the appropriate functional activity (though not necessarily to the same degree).
  • the present invention also includes all suitable isotopic variations of the agent or pharmaceutically acceptable salts thereof.
  • An isotopic variation of an agent of the present invention or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature.
  • isotopes that can be incorporated into the agent and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as 2H, 3H, 13C, 14C, 15N, 17O, 180, 31P, 32P, 35S, 18F and 36Cl, respectively.
  • isotopic variations of the agent and pharmaceutically acceptable salts thereof are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., 3H, and carbon-14, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., 2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. Isotopic variations of the agent of the present invention and pharmaceutically acceptable salts thereof of this invention can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.
  • the invention furthermore relates to agents of the present invention in their various crystalline forms, polymorphic forms and (an)hydrous forms. It is well established within the pharmaceutical industry that chemical compounds may be isolated in any of such forms by slightly varying the method of purification and or isolation form the solvents used in the synthetic preparation of such compounds.
  • the invention further includes agents of the present invention in prodrug form.
  • prodrugs are generally compounds wherein one or more appropriate groups have been modified such that the modification may be reversed upon administration to a human or mammalian subject.
  • Such reversion is usually performed by an enzyme naturally present in such subject, though it is possible for a second agent to be administered together with such a prodrug in order to perform the reversion in vivo.
  • Examples of such modifications include ester (for example, any of those described above), wherein the reversion may be carried out be an esterase etc.
  • Other such systems will be well known to those skilled in the art.
  • compositions of the present invention may be adapted for oral, rectal, vaginal, parenteral, intramuscular, intraperitoneal, intraarterial, intrathecal, intrabronchial, subcutaneous, intradermal, intravenous, nasal, buccal or sublingual routes of administration.
  • compositions For oral administration, particular use is made of compressed tablets, pills, tablets, gellules, drops, and capsules. Preferably, these compositions contain from 1 to 2000 mg and more preferably from 50-1000 mg, of active ingredient per dose.
  • compositions of the present invention may also be in form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dusting powders.
  • the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin.
  • the active ingredient can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required.
  • Injectable forms may contain between 10 - 1000 mg, preferably between 10 - 500 mg, of active ingredient per dose.
  • compositions may be formulated in unit dosage, form, i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose.
  • the combination or pharmaceutical composition of the invention is administered intravenously.
  • a person of ordinary skill in the art can easily determine an appropriate dose of one of the instant compositions to administer to a subject without undue experimentation.
  • a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy.
  • the dosages disclosed herein are exemplary of the average case. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
  • the compound of formula I may be administered at a dose of from 0.01 to 30 mg/kg body weight, such as from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
  • the compound of formula II may be administered at a dose of from 0.01 to 30 mg/kg body weight, such as from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
  • one or more doses of 10 to 150 mg/day of the compounds of formula I or formula II will be administered to the patient for the treatment of malignancy.
  • the HDAC inhibitor is typically administered in accordance with a physician's direction at dosages described in the relevant references discussed above.
  • Pivanex is typically administered at about 2.34g/m 2 per day. Pivanex is preferably administered intravenously.
  • Suberoylanilide hydroxamic acid (SAHA) is typically administered from about 100-600mg per day.
  • SAHA is preferably administered orally.
  • the total daily dose of HDAC inhibitor can be administered as a single dose or divided into separate dosages preferably administered two, three or four time a day.
  • the HDAC inhibitor is administered at least 2 hours before the administration of the compound of formula I or formula II. More preferably, the HDAC inhibitor is administered at least 4 hours, or more preferably at least 6 or 8 hours, before the administration of the compound of formula I or formula II. Even more preferably, the HDAC inhibitor is administered at least 12 hours, or more preferably at least 18 or 24 hours, before the administration of the compound of formula I or formula II.
  • the HDAC inhibitor is administered at least 2 hours after the administration of the compound of formula I or formula II. More preferably, the HDAC inhibitor is administered at least 4 hours, or more preferably at least 6 or 8 hours, after the administration of the compound of formula I or formula II. Even more preferably, the HDAC inhibitor is administered at least 12 hours, or more preferably at least 18 or 24 hours, after the administration of the compound of formula I or formula II. KIT OF PARTS
  • a further aspect of the invention relates to a kit of parts comprising:
  • a HDAC inhibitor optionally admixed with a pharmaceutically acceptable diluent, excipient or carrier, optionally admixed with a pharmaceutically acceptable diluent, excipient or carrier.
  • the HDAC inhibitor is selected from sodium butyrate, or a prodrug thereof, suberoylanilide hydroxamic acid (SAHA), sodium valproate and trichostatin A (TSA).
  • SAHA suberoylanilide hydroxamic acid
  • TSA trichostatin A
  • the HDAC inhibitor is selected from sodium butyrate, or a prodrug thereof, suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA).
  • SAHA suberoylanilide hydroxamic acid
  • TSA trichostatin A
  • the compound of formula I or formula II and the HDAC inhibitor are each in unit dosage form.
  • the kit of parts contains a plurality of unit dosage forms of each component, i.e. of components (i) and (ii) above.
  • the kit of parts may further comprise a means for facilitating compliance with a particular dosing regimen, for example, instructions indicating when, how, and how frequently the unit dosage forms of each component should be taken.
  • Figure 1 shows that concomitant treatment of seliciclib and sodium butyrate leads to a synergistic increase in apoptosis in A549 cells, as determined by an increase in sub- Gl cell fragments, following 72 hours treatment.
  • Figure 2 shows that concomitant treatment of seliciclib and sodium butyrate in H460 cells leads to a synergistic increase in apoptosis following 72 hours treatment, as determined by annexin V staining.
  • Figure 3 shows that in respect of the seliciclib/sodium butyrate combination there is an increase in apoptosis at the IC50 concentration, as determined by caspase cleavage of cytokeratin 18 (M30 ELISA) and PARP cleavage.
  • Figure 3 also shows a synergistic decrease in McIl levels which probably relates to the loss of this anti- apoptotic protein, pushing the cells into apoptosis.
  • Figure 4 shows the molecular pathways of apoptosis with regard to the seliciclib/sodium butyrate combination in more detail.
  • Loss of McIl and Bcl2 both anti-apoptotic proteins pushes the cells towards apoptosis.
  • Both XIAP and survivin are inhibitors of the apoptotic process therefore the loss of these proteins again push the cells towards apoptosis.
  • This effect may explain the synergistic induction of apoptosis as shown by the appearance of PARP.
  • the Histone Western blot shows that the HDAC inhibitor increases the amount of acetylated histone (since deacetylation is inhibited).
  • Figure 5 shows the time course of cellular events at the IC50 in respect of the seliciclib/sodium butyrate combination. At later time points, the synergistic activation of caspases 3 and 9 (indicating apoptosis is induced) can now be seen.
  • Figure 6 shows the cell cycle distribution after treatment with DMSO (control), sodium valproate, seliciclib and sodium valproate/seliciclib in combination.
  • H460 cells were treated with the indicated drug(s) for the times shown prior to PI analysis on the flow cytometer. The results are the average of two duplicate samples.
  • Figure 7 shows the cell cycle distribution after treatment with DMSO (control), sodium valproate, the indicated CDK inhibitor and sodium valproate/CDK inhibitor in combination.
  • H460 cells were treated with the indicated drug(s) for the times shown prior to PI analysis on the flow cytometer. The results are the average of two duplicate samples.
  • Compound [1] referred to herein is (2R,3S)-3-( ⁇ 9-isopropyl-6-[(pyridin-3- ylmethyl)amino]-9H-purin-2-yl ⁇ amino)pentan-2-ol.
  • Sodium butyrate and sodium valproate were obtained from Sigma; TSA was obtained from AG Scientific, Inc.; SAHA was obtained from Toronto Research Chemicals, Inc.
  • the concomitant treatment 1.5-fold serial dilutions of the CDK inhibitor (seliciclib, compounds [l]-[4]), HDAC inhibitor, or both drugs simultaneously were added to cells 24h after plating, and left for 72h at 37 0 C.
  • the drug concentrations selected were chosen to span the IC 50 values for the drugs tested.
  • the first drug was added 2h after cells were plated, and left for 24h.
  • Medium was aspirated and replaced with fresh medium containing the second drug, and incubated for 72h.
  • the two controls for each sequential treatment involved substituting one of the drug treatments with medium.
  • Protein lysates were generated from 10cm plates that were seeded at approximately 5 x 10 5 cells/well, in medium containing 10% FCS. Cells were incubated with HDAC inhibitor and/or seliciclib at the indicated concentrations and times prior to harvest. After incubation, the supernatants were removed and centrifuged at 2000rpm for 5 min to pellet any floating cells.
  • Flow Cytometry H460 cells were seeded in 10cm plates at approximately 3x10 5 cells/plate and left to settle overnight. Next day, seliciclib, sodium butyrate or both drugs were added at the indicated concentrations. After either 24h or 72h treatment, cells were harvested by trypsinisation. Cell cycle analysis by propidium iodide (PI) staining involved fixing the cells overnight in 70% (v/v) ethanol at -2O 0 C prior to analysis on the flow cytometer. Annexin V staining was performed as indicated in manufacturers instructions, on live, non-fixed cells.
  • PI propidium iodide
  • H460 cells were seeded onto 10cm plates at approximately 0.5 x 10 6 cells/plate and allowed to settle for 24h.
  • Cells were treated with sodium valproate for 24 hours followed by the CDK inhibitor (seliciclib, compounds [l]-[4]) for a further 24 hours.
  • the concentrations of compound used were equivalent to 1 x IC 5 O.
  • Single agent control treatments were also carried out. These involved treating the cells with sodium valproate for 24 hours followed by drug-free medium for a further 24 hours, or drug- free medium for 24 hours followed by the CDK inhibitor for a further 24 hours. All cells were harvested by collecting the media prior to media changes, as well as at 48 hours.
  • Adherent cells were harvested by trypsinisation, pooled with the cells in suspension, washed twice in PBS and fixed by resuspending in ImI ice-cold 70% ethanol. Standard cell cycle analysis by propidium iodide staining was carried out on the flow cytometer. Results are the average of duplicate samples.
  • M30/TPS analysis of time-gap experiment A549 cells were seeded in 96 well plates and left to settle overnight. Cells were treated with seliciclib, sodium butyrate or the combination at the indicated concentrations. After 72h treatment, medium was harvested, retained and stored at - 2O 0 C. Samples were analysed in the M30 ELISA as described in the manufacturers instructions.
  • Seliciclib was tested in combination with the indicated HDAC inhibitors in H460 and A549 cell lines, using three different treatment regimes.
  • the Combination Index values from each drug treatment are shown for ED50, ED75 and ED90 values (the point on the curve where 50%, 75% and 90% of the cells have been killed).
  • Data are the average of at least three independent experiments (Table 1).
  • Table 1 Data for the effect of seliciclib and the HDAC inhibitors on the A549 cell lines are shown in parentheses.
  • seliciclib is synergistic when used in combination with all three HDAC inhibitors tested, demonstrating that combining seliciclib with a HDAC inhibitor is a good concept for treating NSCLC cell lines.
  • butyrate was tested in combination with the indicated CDK inhibitors in H460 cells, using three different treatment regimes.
  • the Combination Index values from each drug treatment are shown for ED50, ED75 and ED90 values (the point on the curve where 50%, 75% and 90% of the cells have been killed).
  • Data are the average of three independent experiments (Table 2).
  • Table 2 Data for the effect of compounds [l]-[4] in combination with sodium butyrate.
  • Seliciclib and Butyrate induce a synergistic increase in sub-Gl A549 cells.
  • A549 cells were incubated with IC50 butyrate, 0.25 - 1.5 X IC50 seliciclib, or 0.25 - 1.5 X IC50 seliciclib in the presence of IC50 butyrate for 72h. Cells were then harvested, stained with propidium iodide and their DNA content analysed by flow cytometry. Data are representative of two independent experiments ( Figure 1).
  • Seliciclib and Butyrate induce a synergistic increase in apoptotic H460 cells.
  • H460 cells were incubated with 0.25 - 1.5X IC50 butyrate, 0.25 - 1.5 X IC50 seliciclib, or 0.25 - 1.5 X IC50 seliciclib and butyrate for 72h. Cells were then harvested, stained with annexin V and analysed on the flow cytometer. Data are representative of two independent experiments ( Figure 2).
  • Annexin V labels live cells that are undergoing apoptosis.
  • butyrate and seliciclib induced a much larger annexin V signal than the two single drug treatments combined, indicating a synergistic increase in apoptotic cells.
  • the highest concentration of butyrate and seliciclib (1.5 X IC50) appears to contain fewer cells undergoing apoptosis than those treated with 0.67 X or 1 X IC50, the reason for this is not clear at present.
  • A549 cells were treated with DMSO (control) or with IC50 concentrations of seliciclib, sodium butyrate, or seliciclib and butyrate for 72h, as indicated.
  • Cell culture supernatants were harvested and tested in the M30 apoptosense ELISA, and the cells harvested and analysed for cleaved PARP and McI-I by western blotting. Data are representative of two independent experiments ( Figure 3).
  • H460 cells were treated with butyrate, seliciclib or seliciclib and butyrate at 1 X or 1.5 X IC50 concentrations for 24h. Cells were harvested and the resulting cell lysates analysed by western blotting with the indicated antibodies. Data are representative of two independent experiments ( Figure 4).
  • H460 cells were treated with IX IC50 butyrate, seliciclib or seliciclib and butyrate for the indicated times. Cells were harvested and the resulting cell lysates analysed by western blotting with the indicated antibodies. Data are representative of two independent experiments ( Figure 5).

Abstract

A first aspect of the invention relates to a combination comprising a HDAC inhibitor and a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, and a HDAC inhibitor. A second aspect of the invention relates to a pharmaceutical product comprising a HDAC inhibitor and a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, as a combined preparation for simultaneous, sequential or separate use in therapy. A third aspect of the invention relates to a method for treating a proliferative disorder, said method comprising simultaneously, sequentially or separately administering a compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, and a HDAC inhibitor to a subject.

Description

COMBINATION
The present invention relates to a pharmaceutical combination suitable for the treatment of proliferative disorders. In particular, the present invention relates to combinations for the treatment of cancer, preferably non-small cell lung cancer (NSCLC).
BACKGROUND TO THE INVENTION
Cyclin-dependent kinases (CDKs) are serine/threonine kinases that play a crucial regulatory role in the cell cycle. CDKs regulate cell cycle progression by phosphorylation of various proteins involved in DNA replication and cell division, including transcription factors and tumour suppressor proteins (Senderowicz, AM. Small-molecule cyclin-dependent kinase modulators, Oncogene, 2003; 22: 6609- 6620). Certain CDKs also play a role in the regulation of RNA synthesis by their involvement in the phosphorylation of the carboxy terminal domain (CTD) of the largest subunit of RNA polymerase II (pol II). It is not surprising, therefore, that CDKs have become attractive therapeutic targets. Consequently, many new pharmacological agents capable of interfering with the activity of CDKs by competing for their ATP binding site are currently being tested in clinical trials (Fischer PM and Gianella-Borradori A, CDK inhibitors in clinical development for the treatment of cancer, Expert Opin Investig Drugs. 2003; 12: 955-970).
The prior art has described several compounds that are capable of regulating the cell cycle by virtue of inhibiting cyclin dependent kinases. These compounds include butyrolactone, flavopiridol and 2-(2-hydroxyethylamino)-6-ben2ylamino-9- methylpurine (olomoucine). Olomoucine and related compounds have been shown to be inhibitors of cdc2. Cdc2 (also known as cdkl) is a catalytic sub-unit of a family of cyclin dependent kinases that are involved in cell cycle regulation.
These kinases comprise at least two sub-units, namely a catalytic sub-unit (of which cdc2 is the prototype) and a regulatory sub-unit (cyclin). The cdks are regulated by transitory association with a member of the cyclin family: cyclin A (cdc2, CDK2),\ : cyclin B1-B3 (cdc2), cyclin C (CDK8), cyclin D1-D3 (CDK2-CDK4- CDK5-CDK6), cyclin E (CDK2), cyclin H (CDK7).
Each of these complexes is involved in a phase of the cellular cycle. CDK activity is regulated by post-translatory modification, by transitory associations with other proteins and by modifications of their intra-cellular localization. The CDK regulators comprise activators (cyclins, CDK7/cyclin H, cdc25 phosphateses), the p9.sup.CKS and pi 5. sup. CDK-BP sub-units, and the inhibiting proteins (pl6.sup.INK4A, pl5.sup.INK4B, p21.sup.Cipl, pl8, p27.sup.Kipl).
There is now considerable support in the literature for the hypothesis that CDKs and their regulatory proteins play a significant role in the development of human tumors. Thus, in numerous tumors a temporal abnormal expression of cyclin-dependent kinases, and a major de-regulation of protein inhibitors (mutations, deletions) has been observed.
Roscovitine has been demonstrated to be a potent inhibitor of cyclin dependent kinase enzymes, particularly CDK2. CDK inhibitors are understood to block passage of cells from the Gl /S and the G2/M phase of the cell cycle. The pure R-enantiomer of roscovitine, seliciclib (R-Roscovitine; CYC202) has recently emerged as a potent inducer of apoptosis in a variety of tumour cells (McClue SJ, Blake D, Clarke R, et al,
In vitro and in vivo antitumor properties of the cyclin dependent kinase inhibitor CYC202 (R-Roscovitine), Int J Cancer. 2002; 102: 463-468) and is already in clinical trials to treat breast cancer and non-small cell lung cancer (Fischer PM and Gianella-
Borradori A, CDK inhibitors in clinical development for the treatment of cancer,
Expert Opin Investig Drugs, 2003; 12: 955-970). Roscovitine has also been shown to be an inhibitor of retinoblastoma phosphorylation and therefore implicated as acting more potently on Rb positive tumors.
It well established in the art that active pharmaceutical agents can often be administered in combination in order to optimise the treatment regime. For example, the use of a CDK inhibitor in combination with a second chemotherapeutic agent is described in WO 03/077999, WO 03/082337, WO 2004/041262, WO 2004/041267, WO 2004/041268, WO 2004/041308, WO 2004/110455 and WO 2005/053699 (all to Cyclacel Limited). The present invention seeks to provide a new combination of known pharmaceutical agents that is particularly suitable for the treatment of proliferative disorders, especially cancer. More specifically, a preferred aspect of the invention centres on combinations useful in the treatment of non-small cell lung cancer (NSCLC).
STATEMENT OF THE INVENTION
A first aspect of the present invention relates to a combination comprising a histone deacetylase inhibitor and a compound of formula I or II, or a pharmaceutically acceptable salt thereof,
wherein said compound of formula I is defined as
or a pharmaceutically acceptable salt thereof, wherein one of R1 and R2 is methyl, ethyl or isopropyl, and the other is H;
R3 and R4 are each independently H, branched or unbranched C1-C6 alkyl, or aryl, and wherein at least one of R3 and R4 is other than H;
R5 is a branched or unbranched Cj -C5 alkyl group or a Ci-C6 cycloalkyl group, each of which may be optionally substituted with one or more OH groups;
R6, R7, R8 and R9 are each independently H, halogen, NO2, OH, OMe, CN,
NH2, COOH, CONH2, or SO2NH2;
and said compound of formula II is defined as
II
wherein
R10 and R14 are each independently H, C(OR' ) or a hydrocarbyl group optionally substituted by one or more R15 groups;
R11, R12, and R13 are each independently H, alkyl or alkenyl, each of which may be optionally substituted with one or more R1 groups;
R15 and R16 are each independently halogen, NO2, CN, (CH2)mORa,
O(CH2)nORb, (CH2)pNRcRd, CF3, COORe, C0NRfRs, CORh, SO3H5 SO2R1, SO2NRjRk, (CH2)qNRa'COR8>, Rf, (CH2)rNRb'SO2Rh', SO2NRd'Rr,
SO2NR6 (CH2)SORC , heterocycloalkyl or heteroaryl, wherein said heterocycloalkyl and heteroaryl may be optionally substituted by one or more substituents selected from aralkyl, sulfonyl, Rm and COR";
R8 , Rh , R1 and Rj are each independently selected from alkyl, aryl, aralkyl and heteroaryl, each of which may be optionally substituted with one or more substituents selected from halogen, OH, NO2, NH2 CF3 and COOH; m, p, q and r are each independently O, 1, 2 or 3; n and s are each independently 1, 2, or 3; and
Ra'n and Ra "F are each independently H or alkyl.
Although the compounds of formula I and formula II, and the above-mentioned HDAC inhibitor are well established in the art as individual therapeutic agents, there has been no suggestion that the specific combinations claimed in the present invention would be effective in the treatment of cancer. Moreover, there has been no suggestion in the art that the specifically claimed combinations would be useful in the treatment of NSCLC, which is known to be particularly difficult to treat. A second aspect relates to a pharmaceutical composition comprising a combination according to the invention and a pharmaceutically acceptable carrier, diluent or excipient.
A third aspect relates to the use of a combination according to the invention in the preparation of a medicament for treating a proliferative disorder.
A fourth aspect relates to a pharmaceutical product comprising a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, and a HDAC inhibitor, as a combined preparation for simultaneous, sequential or separate use in therapy.
A fifth aspect relates to a method of treating a proliferative disorder, said method comprising simultaneously, sequentially or separately administering a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, and a HDAC inhibitor.
A sixth aspect relates to the use of a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment of a proliferative disorder, wherein said treatment comprises simultaneously, sequentially or separately administering a HDAC inhibitor.
A seventh aspect relates to the use of a HDAC inhibitor in the preparation of a medicament for the treatment of a proliferative disorder, wherein said treatment comprises simultaneously, sequentially or separately administering a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof.
An eighth aspect relates to the use of (i) a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, and (ii) a HDAC inhibitor, in the preparation of a medicament for treating a proliferative disorder.
A ninth aspect relates to the use of a HDAC inhibitor, in the preparation of a medicament for treating a proliferative disorder, wherein said medicament is for use in combination therapy with a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof. A tenth aspect relates to the use of a HDAC inhibitor, in the preparation of a medicament for treating a proliferative disorder, wherein said medicament is for use in pretreatment therapy with a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof.
An eleventh aspect relates to the use of a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating a proliferative disorder, wherein said medicament is for use in combination therapy with a HDAC inhibitor.
A twelfth aspect relates to the use of a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating a proliferative disorder, wherein said medicament is for use in pretreatment therapy with a HDAC inhibitor.
A thirteenth aspect relates to the use of a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, and a HDAC inhibitor, in the preparation of a medicament for the treatment of non small cell lung cancer (NSCLC).
DETAILED DESCRIPTION
The preferred embodiments set out below are applicable to all the above-mentioned aspects of the invention.
In one preferred embodiment, the invention relates to a combination which comprises a histone deacetylase (HDAC) inhibitor and a compound of formula I, or a pharmaceutically acceptable salt thereof.
In one highly preferred embodiment, the invention provides a combination comprising a 2,6,9-substituted purine derivative of formula I, which exhibits improved resistance to metabolic deactivation, and a HDAC inhibitor.
In one preferred embodiment of the invention, one of R1 and R2 is ethyl or isopropyl, and the other is H.
In another preferred embodiment of the invention, Rs is isopropyl or cyclopentyl. In one preferred embodiment, R6, R7, R8 and R9 are all H.
In one preferred embodiment, R1 or R2 is ethyl and the other is H.
In one preferred embodiment, R3 and R4 are each independently H5 methyl, ethyl, propyl, butyl or phenyl.
Thus, in one preferred embodiment, R3 and R4 are each independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, t-butyl or phenyl.
In a more preferred embodiment, R3 and R4 are each independently H, methyl, ethyl, propyl or butyl.
Thus, in one preferred embodiment, R3 and R4 are each independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl or t-butyl.
In an even more preferred embodiment, R3 and R4 are each independently H, methyl, ethyl, isopropyl or t-butyl.
In one especially preferred embodiment, said compound of formula I is selected from the following:
(25'3i?)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-pentan- 2-ol;
(2R3S)-3 ~ { 9-Isopropyl-6- [(pyridin-3 -ylmethyl)-amino] -9H-purin-2-ylamino } -pentan- 2-ol;
(3i?jS',4i?)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-hexan- 3-ol;
(3 RS,4S)-4- { 9-Isopropyl-6- [(pyridin-3 -ylmethyl)-amino] -9H-purin-2-ylamino } -hexan- 3-ol;
(3i?iSr,4i?)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2- methyl-hexan-3-ol;
(3i?5',4S)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2- methyl-hexan-3-ol; (3i25',4i?)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2,2- dimethyl-hexan-3 -ol;
(3i?5,41S)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2,2- dimethyl-hexan-3-ol;
(3i?)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl- pentan-2-ol;
(3<S)-3 - { 9-Isopropy 1-6- [(pyridin-3 -ylmethyl)-amino] -9H-purin-2-ylamino } -2-methyl- pentan-2-ol;
(3 S)-3 - { 9-Isopropyl-6- [(pyridin-3 -ylmethyl)-amino] -9H-purin-2-ylamino } -2-methyl- pentan-2-ol; and
(3R)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2 -methyl- pentan-2-ol.
Even more preferably, said compound of formula I is selected from the following: (26'3J?)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-pentan-2- ol; (2i?3jS)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purm-2-ylamino}-pentan-2- ol;
(3i?iS',4i2)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-hexan-
3-ol;
(3i?5',45)-4-{9-Isopropyl-6-[(pyridin.-3-ylmethyl)-amino]-9H-purin-2-ylammo}-hexan-
3-ol;
(3i?5',45}-4-{9-Isopropyl-6-[(ρyridin-3-ylmethyl)-amino]-9H-ρurin-2-ylamino}-2,2- dimethyl-hexan-3 -ol ;
(3i?)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl- pentan-2-ol; and
(31S)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-3-ylamino}-2-methyl- pentan-2-ol.
More preferably still, said compound of formula I is selected from the following: (3R)-3-{9-isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl- pentan-2-ol; (3 S)-3 - { 9-isopropy l-6-[(pyridin-3 -ylmethy l)-amino] -9H-purin-2-ylamino } -2-methyl- pentan-2-ol;
(2S3R)-3-{9-isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylammo}-pentan- 2-ol; (2R3 S)-3 - { 9-isopropy 1-6- [(pyridin-3 -ylmethy l)-amino] -9H-purin-2-ylamino } -pentan- 2-ol; and any optical isomer of 3-{9-isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2- ylamino} -pentan-2-ol.
In one especially preferred embodiment, said compound of formula I is selected from the following:
3 -( { 9-isopropyl-6- [(pyridin-3 -y lmethyl)amino] -9H-purin-2-yl } amino)pentan-2-ol;
(2R,3 S)-3 -( { 9-isopropy 1-6- [(pyridin-3 -ylmethyl)amino] -9H-purin-2-yl } amino)pentan-
2-ol; (3R)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2-methyl- pentan-2-ol; and
(3 S)-3 - { 9-Isopropyl-6- [(pyridin-3 -ylmethy l)-amino] -9H-purin-2-ylamino } -2-methyl- pentan-2-ol).
In another aspect, the present invention provides a combination comprising a compound of formula II and a HDAC inhibitor.
Previous studies by the applicant disclosed novel 2-anilino-4-(thiazol-5-yl)- pyrimidine compounds as ATP-competitive inhibitors of various protein kinases (S. Y. Wu et al, 2003, Structure, 11, 399; WO 2001072745, WO 2002079193, and WO 2003029248). Recent studies have now revealed that corresponding compounds containing a 3H-thiazol-2-one-5-yl group are also biologically active as kinase inhibitors (WO 2005042525).
One embodiment of the present invention relates to a combination comprising a compound of formula Ha, or a pharmaceutically acceptable salt thereof,
Ha wherein
R10 and R14 are each independently H or a hydrocarbyl group optionally substituted by one or more R15 groups;
R11, R12, and R13 are each independently H, alkyl or alkenyl, each of which may be optionally substituted with one or more R groups;
R15 and R16 are each independently halogen, NO2, CN, (CH2)mORa, where m is 0, 1, 2 or 3, O(CH2)nORb, where n is 1, 2, or 3, NRcRd, CF3, COORe, CONRfRg, CORh, SO3H5 SO2R1, SO2NRjRk, heterocycloalkyl or heteroaryl, wherein said heterocycloalkyl and heteroaryl may be optionally substituted by one or more substituents selected from Rm and CORn; and
Ra"n are each independently H or alkyl.
As used herein, the term "hydrocarbyl" refers to a group comprising at least C and H. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain heteroatoms. Suitable heteroatoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen, oxygen, phosphorus and silicon. Preferably, the hydrocarbyl group is an aryl, heteroaryl, alkyl, cycloalkyl, aralkyl or alkenyl group.
As used herein, the term "alkyl" includes both saturated straight chain and branched alkyl groups which may be substituted (mono- or poly-) or unsubstituted. Preferably, the alkyl group is a Ci-20 alkyl group, more preferably a Ci.15, more preferably still a Ci-J2 alkyl group, more preferably still, a Cj-6 alkyl group, more preferably a C1.3 alkyl group. Particularly preferred alkyl groups include, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl and hexyl. Suitable substituents include, for example, one or more R 5 groups.
As used herein, the term "cycloalkyl" refers to a cyclic alkyl group which may be substituted (mono- or poly-) or unsubstituted. Preferably, the cycloalkyl group is a C3-12 cycloalkyl group. Suitable substituents include, for example, one or more R15 groups.
The term "heterocycloalkyl" refers to a cycloalkyl group containing one or more heteroatoms selected from O, N and S. Examples of heterocycloalkyl include 1- (1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, pyrrolidinyl, dihydrofuranyl, tetrahydropyranyl, pyranyl, thiopyranyl, aziridinyl, oxiranyl, methylenedioxyl, chromenyl, isoxazolidinyl, l,3-oxazolidin-3-yl, isothiazolidinyl, l,3-thiazolidin-3-yl, 1 ,2-pyrazolidin-2-yl, 1,3-pyrazolidin-l-yl, thiomorpholinyl, l,2-tetrahydrothiazin-2- yl, l,3-tetrahydrothiazin-3-yl, tetrahydrothiadiazinyl, l,2-tetrahydrodiazin-2-yl, 1,3- tetrahydrodiazin-1-yl, tetrahydroazepinyl, piperazinyl, chromanyl, etc. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Thus, one of ordinary skill in the art will understand that the connection of said heterocycloalkyl rings is through a carbon or a sp3 hybridized nitrogen heteroatom. Preferred heterocycloalkyl groups include piperazine, morpholine, piperidine and pyrrolidine.
As used herein, the term "alkenyl" refers to a group containing one or more carbon- carbon double bonds, which may be branched or unbranched, substituted (mono- or poly-) or unsubstituted. Preferably the alkenyl group is a C2-20 alkenyl group, more preferably a C2-15 alkenyl group, more preferably still a C2-I2 alkenyl group, or preferably a C2-6 alkenyl group, more preferably a C2-3 alkenyl group. Suitable substituents include, for example, one or more R15 groups as defined above. As used herein, the term "aryl" refers to a Qβ-n aromatic group which may be substituted (mono- or poly-) or unsubstituted. Typical examples include phenyl and naphthyl etc. Suitable substituents include, for example, one or more R15 groups.
As used herein, the term "heteroaryl" refers to a C4-12 aromatic, substituted (mono- or poly-) or unsubstituted group, which comprises one or more heteroatoms. Preferred heteroaryl groups include pyrrole, pyrazole, pyrimidine, pyrazine, pyridine, quinoline, triazole, tetrazole, thiophene and furan. Again, suitable substituents include, for example, one or more R15 groups.
Preferably, R8 , Rh , R1 and R* are each independently selected from alkyl, phenyl, benzyl and pyridyl, each of which may be optionally substituted with one or more substituents selected from halogen, OH, NO2, NH2 CF3 and COOH;
Preferably, Ra"n and Ra "f are each independently H, methyl, ethyl or isopropyl.
In one preferred embodiment of the invention, R10 and R14 are each independently H or a C1-20 hydrocarbyl group optionally comprising up to six heteroatoms selected from from N, O, and S, and which is optionally substituted by one, two or three R15 groups;
In another preferred embodiment, R14 is aryl or heteroaryl, each of which may be optionally substituted by one or more R15 groups.
In another preferred embodiment, R14 is H, CO(RJ ), aryl or heteroaryl, wherein said aryl or heteroaryl groups may be optionally substituted by one or more R15 groups.
More preferably, R14 is H, COMe, phenyl or pyridyl, wherein said phenyl or pyridyl groups may be optionally substituted by one or more R15 groups.
More preferably still, R14 is phenyl or pyridinyl, each of which may be optionally substituted by one or more R15 groups. In a preferred embodiment, R10 is H or alkyl. More preferably, R10 is H, methyl, ethyl or 3-methylbutyl.
Preferably, R11, R12, and R13 are each independently H, C1-C6 alkyl or C2-C6 alkenyl, each of which may be optionally substituted with one, two or three R16 groups.
More preferably, R is C1-6 alkyl. More preferably still, R 11 is methyl.
Preferably, R12 and R13 are both H.
Preferably, R15 and R16 are each independently F, Cl, Br, I, NO2, CN3 OH, OMe, OEt, CH2OH, O(CH2)2OMe, NH2, NHMe, NMe2, CF3, COOH, CONH2, CONHMe, CONMe2, COMe, SO3H, SO2Me, SO2NH2, SO2NHMe, SO2NMe2, morpholine, piperidine, piperazine, N-acetylpiperazine, N-methylpiperazine, triazole, or tetrazole.
In one preferred embodiment, R12 and R13 are both H and R11 is Me.
In one particularly preferred embodiment, the compound of the invention is of formula III, or a pharmaceutically acceptable salt thereof,
III wherein
R10 is as defined above in claim 1 or claim 12;
X is C; or X is N and R17 is absent;
R , 117 ', τ R> 1l8iS τ R» 1i9y and R >2z0u are each independently H or as defined for R15 and R , 16
More preferably, for said compound of formula III, R10 is H or alkyl;
R17 is H, NO2, OR", halogen, CF3, CN, CORq, alkyl, NRrRs, 0(CHa)1OR1; R18 is H, ORU, halogen, alkyl, NRVRW, or a heterocycloalkyl optionally substituted with one or more substituents selected from Rm and C0Rn; t is O, 1, 2 or 3; R19 is H, alkyl or NRxRy; and Rp"y are each independently H or alkyl.
In one particularly preferred embodiment, R10 is H, Me, Et or 3-methylbutyl.
More preferably still, for said compound of formula III,
R17 is H, NO2, OH, Me, I, CF3, CN, CH2OH, CO2H, CO2Me OrNH2;
R18 is H, F, OH, I, Cl, Br, OMe, NMe2, morpholine, Me, N-methylpiperazine, N- acetylpiperazine or piperazine; and R19 is H, Me or NMe2.
In one preferred embodiment, for said compound of formula III, R17 is selected from H, NO2, halogen, CN, CF3, SO3H, (CH2)mORa, COORe, (CH2)pNRcRd, (CH2)rNRb'SO2Rh', (CH2)qNRa'C0Rgl, SO2NRjRk, C0NRfRg, SO2NRe'(CH2)sORc>, S02NRd R' and heterocycloalkyl optionally substituted by one or more COR" or sulfonyl groups.
More preferably, R17 is selected from H, NO2, OH, Me, I, CN, CH2OH, CF3, CO2H, CO2Me, NH2, Cl, 4-acetylpiperazin-l-yl, OMe, SO3H, CH2NHSO2Me, CH2NHCOPh, CH2NHSO2CF3, SO2NH2, CONH1Pr, SO2NHEt, SO2NH(CH2)2OMe, SO2NH1Pr, SO2NH(CH2)2OH, NHMe, SO2NH-benzyl and morpholin-4-sulfonyl.
In one preferred embodiment, for said compound of formula III, R18 is selected from H, NO2, SO3H, halogen, (CH2)mORa, (CH2)pNRcRd, (CH2)qNRa'CORs>, SO2NRe'(CH2)sORc>, SO2NRd>R'' and heterocycloalkyl optionally substituted by one or more COR", Rm or aralkyl groups. More preferably, R18 is selected from H, F, OH, Cl, Br, OMe, NMe2, morpholin-4-yl, 4-methylpiperazin-l-yl, Me, 4-acetyl-piperazin-l-yl, I, CH2NHCOMe, NO2, SO3H, SO2NH(CH2)2OMe, 4-benzylpiperazin-l-yl, SO2NH(CH2)2OH, SO2NH-benzyl, CH2NH2, CH2NHCO-(pyrid-2-yl) and piperazin-1-yl.
In one preferred embodiment, for said compound of formula III, R19 is selected from H, Rf and (CH2)pNR0Rd.
More preferably, R19 is selected from H, Me and NMe2.
In one preferred embodiment, for said compound of formula III, R20 is selected from H5 Rf , CF3, halogen and (CH2)qNRa CORg>.
More preferably, R20 is selected from H, NHCOMe, CF3, Br and Me.
In one preferred embodiment, X is N and R1 is absent.
In another preferred embodiment, X is C.
In one preferred embodiment of the invention, the compound of formula II is selected from the following:
3,4-Dimethyl-5-[2-(3-nitro-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one;
5-[2-(4-Fluoro-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one;
5-[2-(4-Hydroxy-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one;
5-[2-(4-Chloro-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one;
5-[2-(4-Bromo-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one;
5-[2-(4-Methoxy-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one;
5 - [2-(3 -Hydroxy-phenylamino)-pyrimidin-4-yl] -3 ,4-dimethyl-3 H-thiazol-2-one;
5-[2-(4-Dimethylamino-phenylamino)-ρyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one;
3,4-Dimethyl-5-[2-(4-moφholin-4-yl-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one;
5-[2-(4-Fluoro-3-nitro-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one;
3,4-Dimethyl-5-[2-(4-methyl-3-nitro-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one; 5-[2-(4-Fluoro-3-methyl-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one; 3 ,4-Dimethyl-5 - { 2-[4-(4-methyl-piperazin- 1 -yl)-phenylamino] -pyrimidin-4-yl } -3 H-thiazol-2- one;
5 -[2-(3 -Iodo-4-methy l-phenylamino)-pyrimidin-4-yl] -3 ,4-dimethyl-3 H-thiazol-2-one; 5-[2-(4-Chloro-3-methyl-phenylamino)-pyrimidin-4-yl]-354-dimethyl-3H-thiazol-2-one; 3-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-benzonitrile; 5-{2-[4-(4-Acetyl-piperazin-l-yl)-phenylamino]-pyrimidin.-4-yl}-3,4-dimethyl-3H-thiazol-2- one;
5-[2-(4-Chloro-3-hydroxymethyl-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2- one;
3,4-Dimethyl-5-[2-(3-trifluoromethyl-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one; 3,4-Dimethyl-5-[2-(2-methyl-5-nitro-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one; 3,4-Dimethyl-5-[2-(4-methyl-3-trifluoromethyl-phenylamino)-pyrimidin-4-yl]-3H-t]:iiazol-2- one;
5-[2-(4-Dimethylamino-3-nitro-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2- one;
3-Ethyl-4-methyl-5-[2-(3-nitro-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one; 2-Chloro-5-[4-(3-ethyl-4-methyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]- benzoic acid;
2-Chloro-5-[4-(3-ethyl-4-methyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]- benzoic acid methyl ester;
5-[2-(4-Dimethylamino-phenylamino)-pyrimidin-4-yl]-3-ethyl-4-methyl-3H-thiazol-2-one; 3-Ethyl-4-methyl-5-[2-(4-morρholin-4-yl-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one; 3-Ethyl-4-methyl-5-[2-(4-methyl-3-nitro-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one; 5-[2-(4-Dimethylamino-3-nitro-phenylamino)-pyrimidin-4-yl]-3-ethyl-4-methyl-3H-thiazol- 2-one;
4-Methyl-3-(3-methyl-butyl)-5-[2-(3-nitro-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one; 5-[2-(4-Chloro-ρhenylamino)-pyrimidin-4-yl]-4-methyl-3-(3-methyl-butyl)-3H-thiazol-2-one; 5-[2-(6-Chloro-pyridin-3-ylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one; 3-Ethyl-5-[2-(6-methoxy-pyridin-3-ylamino)-pyrimidin-4-yl]-4-methyl-3H-thiazol-2-one; 5-[2-(6-Chloro-pyridin-3-ylamino)-pyrimidin-4-yl]-4-methyl-3-(3-methyl-butyl)-3H-thiazol- -one; 5-[2-(6-Methoxy-pyridin-3-ylamino)-pyrimidin-4-yl]-4-methyl-3-(3-methyl-butyl)-3H- thiazol-2-one;
5-[2-(4-Iodo-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one; 5-[2-(2-Dimethylamino-5-nitro-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2- one;
3,4-Dimethyl-5-[2-(4-piperazin-l-yl-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one; 5-[2-(3-Amino-4-methyl-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one; 4-Methyl-5 - [2-(3 -nitro-phenylamino)-pyrimidin-4-yl] -3 H-thiazol-2-one ; 4-Methyl-5-[2-(4-methyl-3-nitro-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one; N-{3-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-benzyl}- acetamide;
3-Ethyl-5-[2-(3-hydroxy-phenylamino)-pyrimidin-4-yl]-4-methyl-3H-thiazol-2-one; 5-[2-(3-Chloro-4-piperazin-l-yl-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2- one;
3 -Ethyl-5 - [2-(4-fluoro-phenylamino)-pyrimidin-4-yl] -4-methyl-3 H-thiazol-2-one ; 5-[2-(4-Chloro-phenylamino)-pyrimidin-4-yl]-3-ethyl-4-methyl-3H-thiazol-2-one; 3 -Ethyl-5 - [2-(3 -hydroxy-4-methyl-phenylamino)-pyriniidin-4-yl] -4-methyl-3H-thiazol-2-one; 5-[2-(4-Chloro-3-trifluoromethyl-phenylamino)-pyrimidin-4-yl]-3-ethyl-4-methyl-3H-thiazol- 2-one;
5-{2-[3-(4-Acetyl-piperazin-l-yl)-phenylamino]-pyrimidin-4-yl}-3,4-dimethyl-3H-thiazol-2- one;
3-Ethyl-5-[2-(3-methoxy-phenylamino)-pyrimidin-4-yl]-4-methyl-3H-thiazol-2-one; 5-[2-(4-Chloro-3-methyl-phenylamino)-pyrimidin-4-yl]-3-ethyl-4-methyl-3H-thiazol-2-one; 3-Ethyl-4-methyl-5-[2-(4-nitro-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one; 4-[4-(3-Ethyl-4-methyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]- benzenesulfonic acid;
3-[4-(3-Ethyl-4-methyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]- benzenesulfonic acid;
N-{3-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-benzyl}- methane-sulfonamide;
5-[2-(5-Methoxy-2-methyl-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one N-{3-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-benzyl}- benzamide; N- { 3-[4-(3 ,4-Dimethyl-2-oxo-2,3 -dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-benzyl } - C,C,C-trifluoro-methanesulfonamide;
N-{4-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-benzyl}- acetamide;
3 - [4-(3 ,4-Dimethyl-2-oxo-2,3 -dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]- benzenesulfonamide;
3-[4-(3,4-Diniethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-N-isopropyl-4- methyl-benzamide;
3-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-N-ethyl- benzenesulfonamide;
5-[2-(5-Hydroxymethyl-2-methyl-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2- one;
N- { 3 -[4-(3 ,4-Dimethyl-2-oxo-2,3 -dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-5- trifluoromethy 1-phenyl } -acetamide;
4-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-N-(2-methoxy- ethyl)-benzenesulfonamide;
5 - [2-(4-Chloro-3 -trifluoromethyl-phenylamino)-pyrimidin-4-yl] -3 ,4-dimethyl-3 H-thiazol-2- one;
3-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-N-(2-methoxy- ethyl)-benzenesulfonamide;
5-[2-(3-Bromo-54rifluoromethyl-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2- one;
5 - { 2- [4-(4-Benzyl-piperazin- 1 -yl)-phenylamino] -pyrimidin-4-yl } -3 ,4-dimethyl-3 H-thiazol-2- one;
4-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-2-trifluoromethyl- benzonitrile;
5 - [2-(3 -Amino-5 -trifluoromethyl-phenylamino)-pyrimidin-4-yl] -3 ,4-dimethyl-3 H-thiazol-2- one;
4-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-N-(2-hydroxy- ethyl)-benzenesulfonamide;
N-Benzyl-4-[4-(3,4-dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]- benzenesulfonamide;
3-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-N-isopropyl- benzenesulfonamide; 3-[4-(3,4-Dimethyl-2-oxo-253-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-N-(2-hydroxy- ethyl)-benzenesulfonarαide;
3,4-Dimethyl-5-[2-(3-methylatnino-5-trifluoromethyl-phenylaniino)-pyrimidin-4-yl]-3H- thiazol-2-one;
N-Benzyl-3-[4-(3,4-dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]- benzenesulfonamide;
3,4-Dimeth.yl-5-{2-[4-methyl-3-(morpholine-4-sulfonyl)-phenylarQino]-pyrimidin-4-yl}-3H- thiazol-2-one;
3,4-Dimethyl-5-{2-[3-(morpholine-4-sulfonyl)-phenylamino]-pyrimidin-4-yl}-3H-thiazol-2- one;
5-[2-(4-Aminomethyl-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one; 5-[2-(6-Chloro-5-methyl-pyridin-3-ylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one; Pyridine-2-carboxylic acid 4-[4-(3,4-dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2- ylamino]-benzylamide;
3 ,4-Dimethyl-5 - {2- [(pyridin-3 -y lmethyl)-amino] -pyrimidin-4-yl } ~3H-thiazol-2-one; 5-(2-Amino-pyrimidin-4-yl)-3,4-dimethyl-3H-thiazol-2-one; N-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-yl]-acetamide;
In one especially preferred embodiment, the compound of formula II is selected from the following:
3,4-Dimethyl-5-[2-(3-nitro-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one [I]; 5-[2-(4-Fluoro-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one [2]; 5-[2-(4-Hydroxy-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one [3]; 5-[2-(4-Chloro-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one [4]; 5-[2-(4-Bromo-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one [5]; 5-[2-(4-Methoxy-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one [6]; 5-[2-(3-Hydroxy-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one [7]; 5-[2-(4-Dimethylamino-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one [8]; 3,4-Dimethyl-5-[2-(4-morpholin-4-yl-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one [9]; 5-[2-(4-Fluoro-3-nitro-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one [10]; 3,4-Dimethyl-5-[2-(4-methyl-3-nitro-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one [l l]; 5-[2-(4-Fluoro-3-methyl-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one [12];
354-Dimethyl-5-{2-[4-(4-methyl-piperazin-l-yl)-phenylamino]-pyrimidin-4-yl}-3H-thiazol-
2-one [13];
5-[2-(3-Iodo-4-methyl-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one [14];
5-[2-(4-Chloro-3-methyl-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one
[15];
3-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-benzonitrile
[16];
5-{2-[4-(4-AcetyI-piperazin-l-yl)-phenylamino]-pyrimidin-4-yl}-3,4-dimethyl-3H-thiazol-
2-one [17];
5 - [2-(4-Chloro-3 -hydroxymethyl-phenylamino)-pyrimidin-4-yl] -3 ,4-dimethyl-3 H-thiazol-
2-one [18];
3,4-Dimethyl-5-[2-(3-trifluoiOmethyl-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one [19];
3,4-Dimethyl-5-[2-(2-methyl-5-nitro-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one [20];
3,4-Dimethyl-5-[2-(4-methyl-3-trifluoromethyl-phenylamino)-pyrimidin-4-yl]-3H-thiazol-
2-one [21];
5-[2-(4-Dimethylamino-3-nitro-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2- one [22];
3 -Ethyl-4-methyl-5 -[2-(3 -nitro-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one [23 ] ;
2-Chloro-5-[4-(3-ethyl-4-methyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]- benzoic acid [24];
2-Chloro-5-[4-(3-ethyl-4-methyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]- benzoic acid methyl ester [25];
5 - [2-(4-Dimethylamino-phenylamino)-pyrimidin-4-yl] -3 -ethyl-4-methyl-3 H-thiazol-2-one
[26];
3-Ethyl-4-methyl-5-[2-(4-morpholin-4-yl-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one
[27];
3-Ethyl-4-methyl-5-[2-(4-methyl-3-nitro-ρhenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one
[28];
5-[2-(4-Dimethylamino-3-nitro-phenylamino)-pyrimidin-4-yl]-3-ethyl-4-methyl-3H- thiazol-2-one [29];
4-Methyl-3-(3-methyl-butyl)-5-[2-(3-nitro-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one
[30]; 5-[2-(4-Chloro-phenylamino)-pyrimidin-4-yl]-4-methyl-3-(3-methyl-butyl)-3H-thiazol-2- one [31];
5-[2-(6-Chloro-pyridin-3-ylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one [32]; 3-Ethyl-5-[2-(6-methoxy-pyridin-3-ylamino)-pyrimidin-4-yl]-4-methyl-3H-thiazol-2-one [33];
5-[2-(6-Chloro-pyridin-3-ylamino)-pyrimidin-4-yl]-4-methyl-3-(3-methyl-butyl)-3H- thiazol-2-one [34];
5 - [2-(6-Methoxy-pyridin-3 -ylamino)-pyrimidin-4-yl] -4-methyl-3 -(3 -methy l-butyl)-3H- thiazol-2-one [35];
5-[2-(4-Iodo-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one [36]; 5-[2-(2-Dimethylamino-5-nitro-phenylamino)-pyrimidin-4-yl]-3,4-diniethyl-3H-thiazol-2- one [37];
3,4-Dimethyl-5-[2-(4-piperazin-l-yl-phenylammo)-pyrimidin-4-yl]-3M-thiazol-2-one [38]; 5-[2-(3-Amino-4-methyl-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one [39];
4-Methyl-5-[2-(3-nitro-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one [40]; and 4-Methyl-5-[2-(4-methyl-3-nitro-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one [41].
In one highly preferred embodiment, said compound of formula II is 3,4-dimethyl-5~ [2-(4-piperazin-l-yl-phenylamino)pyrimidin-4-yl]-3H-thiazol-2-one.
As mentioned above, the presently claimed combinations comprise a compound of formula I or formula II and a histone deacetylase (HDAC) inhibitor.
Histones are small positively charged proteins that are rich in basic amino acids (positively charged at physiological pH). There are five main types of histones namely, Hl, H2A, H2B, H3, and H4 which exhibit a high degree of structural similarity. Histones are not found in eubacteria (e.g., E. coli), although the DNA of these bacteria is associated with other proteins that presumably function like histones to package the DNA within the bacterial cell. Archaebacteria, however, do contain histones that package their DNAs in structures similar to eukaryotic chromatin (G. M. Cooper, "The Cell - A Molecular Approach", 2nd Edition, Chapter II). The majority of histones are synthesized during the S phase of the cell cycle, and newly synthesized histones quickly enter the nucleus to become associated with DNA. Within minutes of its synthesis, new DNA becomes associated with histones in nucleosomal structures.
The amino-terminal tail domains of histones may be enzymatically modified by post- translational addition of methyl (to lysine and arginine groups), acetyl (to lysine groups), or phosphate groups (to serine groups) (Spencer et al, Gene, 1999, 240(1), 1). This results in a reduction of the net positive charge of the histone which, consequently, may weaken the binding of the histone to DNA.
Studies of histone deacetylators (HDACs), as well as the compounds which inhibit HDACs, have elucidated the mechanisms through which some disease states act. For example, in the search for novel anti-malarial compounds, the naturally occurring apicidin was shown to inhibit the in vitro growth of P. falciparum by hyperacetylating histones (K. T. Andrews et al, Int. J. Parasitol., 2000, 30(6), 761).
HDACs, therefore, are believed to be associated with a number of different diseases which include proliferative disorders such as leukemia (Lin et al, Nature, 1998, 391, 811), melanomas/squamous cell carcinomas (Gillenwater et al, Int. J. Cancer, 1998, 75217; Saunders et al, Cancer Res., 1999, 59, 399), breast cancer, prostrate cancer, bladder cancer (Gelmetti et al, MoI. Cell Biol., 1998, 18, 7185; Wang et al, PNAS, 1998, 951, 10860) and colon cancer (C. A. Hassig, et al, 1997, Chem. Biol., 4, 783; S. Y. Archer et al, PNAS, 1998, 95(12), 6791).
US 2005/0004007 discloses a method for promoting apoptosis in cancer cells which involves administering a cyclin dependent kinase inhibitor and an agent which induces cellular differentiation. Several categories of agent which induce cellular differention are given namely, histone deacetylase inhibitors, protein kinase C, retinoids and vitamin D3. However, combinations comprising a compound of formula I or formula II and a HDAC inhibitor are not specifically disclosed, nor is the use of this combination in the treatment of solid tumours, such as NSCLC. On the contrary, the exemplification of US 2005/0004007 is limited to combinations of flavopiridol with selected HDAC inhibitors tested on leukemia cell lines. Accordingly, to date, there has been no disclosure of the specific combinations claimed in the present application, let alone any suggestion that they would be therapeutically useful in the treatment of lung cancers such as NSCLC.
In one preferred embodiment of the invention, the HDAC inhibitor is sodium butyrate.
Sodium butyrate is formed on the fermentation of dietary fibres in the lumen of the large intestine (G. J. Kelloff et al, Cancer Chemoprevention: Volume 1, page 665). It has been found to increase the expression of exon 7-containing SMN protein from the SMN2 gene in spinal muscular atrophy lymphoid cell lines. It was proposed that sodium butyrate worked by acetylating nucleosomal DNA and other factors that control alternating splicing of exon 7 of the SMN2 gene (J.-G. Chang et al, PNAS, 2001, 98(17), 9809).
Sodium butyrate has also been shown to induce differentiation in cultured erythroleukemic cells (A. Leder et al, Cell, 1975, 5(3), 319) and PC 12 pheochromocytoma cells (J. C. Byrd et al, Brain Res., 1987, 428(1), 151), along with increasing the expression of fetal hemoglobin genes in humans and other animals (S. P. Perrine et al, Adv. Exp. Med. Biol., 1989, 271, 177 and S. P. Perrine et al, PNAS, 1988, 85(22), 8540).
In another preferred embodiment, the HDAC inhibitor is a prodrug of sodium butyrate.
In a particularly preferred embodiment, the prodrug is pivaloyloxymethyl butyrate. Pivaloyloxymethyl butyrate (Pivanex®) is an acyloxyalkyl ester prodrug of butyric acid and has been shown to induce the instrinsic pathway of apoptosis in leukemia and neuroblastoma cells (S. Mei et al, International Journal of Oncology, 2004, 25, 1509).
In another preferred embodiment of the invention, the HDAC inhibitor is trichostatin A (TSA).
The antifungal antibiotic trichostatin was first isolated from the metabolites of strains of Streptomyces hygroscopicus (N. Tsuji et al, J. Antibiot, 1976, 29, 1). Trichostatin A (TSA) is a specific and reversible inhibitor of HDAC. At nanomolar concentrations, TSA causes a marked accumulation of highly acetylated histones in vivo and strongly inhibits the activity of the partially purified histone deacetylase in vitro (M. Yoshida et al, J. Biol. Chem., 1990, 265(28), 17174). In human Jerkat T cells, TSA arrests cell cycle progression in Gl and inhibits the activity of the HDl deacetylase with an IC50 of 70 nM (Y. Hoshikawa et al, Exp. Cell Res., 1994, 214, 189).
TSA can also concomitantly modify the expression of genes. Mishra et al demonstrated that TSA significantly downregulated CDl 54 and IL-IO and up- regulated IFN-γ gene expression in systemic lupus erythematosis (SLE) T cells. SLE is an autoimmune disease characterised by dysregulated production of antibodies which leads to irreversible, immune complex-mediated end-organ failure ( N. Mishra et al, PNAS, 2001, 98(5), 2628).
In another preferred embodiment of the invention, the HDAC inhibitor is suberoylanilide hyroxamic acid (SAHA).
Suberoylanilide hyroxamic acid (SAHA) is a synthetic derivative of TSA which inhibits HDAC activity at micromolar concentrations. SAHA is currently undergoing Phase II clinical trials in the US for its use in the treatment of relapsed or refractory advanced Hodgkin's lymphoma.
The antiproliferative effect of SAHA is well documented. WO 2005/097747 (Aton Pharma) discloses the use of prodrugs of hydroxamic based HDAC inhibitors, such as SAHA, in the treatment of neoplasms, thioredoxin (TRX)-mediated diseases and in the prevention and/or treatment of CNS diseases.
US 2004/4127525 (Bacopoulos et al) discloses the use of SAHA in the treatment of lymphomas, such as diffuse large B-cell lymphoma. WO 2005/039498 and WO 2005/018578 (both to Aton Pharma) disclose further methods for the treatment of neoplasms, wherein WO2005/039498 relates to leukemia and WO 2005/018578 relates to mesothelioma or lymphoma. In one preferred embodiment of the invention, the HDAC inhibitor is sodium valproate (otherwise known as sodium 2-propylpentanoate). Sodium valproate is the sodium salt of valproic acid and is a NICE-approved anticonvulsant drug used in the treatment of epilepsy. More recently, studies have investigated the use of sodium valproate for the treatment of advanced solid tumour malignancies and cancer-related neuropathic pain. Combination studies involving valproic acid and UCN-Ol have also been undertaken. In this regard, although valproic acid itself has only a weak anticancer effect, studies have shown that it becomes highly effective against cancer cells when used in combination.
Another aspect of the present invention relates to a pharmaceutical composition comprising a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, and a HDAC inhibitor.
Another aspect relates to a pharmaceutical product comprising the combination of the present invention for use in the treatment of a proliferative disorder, wherein the disorder is preferably cancer, and more preferably, NSCLC.
A further aspect of the present invention relates to a pharmaceutical product comprising a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, and a HDAC inhibitor as a combined preparation for simultaneous, sequential or separate use in therapy.
Preferably, the HDAC inhibitor is selected from sodium butyrate, or a prodrug thereof, suberoylanilide hydroxamic acid (SAHA), sodium valproate and trichostatin (TSA).
Yet another aspect relates to a method of treating a proliferative disorder, said method comprising simultaneously, sequentially or separately administering a combination of the present invention.
As used herein, "simultaneously" is used to mean that the two agents are administered concurrently, whereas the term "in combination" is used to mean that they are administered, if not simultaneously, then "sequentially" with a timeframe that they are able to act therapeutically within the same time frame. Thus, administration "sequentially" may permit one agent to be administered within 5 minutes, 10 minutes or a matter of hours after the other provided that they are both concurrently present in therapeutic amounts. The time delay between administration of the components will vary depending on the exact nature of the components, the interaction therebetween and their respective half-lives.
In contrast to "in combination" or " sequentially", "separately" is used herein to mean that the gap between administering one agent and the other is significant i.e. the first administered agent may no longer be present in the bloodstream in a therapeutically effective amount when the second agent is administered.
In one preferred embodiment, the HDAC inhibitor is administered sequentially or separately prior to the compound of formula I or formula II, or a pharmaceutically acceptable salt thereof.
In another preferred embodiment, the compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, is administered sequentially or separately prior to the HDAC inhibitor.
In another preferred embodiment, the compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, and the HDAC inhibitor are administered simultaneously.
Where the HDAC inhibitor is sodium butyrate, or a prodrug thereof, preferably the compound of formula I or formula II is administered simultaneously with, or separately or sequentially with, the HDAC inhibitor, irrespective of the order of administration. More preferably, the sodium butyrate and compound of formula I or formula II are administered separately or sequentially.
In one preferred embodiment, the HDAC inhibitor is sodium butyrate and the compound of formula I is compound [I]. Preferably, for this embodiment, the sodium butyrate and compound [1] are administered separately or sequentially, irrespective of the order of administration.
In one preferred embodiment, the HDAC inhibitor is sodium butyrate and the compound of formula II is compound [2]. Preferably, for this embodiment, the sodium butyrate and compound [2] are administered separately or sequentially, irrespective of the order of administration, or simultaneously.
Where the HDAC inhibitor is sodium valproate, preferably the compound of formula I or formula II is administered simultaneously with, or separately or sequentially with, the HDAC inhibitor, irrespective of the order of administration. More preferably, the sodium valproate and compound of formula I or formula II are administered separately or sequentially. Even more preferably, the sodium valproate is administered separately or sequentially prior to the compound of formula I or II.
In one highly preferred embodiment, the HDAC inhibitor is sodium valproate, and the compound of formula I is compound [I]. Preferably for this embodiment, sodium valproate and compound [1] are administered separately or sequentially, irrespective of the order of administration.
In another highly preferred embodiment, the HDAC inhibitor is sodium valproate, and the compound of formula II is compound [2]. Preferably for this embodiment, sodium valproate and compound [2] are administered separately or sequentially, irrespective of the order of administration.
In another highly preferred embodiment, the HDAC inhibitor is sodium valproate, and the compound of formula I is compound [3]. Preferably for this embodiment, the sodium valproate is administered separately or sequentially prior to compound [3], i.e. the subject is pretreated with sodium valproate.
In another highly preferred embodiment, the HDAC inhibitor is sodium valproate, and the compound of formula I is compound [4]. Preferably for this embodiment, the sodium valproate is administered separately or sequentially prior to compound [4], i.e. the subject is pretreated with sodium valproate.
In one preferred embodiment, the compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, and the HDAC inhibitor are each administered in a therapeutically effective amount with respect to the individual components. In another preferred embodiment, the compound of formula I or formula II, or pharmaceutically acceptable salt thereof, and the HDAC inhibitor are each administered in a sub-therapeutic amount with respect to the individual components.
The term "sub-therapeutic amount" means an amount that is lower than that typically required to produce a therapeutic effect with respect to treatment with the compound of formula I or formula II alone or the HDAC inhibitor alone.
A further aspect relates to the use of the combination of the present invention in the preparation of a medicament for treating a proliferative disorder.
As used herein the phrase "preparation of a medicament" includes the use of one or more of the above described components directly as the medicament or in any stage of the manufacture of such a medicament.
Another aspect relates to the use of a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment of a proliferative disorder, wherein said treatment comprises simultaneously, sequentially or separately administering a HDAC inhibitor to a subject.
Yet another aspect relates to the use of a HDAC inhibitor, in the preparation of a medicament for the treatment of a proliferative disorder, wherein said medicament is for use in combination therapy with the compound of formula I or formula II, or a pharmaceutically acceptable salt thereof. Alternatively, the therapy can be pretreatment therapy.
A further aspect relates to the use of the compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment of a proliferative disorder, wherein said medicament is for use in combination therapy with a HDAC inhibitor. Alternatively, the therapy can be pretreatment therapy.
As used herein, the term "combination therapy" refers to therapy in which the HDAC inhibitor and a compound of formula I or formula II are administered, if not simultaneously, then sequentially within a time frame that they both are available to act therapeutically within the same time frame.
As used herein, the term "pretreatment therapy" or "pretreated" means a regimen in which one agent is administered prior to, either separately or sequentially, the second agent. Preferably, the second agent is administered at least 2 hours after the administration of the first agent. More preferably, the second agent is administered at least 4 hours, or more preferably at least 6 or 8 hours, after the administration of the first agent. Even more preferably, the second agent is administered at least 12 hours, or more preferably at least 18 or 24 hours, after the administration of the first agent.
Preferably, the compound of formula I or formula II and the HDAC inhibitor interact in a synergistic manner. As used herein, the term "synergistic" means that the compound of formula I or formula II and the HDAC inhibitor produce a greater effect when used in combination than would be expected from adding the individual effects of the two components. Advantageously, a synergistic interaction may allow for lower doses of each component to be administered to a patient, thereby decreasing the toxicity of chemotherapy, whilst producing and/or maintaining the same therapeutic effect. Thus, in a particularly preferred embodiment, each component can be administered in a sub-therapeutic amount.
In another preferred embodiment, the CDK inhibitor and the HDAC inhibitor interact in a manner so as to alleviate or eliminate adverse side effects associated with use of the individual components in monotherapy, or associated with their use in known combinations.
For all of the above embodiments, preferably the HDAC inhibitor is selected from sodium butyrate, or a prodrug thereof, suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA).
Another aspect of the invention relates to a combination comprising:
(i) a HDAC inhibitor; and (ii) (a) a 2,6,9-substituted purine derivative or a pharmaceutically acceptable salt thereof; or (b) a 2-substituted-4-heteroaryl-pyrimidine derivative, or a pharmaceutically acceptable salt thereof.
The above-described preferred embodiments, methods, compositions and uses apply equally to this combination.
PROLIFERATIVE DISORDER
The term "proliferative disorder" is used herein in a broad sense to include any disorder that requires control of the cell cycle, for example cardiovascular disorders such as restenosis and cardiomyopathy, auto-immune disorders such as glomerulonephritis and rheumatoid arthritis, dermatological disorders such as psoriasis, anti-inflammatory, anti-fungal, antiparasitic disorders such as malaria, emphysema and alopecia. In these disorders, the compounds of the present invention may induce apoptosis or maintain stasis within the desired cells as required.
In respect of all of the above aspects and embodiments, preferably the proliferative disorder is cancer.
NON-SMALL CELL LUNG CANCER
In one particularly preferred embodiment of the invention, the proliferative disorder is lung cancer, more preferably, non-small cell lung cancer (NSCLC).
Lung cancers (bronchogenic carcinomas) may be divided into two broad categories namely, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The distinction between these two types of cancer is based on the appearance of the tumour cells when viewed under a microscope.
SCLC accounts for 20% of lung cancers diagnosed and it is characterised by small cells which are mostly filled with the nucleus (hence the name). It is sometimes also referred to as "oat cell" cancer. SCLC is the most aggressive type of cancer, which metastasizes rapidly to other parts of the body. Diagnosis with SCLC often occurs only after the cancer has spread throughout the body. In general, SCLC is almost always caused as a result of smoking. NSCLC can be subdivided into a group of related lung cancers which include epidermoid or squamous cell carcinoma, adenocarcinoma and large cell carcinoma.
Squamous cell lung cancer accounts for approximately 30% of all lung cancer cases and develops from reserve cells (which have the role of replacing damaged epithelium cells) in the lining of the lungs and bronchi. As a result, the cancer often initially develops in the centre of the chest. Squamous cell lung cancers are frequently slow growing and can take several years to progress from a confined tumour into invasive cancer. In 10-20% of cases, the cancer cavitates within the lungs. On metastasis, it often spreads to the bone, liver, adrenal glands, small intestine and brain.
Adenocarcinoma is the most common form of lung cancer making up 30-40% of all lung cancer cases. Adenocarcinoma develops in the outer part of the lung and develops from mucus-producing cells. The course of this cancer varies widely but often progresses slowly and the patient will present with few or no symptoms. In some cases, however, it can be extremely aggressive and rapidly fatal. In 50% of cases when it metastasises, it spreads only to the brain. Other locations to which adrenocarcinoma spreads include the liver, the adrenal glands, and bone.
The incidence of large cell carcinoma occurs less frequently than that of either adenocarcinoma or squamous cell carcinoma and accounts for 10-20% of lung cancer cases. The cancer is composed of large-sized cells that are anaplastic in nature and often arise in the bronchi. Large cell carcinoma develops on the periphery of the lungs and can spread to the plura.
Currently, lung cancer may be treated by surgery, radiation therapy or chemotherapy. Chemotherapy may be administered either alone or in combination with the other treatment options. Common NSCLC drugs and regimens include camptosar (irinotecan; CPT-I l), camptothecin, carboplatin (paraplatin), cisplatin (platinol), epirubicin, gemcitabine, navelbine (vinorelbine), oxaliplatin, taxol (paclitaxel) and taxotere (docetaxol) (NSCLC Treatment - Chemotherapy, Lung Cancer Online).
However, chemotherapy is not curative. Other disadvantages of this treatment include toxicity, bystander damage to normal tissues and drug resistance (W. Wang et al, Cancer Sci., 2005, 96(10), 706). Furthermore, studies have shown that there is little survival benefit with some of the known treatments, such as vinorelbine (M. A. Socinski et al, Clin. Adv. Hematol. Oncol., 2003, 1(1), 33). Even a novel active such a troxacitabine has been shown to have little activity in NSCLC in 10 mg/m doses administered intravenously over 30 minutes every three weeks (S. F. Dent et al, Lung, 2005, 183(4), 265).
The combination of gemcitabine/cisplatin has become widely used in Europe for the treatment of NSCLC. Cisplatin, however, is acknowledged to have certain disadvantages in that significant non-hematological toxicity (ototoxicity and nephroxicity) occurs in patients, along with emesis (P. Zatloukal et al, Lung Cancer, 2002, 38, S33).
As the outcome for a patient diagnosed with lung cancer is poor - the ten year survival rate for all treated cases is only approximately 8% - there exists a continuing need to develop effective treatments.
PHARMACEUTICAL COMPOSITIONS In a particularly preferred embodiment, the pharmaceutical product of the invention is in the form of a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent or excipient.
Even though the compounds of the present invention (including their pharmaceutically acceptable salts, esters and pharmaceutically acceptable solvates) can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy. The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine.
Examples of such suitable excipients for the various different forms of pharmaceutical compositions described herein may be found in the "Handbook of Pharmaceutical Excipients", 2nd "Edition, (1994), Edited by A Wade and PJ Weller. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).
Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like. Examples of suitable diluents include ethanol, glycerol and water.
The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s).
Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol.
Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.
Preservatives, stabilizers, dyes and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.
SALTS/ESTERS
The agents of the present invention can be present as salts or esters, in particular pharmaceutically acceptable salts or esters.
Pharmaceutically acceptable salts of the agents of the invention include suitable acid addition or base salts thereof. A review of suitable pharmaceutical salts may be found in Berge et al, J Pharm Sci, 66, 1-19 (1977). Salts are formed, for example with strong inorganic acids such as mineral acids, e.g. sulphuric acid, phosphoric acid or hydrohalic acids; with strong organic carboxylic acids, such as alkanecarboxylic acids of 1 to 4 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acids, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (Cl-C4)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid.
Esters are formed either using organic acids or alcohols/hydroxides, depending on the functional group being esterified. Organic acids include carboxylic acids, such as alkanecarboxylic acids of 1 to 12 carbon atoms which are unsubstituted or substituted (e.g., by halogen), such as acetic acid; with saturated or unsaturated dicarboxylic acid, for example oxalic, malonic, succinic, maleic, fumaric, phthalic or tetraphthalic; with hydroxycarboxylic acids, for example ascorbic, glycolic, lactic, malic, tartaric or citric acid; with aminoacids, for example aspartic or glutamic acid; with benzoic acid; or with organic sulfonic acids, such as (Cl-C4)-alkyl- or aryl-sulfonic acids which are unsubstituted or substituted (for example, by a halogen) such as methane- or p-toluene sulfonic acid. Suitable hydroxides include inorganic hydroxides, such as sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminium hydroxide. Alcohols include alkanealcohols of 1-12 carbon atoms which may be unsubstituted or substituted, e.g. by a halogen).
ENANTIOMERS/TAUTOMERS
The invention also includes where appropriate all enantiomers and tautomers of the agents. The man skilled in the art will recognise compounds that possess optical properties (one or more chiral carbon atoms) or tautomeric characteristics. The corresponding enantiomers and/or tautomers may be isolated/prepared by methods known in the art. STEREO AND GEOMETRIC ISOMERS
Some of the agents of the invention may exist as stereoisomers and/or geometric isomers — e.g. they may possess one or more asymmetric and/or geometric centres and so may exist in two or more stereoisomeric and/or geometric forms. The present invention contemplates the use of ail the individual stereoisomers and geometric isomers of those inhibitor agents, and mixtures thereof. The terms used in the claims encompass these forms, provided said forms retain the appropriate functional activity (though not necessarily to the same degree).
The present invention also includes all suitable isotopic variations of the agent or pharmaceutically acceptable salts thereof. An isotopic variation of an agent of the present invention or a pharmaceutically acceptable salt thereof is defined as one in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into the agent and pharmaceutically acceptable salts thereof include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine and chlorine such as 2H, 3H, 13C, 14C, 15N, 17O, 180, 31P, 32P, 35S, 18F and 36Cl, respectively. Certain isotopic variations of the agent and pharmaceutically acceptable salts thereof, for example, those in which a radioactive isotope such as 3H or 14C is incorporated, are useful in drug and/or substrate tissue distribution studies. Tritiated, i.e., 3H, and carbon-14, i.e., 14C, isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with isotopes such as deuterium, i.e., 2H, may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements and hence may be preferred in some circumstances. Isotopic variations of the agent of the present invention and pharmaceutically acceptable salts thereof of this invention can generally be prepared by conventional procedures using appropriate isotopic variations of suitable reagents.
SOLVATES
The present invention also includes solvate forms of the agents of the present invention. The terms used in the claims encompass these forms. POLYMORPHS
The invention furthermore relates to agents of the present invention in their various crystalline forms, polymorphic forms and (an)hydrous forms. It is well established within the pharmaceutical industry that chemical compounds may be isolated in any of such forms by slightly varying the method of purification and or isolation form the solvents used in the synthetic preparation of such compounds.
PRODRUGS
The invention further includes agents of the present invention in prodrug form. Such prodrugs are generally compounds wherein one or more appropriate groups have been modified such that the modification may be reversed upon administration to a human or mammalian subject. Such reversion is usually performed by an enzyme naturally present in such subject, though it is possible for a second agent to be administered together with such a prodrug in order to perform the reversion in vivo. Examples of such modifications include ester (for example, any of those described above), wherein the reversion may be carried out be an esterase etc. Other such systems will be well known to those skilled in the art.
ADMINISTRATION
The pharmaceutical compositions of the present invention may be adapted for oral, rectal, vaginal, parenteral, intramuscular, intraperitoneal, intraarterial, intrathecal, intrabronchial, subcutaneous, intradermal, intravenous, nasal, buccal or sublingual routes of administration.
For oral administration, particular use is made of compressed tablets, pills, tablets, gellules, drops, and capsules. Preferably, these compositions contain from 1 to 2000 mg and more preferably from 50-1000 mg, of active ingredient per dose.
Other forms of administration comprise solutions or emulsions which may be injected intravenously, intraarterially, intrathecally, subcutaneously, intradermally, intraperitoneally or intramuscularly, and which are prepared from sterile or sterilisable solutions. The pharmaceutical compositions of the present invention may also be in form of suppositories, pessaries, suspensions, emulsions, lotions, ointments, creams, gels, sprays, solutions or dusting powders.
An alternative means of transdermal administration is by use of a skin patch. For example, the active ingredient can be incorporated into a cream consisting of an aqueous emulsion of polyethylene glycols or liquid paraffin. The active ingredient can also be incorporated, at a concentration of between 1 and 10% by weight, into an ointment consisting of a white wax or white soft paraffin base together with such stabilisers and preservatives as may be required.
Injectable forms may contain between 10 - 1000 mg, preferably between 10 - 500 mg, of active ingredient per dose.
Compositions may be formulated in unit dosage, form, i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose.
In a particularly preferred embodiment, the combination or pharmaceutical composition of the invention is administered intravenously.
DOSAGE
A person of ordinary skill in the art can easily determine an appropriate dose of one of the instant compositions to administer to a subject without undue experimentation. Typically, a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. The dosages disclosed herein are exemplary of the average case. There can of course be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
Depending upon the need, the compound of formula I may be administered at a dose of from 0.01 to 30 mg/kg body weight, such as from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight. Depending upon the need, the compound of formula II may be administered at a dose of from 0.01 to 30 mg/kg body weight, such as from 0.1 to 10 mg/kg, more preferably from 0.1 to 1 mg/kg body weight.
In an exemplary embodiment, one or more doses of 10 to 150 mg/day of the compounds of formula I or formula II will be administered to the patient for the treatment of malignancy.
By way of guidance, the HDAC inhibitor is typically administered in accordance with a physician's direction at dosages described in the relevant references discussed above. Pivanex is typically administered at about 2.34g/m2 per day. Pivanex is preferably administered intravenously. Suberoylanilide hydroxamic acid (SAHA) is typically administered from about 100-600mg per day. Suberoylanilide hydroxamic acid (SAHA) is preferably administered orally. The total daily dose of HDAC inhibitor can be administered as a single dose or divided into separate dosages preferably administered two, three or four time a day.
Preferably, the HDAC inhibitor is administered at least 2 hours before the administration of the compound of formula I or formula II. More preferably, the HDAC inhibitor is administered at least 4 hours, or more preferably at least 6 or 8 hours, before the administration of the compound of formula I or formula II. Even more preferably, the HDAC inhibitor is administered at least 12 hours, or more preferably at least 18 or 24 hours, before the administration of the compound of formula I or formula II.
In another preferred embodiment, the HDAC inhibitor is administered at least 2 hours after the administration of the compound of formula I or formula II. More preferably, the HDAC inhibitor is administered at least 4 hours, or more preferably at least 6 or 8 hours, after the administration of the compound of formula I or formula II. Even more preferably, the HDAC inhibitor is administered at least 12 hours, or more preferably at least 18 or 24 hours, after the administration of the compound of formula I or formula II. KIT OF PARTS
A further aspect of the invention relates to a kit of parts comprising:
(i) a compound of formula I or formula II, or a pharmaceutically acceptable salt thereof, optionally admixed with a pharmaceutically acceptable diluent, excipient or carrier; and
(ii) a HDAC inhibitor, optionally admixed with a pharmaceutically acceptable diluent, excipient or carrier, optionally admixed with a pharmaceutically acceptable diluent, excipient or carrier.
Preferably, the HDAC inhibitor is selected from sodium butyrate, or a prodrug thereof, suberoylanilide hydroxamic acid (SAHA), sodium valproate and trichostatin A (TSA).
More preferably, the HDAC inhibitor is selected from sodium butyrate, or a prodrug thereof, suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA).
Preferably, the compound of formula I or formula II and the HDAC inhibitor are each in unit dosage form. Preferably, the kit of parts contains a plurality of unit dosage forms of each component, i.e. of components (i) and (ii) above.
Optionally, the kit of parts may further comprise a means for facilitating compliance with a particular dosing regimen, for example, instructions indicating when, how, and how frequently the unit dosage forms of each component should be taken.
The present invention is further described by way of example, and with reference to the following figures, wherein:
Figure 1 shows that concomitant treatment of seliciclib and sodium butyrate leads to a synergistic increase in apoptosis in A549 cells, as determined by an increase in sub- Gl cell fragments, following 72 hours treatment.
Figure 2 shows that concomitant treatment of seliciclib and sodium butyrate in H460 cells leads to a synergistic increase in apoptosis following 72 hours treatment, as determined by annexin V staining. Figure 3 shows that in respect of the seliciclib/sodium butyrate combination there is an increase in apoptosis at the IC50 concentration, as determined by caspase cleavage of cytokeratin 18 (M30 ELISA) and PARP cleavage. In addition, Figure 3 also shows a synergistic decrease in McIl levels which probably relates to the loss of this anti- apoptotic protein, pushing the cells into apoptosis.
Figure 4 shows the molecular pathways of apoptosis with regard to the seliciclib/sodium butyrate combination in more detail. Loss of McIl and Bcl2 (both anti-apoptotic proteins) pushes the cells towards apoptosis. Both XIAP and survivin are inhibitors of the apoptotic process therefore the loss of these proteins again push the cells towards apoptosis. As the decreases in XIAP and McIl are synergistic, this effect may explain the synergistic induction of apoptosis as shown by the appearance of PARP. The Histone Western blot shows that the HDAC inhibitor increases the amount of acetylated histone (since deacetylation is inhibited).
Figure 5 shows the time course of cellular events at the IC50 in respect of the seliciclib/sodium butyrate combination. At later time points, the synergistic activation of caspases 3 and 9 (indicating apoptosis is induced) can now be seen.
Figure 6 shows the cell cycle distribution after treatment with DMSO (control), sodium valproate, seliciclib and sodium valproate/seliciclib in combination. H460 cells were treated with the indicated drug(s) for the times shown prior to PI analysis on the flow cytometer. The results are the average of two duplicate samples.
Figure 7 shows the cell cycle distribution after treatment with DMSO (control), sodium valproate, the indicated CDK inhibitor and sodium valproate/CDK inhibitor in combination. H460 cells were treated with the indicated drug(s) for the times shown prior to PI analysis on the flow cytometer. The results are the average of two duplicate samples. EXAMPLES
Methods
Compounds of formula I and formula II Compounds of formula I and formula II were prepared in accordance with the methods disclosed in WO 2004/016612 and WO 2005/042525 (both to Cyclacel Limited).
Compound [1] referred to herein is (2R,3S)-3-({9-isopropyl-6-[(pyridin-3- ylmethyl)amino]-9H-purin-2-yl } amino)pentan-2-ol.
Compound [2] referred to herein is 3,4-dimethyl-5-[2-(4-piperazin-l-yl- phenylamino)pyrimidin-4-yl]-3H-thiazol-2-one, having the structure shown below:
Compound [3] referred to herein is (3R)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)- amino]-9H-purin-2-ylamino}-2-methyl-pentan-2-ol, having the structure shown below:
Compound [4] referred to herein is (3S)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)- amino]-9H-purin-2-ylamino}-2-methyl-pentan-2-ol, having the structure shown below:
HDAC inhibitors
Sodium butyrate and sodium valproate were obtained from Sigma; TSA was obtained from AG Scientific, Inc.; SAHA was obtained from Toronto Research Chemicals, Inc.
Cell Culture
Experiments were carried out in 96-well plates and the cell lines seeded at a density of 2000/well for A549 and 3000/well for H460. The IC50 values after 24h treatment and 72h treatment were determined for sodium butyrate in each cell line and SAHA, sodium valproate and TSA in H460 cells, using the Alamar blue assay. Each HDAC inhibitor was then tested in combination with a CDK inhibitor (seliciclib or compounds [l]-[4]) using three different treatment regimes: concomitant, CDK inhibitor pre-treatment followed by HDAC inhibitor and HDAC inhibitor pretreatment followed by CDK inhibitor. Calcusyn drug combination protocol
For the concomitant treatment, 1.5-fold serial dilutions of the CDK inhibitor (seliciclib, compounds [l]-[4]), HDAC inhibitor, or both drugs simultaneously were added to cells 24h after plating, and left for 72h at 370C. The drug concentrations selected were chosen to span the IC50 values for the drugs tested. In the pre-treatment regimes, the first drug was added 2h after cells were plated, and left for 24h. Medium was aspirated and replaced with fresh medium containing the second drug, and incubated for 72h. The two controls for each sequential treatment involved substituting one of the drug treatments with medium. After drug treatment, the cell number in each well was then estimated by incubating the cells for Ih in medium containing 10% alamar blue (Roche, Lewes, East Sussex, U.K.) and reading the absorbance at 544-595 nm. Drug interactions were analysed using the commercial software package Calcusyn, which is based on the median effect model of Chou and Talalay (Chou, T.C. & Talalay, P. (1984) Adv. Enzyme Regul. 22, 27-55. Quantatative analysis of dose-effect relationships: the combined effects of multiple drags or enzyme inhibitors). A Combination Index (CI.) of 1 indicated an additive drug interaction, whereas a C.I. greater than 1 was antagonistic and a score lower than 1 was synergistic.
Western blotting analysis Protein lysates were generated from 10cm plates that were seeded at approximately 5 x 105 cells/well, in medium containing 10% FCS. Cells were incubated with HDAC inhibitor and/or seliciclib at the indicated concentrations and times prior to harvest. After incubation, the supernatants were removed and centrifuged at 2000rpm for 5 min to pellet any floating cells. Cells on the plates were washed once with ice-cold buffer A (5OmM HEPES, pH 7.0, containing 2OmM NaCl), then each plate was scraped into 0.15ml buffer A containing ImM DTT, protease inhibitors (diluted 1 :1000 into buffer A) and phosphatase inhibitors (1OmM Sodium pyrophosphate, 1OmM Sodium Fluoride and ImM Sodium Ortho vanadate). The supernatant cell pellets were resuspended with 50μl buffer A containing DTT, protease and phosphatase inhibitors and pooled with the appropriate sample from the plates. Cells were lysed by sonication (2 x 3s bursts with probe sonicator), and the protein concentration of each tube determined using the BCA assay. Lysates (20-3 Oμg protein loaded/well) were resolved on Bis-Tris gels containing 12% acrylamide and transferred to nitrocellulose for analysis by western blotting. Membranes were blocked for Ih at room temperature in PBS containing 0.02% (v/v) Tween 20 and 5% (w/v) fat-free dried milk. Antibody incubations were carried out overnight at 2-80C in PBS containing 0.02% (v/v) Tween 20 and 3% (w/v) dried milk. Nitrocellulose membranes were probed with the following antibodies:
Antibody Source Target protein Dilution used
Actin (A5441) Sigma β-Actin 1:15000
Cleaved PARP BD Pharmingen Cleaved PARP 1:500
Acetyl-Histone H4 Upstate Acetylated Histone H4 1:1000
XIAP Cell Signalling XIAP 1:1000
Bcl-2 (clonelOO) Upstate Bcl-2 1:1000
McM (S-19) Santa Cruz McI-I 1:1000
Bax (06-499) Upstate Bax 1:1000
Survivin AbCam Survivin 1:500
Cleaved Caspase 3 Cell Signalling Cleaved Caspase 3 1:1000
Cleaved Caspase 9 Cell Signalling Cleaved Caspase 9 1:1000
Flow Cytometry H460 cells were seeded in 10cm plates at approximately 3x105 cells/plate and left to settle overnight. Next day, seliciclib, sodium butyrate or both drugs were added at the indicated concentrations. After either 24h or 72h treatment, cells were harvested by trypsinisation. Cell cycle analysis by propidium iodide (PI) staining involved fixing the cells overnight in 70% (v/v) ethanol at -2O0C prior to analysis on the flow cytometer. Annexin V staining was performed as indicated in manufacturers instructions, on live, non-fixed cells.
Flow cytometry: CDK inhibitor/sodium valproate combination
H460 cells were seeded onto 10cm plates at approximately 0.5 x 106 cells/plate and allowed to settle for 24h. Cells were treated with sodium valproate for 24 hours followed by the CDK inhibitor (seliciclib, compounds [l]-[4]) for a further 24 hours. The concentrations of compound used were equivalent to 1 x IC5O. Single agent control treatments were also carried out. These involved treating the cells with sodium valproate for 24 hours followed by drug-free medium for a further 24 hours, or drug- free medium for 24 hours followed by the CDK inhibitor for a further 24 hours. All cells were harvested by collecting the media prior to media changes, as well as at 48 hours. Adherent cells were harvested by trypsinisation, pooled with the cells in suspension, washed twice in PBS and fixed by resuspending in ImI ice-cold 70% ethanol. Standard cell cycle analysis by propidium iodide staining was carried out on the flow cytometer. Results are the average of duplicate samples.
M30/TPS analysis of time-gap experiment A549 cells were seeded in 96 well plates and left to settle overnight. Cells were treated with seliciclib, sodium butyrate or the combination at the indicated concentrations. After 72h treatment, medium was harvested, retained and stored at - 2O0C. Samples were analysed in the M30 ELISA as described in the manufacturers instructions.
Results
Seliciclib and HDAC inhibitors in combination in NSCLC cell lines.
Seliciclib was tested in combination with the indicated HDAC inhibitors in H460 and A549 cell lines, using three different treatment regimes. The Combination Index values from each drug treatment are shown for ED50, ED75 and ED90 values (the point on the curve where 50%, 75% and 90% of the cells have been killed). Data are the average of at least three independent experiments (Table 1).
Table 1: Data for the effect of seliciclib and the HDAC inhibitors on the A549 cell lines are shown in parentheses.
These results demonstrate that seliciclib and butyrate are synergistic in H460 and A549 cells, with all three treatment regimes tested. Seliciclib and trichostatin A are moderately synergistic in H460 cells, with all three treatment regimes tested. Seliciclib and SAHA are moderately synergistic in H460 cells when SAHA pre- treatment is followed by seliciclib, and additive with the other two treatment regimes. Synergy was observed for sequential treatment with seliciclib and sodium valproate, irrespective of the order of administration. Therefore, if cells are pretreated with the HDAC inhibitor, seliciclib is synergistic when used in combination with all three HDAC inhibitors tested, demonstrating that combining seliciclib with a HDAC inhibitor is a good concept for treating NSCLC cell lines.
Compounds [l]-[4] are synergistic with butyrate in H460 cells
In accordance with the methods described above, butyrate was tested in combination with the indicated CDK inhibitors in H460 cells, using three different treatment regimes. The Combination Index values from each drug treatment are shown for ED50, ED75 and ED90 values (the point on the curve where 50%, 75% and 90% of the cells have been killed). Data are the average of three independent experiments (Table 2).
Table 2: Data for the effect of compounds [l]-[4] in combination with sodium butyrate.
These results demonstrate that compound [2] and butyrate are synergistic in H460 cells, with all three treatment regimes tested. Compound [1] and butyrate are synergistic in H460 cells, with sequential treatment regimes and additive with concomitant treatment. Therefore, compounds [1] and [2] are synergistic when used in combination with butyrate in H460 cells, indicating that combining such CDK inhibitors with HDAC inhibitors is a promising development for treating NSCLC.
Compounds [l]-[4] are synergistic with sodium valproate in H460 cells
The results for combination analysis of sodium valproate and compounds [l]-[4] are shown below in Table 3.
Table 3: Data for the effect of compounds [l]-[4] in combination with sodium valproate
Combination analysis indicated that an additive to synergistic interaction was observed with compounds [I]- [4] when they were combined with sodium valproate (Table 3). As had been observed in previous experiments, synergy could be obtained irrespective of the order of administration. However, the best synergy was obtained by pre-treatment with the HDAC inhibitor, sodium valproate.
Flow Cytometry Studies
Seliciclib and Butyrate induce a synergistic increase in sub-Gl A549 cells.
A549 cells were incubated with IC50 butyrate, 0.25 - 1.5 X IC50 seliciclib, or 0.25 - 1.5 X IC50 seliciclib in the presence of IC50 butyrate for 72h. Cells were then harvested, stained with propidium iodide and their DNA content analysed by flow cytometry. Data are representative of two independent experiments (Figure 1).
Butyrate alone induced a small increase in sub-Gl cells (<2n DNA), which are dead or undergoing apoptosis. Seliciclib treatment induced a dose-dependent increase in sub-Gl cells, which was synergistically enhanced by inclusion of butyrate. These data indicate that seliciclib and butyrate induce a synergistic increase in cells that are dead or dying.
Seliciclib and Butyrate induce a synergistic increase in apoptotic H460 cells.
H460 cells were incubated with 0.25 - 1.5X IC50 butyrate, 0.25 - 1.5 X IC50 seliciclib, or 0.25 - 1.5 X IC50 seliciclib and butyrate for 72h. Cells were then harvested, stained with annexin V and analysed on the flow cytometer. Data are representative of two independent experiments (Figure 2).
Annexin V labels live cells that are undergoing apoptosis. At 0.67 X and 1 X IC50 concentrations, butyrate and seliciclib induced a much larger annexin V signal than the two single drug treatments combined, indicating a synergistic increase in apoptotic cells. The highest concentration of butyrate and seliciclib (1.5 X IC50) appears to contain fewer cells undergoing apoptosis than those treated with 0.67 X or 1 X IC50, the reason for this is not clear at present. Seliciclib and Butyrate synergistically induce apoptosis in A549 cells.
A549 cells were treated with DMSO (control) or with IC50 concentrations of seliciclib, sodium butyrate, or seliciclib and butyrate for 72h, as indicated. Cell culture supernatants were harvested and tested in the M30 apoptosense ELISA, and the cells harvested and analysed for cleaved PARP and McI-I by western blotting. Data are representative of two independent experiments (Figure 3).
The results show that seliciclib and butyrate together give a larger M30 signal than the two individual drug treatments combined, indicating that they synergistic increase apoptosis. This data is supported by the fact that seliciclib and butyrate give a larger cleaved PARP signal than seliciclib or butyrate alone, which is indicative of an additive/synergistic increase in apoptosis. Seliciclib and butyrate also decreased the level of the anti-apoptotic protein McI-I in A549 cells, whereas the single drag treatments has no significant effect on this protein. This could play a role in promoting the increased apoptosis that was detected.
Seliciclib and Butyrate regulate several apoptotic proteins in a dose-dependent manner.
H460 cells were treated with butyrate, seliciclib or seliciclib and butyrate at 1 X or 1.5 X IC50 concentrations for 24h. Cells were harvested and the resulting cell lysates analysed by western blotting with the indicated antibodies. Data are representative of two independent experiments (Figure 4).
The data indicate that by 24h treatment, seliciclib and butyrate synergistically decrease the protein levels of the anti-apoptotic protein McI-I and the caspase inhibitor XIAP. Butyrate treatment reduces the levels of the anti-apoptotic protein Bcl-2 and the caspase inhibitor survivin. Together, these changes will provide a strong pro-apoptotic signal. Indeed, there is a synergistic increase in cleaved PARP at 1 X IC50 concentrations, although the increased cleaved PARP at 1.5X IC50 only appears to be an additive effect at best, which is in agreement with the annexin V data generated at 1.5 X IC50 in Figure 2.
Seliciclib and Butyrate regulate several apoptotic proteins in a time-dependent manner. H460 cells were treated with IX IC50 butyrate, seliciclib or seliciclib and butyrate for the indicated times. Cells were harvested and the resulting cell lysates analysed by western blotting with the indicated antibodies. Data are representative of two independent experiments (Figure 5).
The results confirm the observations in Figure 4, since butyrate reduces the levels of survivin and Bcl-2, and seliciclib and butyrate synergistically reduce McI-I and XIAP levels. These data also demonstrate that the drug combination induces a synergistic increase in the active forms of caspase 3 and 9, which appear at around the same time as the increase in cleaved PARP. Interestingly, the increased apoptosis (as measured by the appearance of cleaved PARP) does not occur until after the decreases in McI-I, Bcl-2, survivin and XIAP, indicating that any or all of these changes may be required to promote the apoptotic effect in this cell line.
CDK inhibitor/sodium valproate combination
Flow cytometry analysis of the various sodium valproate/CDK inhibitor combinations was carried out as described above. The sodium valproate pre-treatment schedule was selected as this was synergistic by Calcusyn. In H460 cells, treatment with any of compounds [l]-[4] or seliciclib induced a small increase in the percentage of cells in sub-Gl (apoptotic cells), but otherwise had no significant impact on the overall cell cycle distribution of H460 cells at the concentrations used (Figures 6 and 7).
Combining sodium valproate with any of the CDK inhibitors tested caused a significant increase in the proportion of sub-Gl (apoptotic) cells that, compared to the sum of the single agent controls, was at least equivalent to an additive interaction, which is in agreement with the data obtained by Calcusyn analysis.
The data illustrated above provides evidence that compounds [l]-[4] and seliciclib appear to be additive-synergistic with the HDAC inhibitor, sodium valproate, in H460 cells in a schedule-independent manner.
Various modifications and variations of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the relevant fields are intended to be covered by the present invention.

Claims

1. A combination comprising (i) a histone deacetylase (HDAC) inhibitor; and (ii) a compound of formula I or II, or a pharmaceutically acceptable salt thereof,
wherein said compound of formula I is defined as
or a pharmaceutically acceptable salt thereof, wherein one of R1 and R2 is methyl, ethyl or isopropyl, and the other is H;
R3 and R4 are each independently H, branched or unbranched C1-C6 alkyl, or aryl, and wherein at least one of R3 and R4 is other than H;
Rs is a branched or unbranched C1-C5 alkyl group or a C1-C6 cycloalkyl group, each of which may be optionally substituted with one or more OH groups;
R6, R7, R8 and R9 are each independently H, halogen, NO2, OH, OMe, CN,
NH2, COOH, CONH2, or SO2NH2;
and said compound of formula II is defined as
II wherein
R10 and R14 are each independently H, C(OR* ) or a hydrocarbyl group optionally substituted by one or more R15 groups;
R11, R12, and R13 are each independently H, alkyl or alkenyl, each of which may be optionally substituted with one or more R16 groups;
R15 and R16 are each independently halogen, NO2, CN, (CH2)mORa,
O(CH2)nORb, (CH2)pNRcRd, CF3, COORe, CONRfRg, CORh, SO3H, SO2R*,
SO2NRjRk, (CH2)qNRa'CORgl, Rf, (CH2)rNRb>SO2Rh' 3 SO2NR^R1',
SO2NR6 (CH2)SORC , heterocycloalkyl or heteroaryl, wherein said heterocycloalkyl and heteroaryl may be optionally substituted by one or more substituents selected from aralkyl, sulfonyl, Rm and COR";
Rg , R , R1 and RJ are each independently selected from alkyl, aryl, aralkyl and heteroaryl, each of which may be optionally substituted with one or more substituents selected from halogen, OH, NO2, NH2 CF3 and COOH; m, p, q and r are each independently O, 1, 2 or 3; n and s are each independently 1, 2, or 3; and
Ra"n and Ra "f are each independently H or alkyl.
2. A combination according to claim 1 which comprises a histone deacetylase (HDAC) inhibitor and a compound of formula I, or a pharmaceutically acceptable salt thereof.
3. A combination according to claim 1 or claim 2 wherein one of R1 and R2 is ethyl or isopropyl, and the other is H.
4. A combination according to any preceding claim wherein wherein R5 is isopropyl or cyclopentyl.
5. A combination according to any preceding claim wherein R6, R7, R8 and R9 are all H.
6. A combination according to any preceding claim wherein one of R1 and R2 is ethyl and the other is H.
7. A combination according to any preceding claim wherein R3 and R4 are each independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl, t-butyl or phenyl.
8. A combination according to any preceding claim wherein R3 and R4 are each independently H, methyl, ethyl, n-propyl, isopropyl, n-butyl, s-butyl or t-butyl.
9. A combination according to claim 8 wherein R3 and R4 are each independently H, methyl, ethyl, isopropyl or t-butyl.
10. A combination according to any preceding claim wherein the compound of formula I is selected from the following:
(2S3R)-3 - { 9-Isopropyl-6- [(pyridin-3 -ylmethyl)-amino] -9H-purin-2-ylamino } - pentan-2-ol;
(2i?35)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}- pentan-2-ol;
(3RS,4R)-4- { 9-Isopropyl-6- [(pyridin-3 -ylmethyl)-amino] -9H-purin-2-ylamino } - hexan-3-ol;
(3i?jS,4iS)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}- hexan-3-ol;
(3Λ5,4i?)-4-{9-Isopropyl-6-[(ρyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2- methyl-hexan-3 -ol ;
(3i?<S',4S)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2- methyl-hexan-3 -ol;
(3RS,4R)-4- { 9-Isopropyl-6- [(pyridin-3 -ylmethyl)-amino] -9H-purin-2-ylamino } -
2,2-dimethyl-hexan-3 -ol;
(3i?5,45)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-
2,2-dimethyl-hexan-3-ol;
(3i?)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2- methyl-pentan-2-ol;
(35)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2- methy l-pentan-2-ol ; (3S)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2- methyl-pentan-2-ol; and
(3R)-3 - { 9-Isopropyl-6- [(pyridin-3 -ylmethyl)-amino] -9H-purin-2-ylamino } -2- tnethyl-ρentan-2-ol.
11. A combination according to any preceding claim wherein the compound of formula I is selected from the following:
(2S3i?)-3 - { 9-Isopropyl-6- [(pyridin-3 -ylmethyl)-amino] -9H-purin-2-ylamino } - pentan-2-ol;
(2i?35)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}- pentan-2-ol;
(3i?<Sr J4i?)-4- { 9-Isopropyl-6- [(pyridin-3 -ylmeth3rl)-amino3-9H-purin-2-ylamino } - hexan-3-ol;
(3/?iS',45)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}- hexan-3-ol;
(3i?5',45)-4-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-
2,2-dimethyl-hexan-3-ol;
(3i?)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2- methyl-pentan-2-ol; and
(35)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-3-ylamino}-2- methyl-pentan-2-ol.
12. A combination according to any preceding claim wherein the compound of formula I is selected from the following:
(3R)-3-{9-isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}-2- methyl-pentan-2-ol ;
(3 S)-3 - { 9-isopropyl-6-[(pyridin-3 -ylmethyl)-amino]-9H-purin-2-ylamino } -2- methyl-pentan-2-ol;
(2S3R)-3-{9-isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}- pentan-2-ol;
(2R3S)-3-{9-isopropyl-6-[(pyridin-3-ylmethyl)-amino]-9H-purin-2-ylamino}- pentan-2-ol; and any optical isomer of 3-{9-isopropyl-6-[(pyridin-3-ylmethyl)-arnino]-9H-purin-2- ylamino } -pentan-2-ol.
13. A combination according to any preceding claim wherein said compound of formula I is selected from:
3-({9-isopropyl-6-[(pyridm-3-ylmethyl)amino]-9H-purin-2-yl}amino)pentan-2-ol; (2R,3S)-3-({9-isopropyl-6-[(pyridin-3-ylmethyl)amino]-9H-purin-2-yl}amino)pentan- 2-ol;
(3R)-3-{9-Isopropyl-6-[(pyridin-3-ylmethyl)-ainino]-9H-purin-2-ylarnmo}-2-methyl- pentan-2-ol; and
(3 S)-3 - { 9-Isopropyl-6- [(pyridin-3 -ylmethyl)-amino]-9H-purin-2-y lamino } -2-methyl- pentan-2-ol).
14. A combination according to claim 1 which comprises a histone deacetylase (HDAC) inhibitor and a compound of formula II, or a pharmaceutically acceptable salt thereof.
15. A combination according to claim 14 wherein R10 and R14 are each independently H or a C1-2O hydrocarbyl group optionally comprising up to six heteroatoms selected from from N, O, and S, and which is optionally substituted by one, two or three R15 groups.
16. A combination according to claim 14 or claim 15 wherein R14 is aryl or heteroaryl, each of which may be optionally substituted by one or more R15 groups.
17. A combination according to claim 16 wherein R14 is phenyl or pyridinyl, each of which may be optionally substituted by one or more R15 groups.
18. A combination according to any one of claims 14 to 17 wherein R10 is H or alkyl.
19. A combination according to any one of claims 14 to 18 wherein R11, R12, and R13 are each independently H, Ci-C6 alkyl or C2-C6 alkenyl, each of which may be optionally substituted with one, two or three R16 groups.
20. A combination according to any one of claims 14 to 19 wherein R15 and R16 are each independently F, Cl5 Br, I, NO2, CN, OH, OMe, OEt, CH2OH, O(CH2)2OMe, NH2, NHMe, NMe2, CF3, COOH, CONH2, CONHMe, CONMe2, COMe, SO3H, SO2Me, SO2NH2, SO2NHMe, SO2NMe2, morpholine, piperidine, piperazine, N- acetylpiperazine, N-methylpiperazine, triazole, or tetrazole.
21. A combination according to any one of claims 14 to 20 wherein R12 and R13 are both H and R1 ! is Me.
22. A combination according any one of claims 14 to 21 which comprises a HDAC inhibitor and a compound of formula III, or a pharmaceutically acceptable salt thereof,
III wherein
R10 is as defined above in claim 1 or claim 12; X is C; or X is N and R17 is absent; R17, R18 R19 and R20 are each independently H or as defined for R15 and R16.
23. A combination according to claim 22 wherein
R10 is H or alkyl;
R17 is H, NO2, ORP, halogen, CF3, CN, C0Rq, alkyl, NRrRs, OCCH^OR1;
R18 is H, ORU, halogen, alkyl, NRVRW, heterocycloalkyl optionally substituted with one or more substituents selected from Rm and COR"; t is O, 1, 2 or 3;
R19 is H, alkyl or NRxRy; and
Rp"yare each independently H or alkyl.
24. A combination according to any one of claims 14 to 23 wherein R10 is H, Me, Et or 3-methylbutyl.
25. A combination according to any one of claims 22 to 24 wherein: R17 is H, NO2, OH, Me, I5 CF3, CN, CH2OH5 CO2H, CO2Me or NH2;
R18 is H, F, OH, I, Cl, Br, OMe, NMe2, morpholine, Me, N-methylpiperazine, N- acetylpiperazine or piperazine; and R19 is H, Me or NMe2.
26. A combination according to claim 22 wherein X is N and R17 is absent.
27. A combination according to claim 22 wherein X is C.
28. A combination according to claim 14 wherein said compound is selected from the following:
3,4-Dimethyl-5-[2-(3-nitro-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one; 5-[2-(4-Fluoro-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one; 5-[2-(4-Hydroxy-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one; 5-[2-(4-Chloro-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one; 5-[2-(4-Bromo-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one; 5-[2-(4-Methoxy-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one; 5-[2-(3-Hydroxy-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one; 5-[2-(4-Dimethylamino-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one; 3,4-Dimethyl-5-[2-(4-morpholin-4-yl-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one; 5-[2-(4-Fluoro-3-nitro-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one; 3,4-Dimethyl-5-[2-(4-methyl-3-nitro-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one; 5-[2-(4-Fluoro-3-methyl-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one;
3 ,4-Dimethyl-5- {2-[4-(4-methyl-piperazin- 1 -yl)-phenylamino]-pyrimidin-4-yl} -3H-thiazol-2- one;
5-[2-(3-Iodo-4-methyl-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one;
5-[2-(4-Chloro-3-methyl-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one;
3-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-ρyrimidin-2-ylamino]-benzonitrile;
5 - { 2- [4-(4- Acetyl-piperazin- 1 -yl)-phenylamino]-pyrimidin-4-yl } -354-dimethyl-3H-thiazol-2- one;
5-[2-(4-Chloro-3-hydroxymethyl-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2- one;
3,4-Dimethyl-5-[2-(3-trifluoromethyl-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one; 3 ,4-Dimethyl-5 - [2-(2-methyl-5-nitro-phenylamino)-pyrimidin-4-y 1] -3 H-thiazol-2-one; 3,4-Dimethyl-5-[2-(4-methyl-3-trifluoromethyl-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2- one;
5-[2-(4-Dimethylamino-3-nitro-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2- one;
3-Ethyl-4-methyl-5-[2-(3-nitro-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one; 2-Chloro-5-[4-(3-ethyl-4-methyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]- benzoic acid;
2-Chloro-5-[4-(3-ethyl-4-methyl-2-oxo-2,3-dihydro~thiazol-5-yl)-pyrimidin-2-ylamino]- benzoic acid methyl ester;
5-[2-(4-Dimethylamino-phenylamino)-pyrimidin-4-yl]-3-ethyl-4-methyl-3H-thiazol-2-one; 3-Ethyl-4-methyl-5-[2-(4-morpholin-4-yl-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one; 3-Ethyl-4-methyl-5-[2-(4-methyl-3-nitro-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one; 5 - [2-(4-Dimethylamino-3 -nitro-phenylamino)-pyrimidin-4-yl] -3 -ethyl-4-methyl-3 H-thiazol- 2-one;
4-Methyl-3-(3-methyl-butyl)-5-[2-(3-nitro-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one; 5 - [2-(4-Chloro-phenylamino)-pyrimidin-4-yl] -4-methyl-3 -(3 -methyl-butyl)-3 H-thiazol-2-one; 5-[2-(6-Chloro-pyridm-3-ylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one; 3 -Ethyl-5 - [2-(6-methoxy-pyridin-3 -y lamino)-pyrimidin-4-yl] -4-methyl-3 H-thiazol-2-one; 5-[2-(6-Chloro-pyridin-3-ylamino)-pyrimidin-4-yl]-4-methyl-3-(3-methyl-butyl)-3H-thiazol- 2-one;
5-[2-(6-Methoxy-pyridin-3-ylamino)-pyrimidin-4-yl]-4-methyl-3-(3-methyl-butyl)-3H- thiazol-2-one;
5-[2-(4-Iodo-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one; 5-[2-(2-Dimethylamino-5-nitro-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2- one;
3 ,4-Dimethyl-5 - [2-(4-piperazin- 1 -yl-phenylamino)-pyrimidin-4-yl] -3 H-thiazol-2-one; 5-[2-(3-Amino-4-methyl-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one; 4-Methyl-5-[2-(3-nitro-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one; 4-Methyl-5-[2-(4-methyl-3-nitro-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one;
N- { 3 -[4-(3 ,4-Dimethyl-2-oxo-2,3 -dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-benzyl} - acetamide;
3-Ethyl-5-[2-(3-hydroxy-phenylamino)-pyrimidin-4-yl]-4-methyl-3H-thiazol-2-one;
5-[2-(3-Chloro-4-piperazin-l-yl-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2- one;
3 -Ethyl-5 - [2-(4-fluoro-pheny lamino)-pyrimidin-4-yl] -4-methyl-3H-thiazol-2-one;
5-[2-(4-Chloro-phenylamino)-pyrimidin-4-yl]-3-ethyl-4-methyl-3H-thiazol-2-one;
3-Ethyl-5-[2-(3-hydroxy-4-methyl-phenylamino)-pyrimidin-4-yl]-4-methyl-3H-thiazol-2-one;
5-[2-(4-Chloro-3-trifluoromethyl-phenylamino)-pyrimidin-4-yl]-3-ethyl-4-methyl-3H-thiazol-
2-one;
5 - { 2- [3 -(4- Acetyl-piperazin- 1 -yl)-phenylamino]-pyrimidin-4-yl } -3 ,4-dimethyl-3 H-thiazol-2- one;
3 -Ethyl-5 -[2-(3 -methoxy-phenylamino)-pyrimidin-4-yl]-4-methyl-3 H-thiazol-2-one;
5-[2-(4-Chloro-3-methyl-phenylamino)-pyrimidin-4-yl]-3-ethyl-4-methyl-3H-thiazol-2-one;
3-Ethyl-4-methyl-5-[2-(4-nitro-phenylamino)-pyrimidin-4-yl]-3H-thiazol-2-one;
4-[4-(3-Ethyl-4-methyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]- benzenesulfonic acid;
3 - [4-(3 -Ethyl -4-methyl-2-oxo-2,3 -dihydro-thiazol- 5 -yl)-pyrimidin-2-ylamino] - benzenesulfonic acid;
N- { 3 - [4-(3 ,4-Dimethyl-2-oxo-2,3 -dihydro-thiazol- 5-yl)-pyrimidin-2-y lamino] -benzyl } - methane-sulfonamide;
5-[2-(5-Methoxy-2-methyl-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one
N-{3-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-benzyl}- benzamide;
N-{3-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-benzyl}-
C,C,C-trifluoro-methanesulfonamide;
N-{4-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-benzyl}- acetamide;
3-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)~pyrimidin-2-ylamino]- benzenesulfonamide;
3-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-N-isopropyl-4- methyl-benzamide; 3-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-N-ethyl- benzenesulfonamide;
5-[2-(5-Hydroxymethyl-2-methyl-phenylamino)-pyrimidin-4-yl]-354-dimethyl-3H-thiazol-2- one;
N-{3-[4-(354-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-5- trifluoromethyl-phenyl } -acetamide;
4-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-N-(2-methoxy- ethyl)-benzenesulfonamide;
5-[2-(4-Chloro-3-trifluoromethyl-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2- one;
3-[4-(354-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-ρyrimidin-2-ylamino]-N-(2-methoxy- ethyl)-benzenesulfonamide;
5 - [2-(3 -Bromo-5-trifluoromethy l-phenylamino)-pyrimidin-4-yl] -3 ,4-dimethyl-3H-thiazol-2- one;
5- {2-[4-(4-Benzyl-piperazin- 1 -yl)-phenylamino]-pyrimidin-4-yl} -3,4-dimethyl-3H-thiazol-2- one;
4-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-2-trifluoromethyl- benzonitrile;
5-[2-(3-Amino-5-trifluoromethyl-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2- one;
4-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-N-(2-hydroxy- ethyl)-benzenesulfonamide;
N-Benzyl-4-[4-(3,4-dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]- benzenesulfonamide;
3-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-N-isopropyl- benzenesulfonamide;
3-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylamino]-N-(2-hydroxy- ethyl)-benzenesulfonamide;
3,4-Dimethyl-5-[2-(3-methylamino-5-trifluoromethyl-phenylamino)-pyrimidin-4-yl]-3H- thiazol-2-one;
N-Benzyl-3-[4-(3,4-dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-ylaniino]- benzenesulfonamide;
3,4-Dimethyl-5-{2-[4-methyl-3-(morpholine-4-sulfonyl)-pb.enylamino]-pyrimidin-4-yl}-3H- thiazol-2-one;
3,4-Dimethyl-5-{2-[3-(morpholine-4-sulfonyl)-phenylamino]-pyrimidin-4-yl}-3H-thiazol-2- one;
5-[2-(4-Aminomethyl-phenylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one;
5-[2-(6-Chloro-5-methyl-pyridin-3-ylamino)-pyrimidin-4-yl]-3,4-dimethyl-3H-thiazol-2-one;
Pyridine-2-carboxylic acid 4-[4-(3,4-dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2- ylaminoj-benzylamide;
3,4-Dimethyl-5-{2-[(pyridin-3-ylmethyl)-amino]-pyrimidin-4-yl}-3H-thiazol-2-one;
5-(2-Amino-pyrimidin-4-yl)-3,4-dimethyl-3H-thiazol-2-one;
N-[4-(3,4-Dimethyl-2-oxo-2,3-dihydro-thiazol-5-yl)-pyrimidin-2-yl]-acetamide;
29. A combination according to claim 28 wherein said compound of formula II is 3 ,4-dimethyl-5-[2-(4-piperazin- 1 "yl-phenylammo)pyrimidin~4-yl]-3H-thiazol-2-one.
30. A combination according to any preceding claim wherein the HDAC inhibitor is selected from sodium butyrate, or a prodrug thereof, suberoylanilide hydroxamic acid (SAHA), sodium valproate and trichostatin A (TSA).
31. A combination according to any preceding claim wherein the HDAC inhibitor is sodium butyrate, or a prodrug thereof.
32. A combination according to claim 31 wherein the prodrug is pivaloyloxymethyl butyrate.
33. A combination according to any of claims 1 to 30 wherein the HDAC inhibitor is suberoylanilide hydroxamic acid (SAHA).
34. A combination according to any of claims 1 to 30 wherein the HDAC inhibitor is trichostatin A (TSA).
35. A combination according to any of claims 1 to 30 wherein the HDAC inhibitor is sodium valproate.
36. A pharmaceutical composition comprising a combination according to any preceding claim and a pharmaceutically acceptable carrier, diluent or excipient.
37. Use of a combination according to any of claims 1 to 35 in the preparation of a medicament for treating a proliferative disorder.
38. A pharmaceutical product comprising a compound of formula I or II as defined in any of claims 1 to 29, or a pharmaceutically acceptable salt thereof, and a HDAC inhibitor, as a combined preparation for simultaneous, sequential or separate use in therapy.
39. A pharmaceutical product according to claim 38 wherein the HDAC inhibitor is selected from sodium butyrate, or a prodrug thereof, suberoylanilide hydroxamic acid (SAHA), sodium valproate and trichostatin A (TSA).
40. A pharmaceutical product according to claim 38 or claim 39 wherein the HDAC inhibitor is sodium butyrate, or a prodrug thereof.
41. A pharmaceutical product according to claim 40 wherein the prodrug is pivaloyloxymethyl butyrate.
42. A pharmaceutical product according to claim 38 or claim 39 wherein the HDAC inhibitor is suberoylanilide hydroxamic acid (SAHA).
43. A pharmaceutical product according to claim 38 or claim 39 wherein the HDAC inhibitor is trichostatin A (TSA).
44. A pharmaceutical product according to claim 38 or claim 39 wherein the HDAC inhibitor is sodium valproate.
45. A pharmaceutical product according to any of claims 38 to 44 in the form of a pharmaceutical composition comprising a pharmaceutical acceptable carrier, diluent or excipient.
46. A pharmaceutical product according to any of claims 38 to 45 for use in the treatment of a proliferative disorder.
47. A pharmaceutical product according to claim 46 wherein the proliferative disorder is cancer.
48. A pharmaceutical product according to claim 47 wherein the cancer is non- small cell lung cancer (NSCLC).
49. A method of treating a proliferative disorder, said method comprising simultaneously, sequentially or separately administering a compound of formula I or II as defined in any of claims 1 to 29, or a pharmaceutically acceptable salt thereof, and a HDAC inhibitor.
50. A method according to claim 49 wherein the HDAC inhibitor is selected from sodium butyrate, or a prodrug thereof, suberoylanilide hydroxamic acid (SAHA), sodium valproate and trichostatin A (TSA).
51. A method according to claim 49 or claim 50 wherein the HDAC inhibitor is sodium butyrate, or a prodrug thereof.
52. A method according to claim 51 wherein the prodrug is pivaloyloxymethyl butyrate.
53. A method according to claim 49 or claim 50 wherein the HDAC inhibitor is suberoylanilide hydroxamic acid (SAHA).
54. A method according to claim 49 or claim 50 wherein the HDAC inhibitor is trichostatin A (TSA).
55. A method according to claim 49 or claim 50 wherein the HDAC inhibitor is sodium valproate.
56. A method according to any of claims 49 to 55 wherein the compound of formula I or II, or pharmaceutically acceptable salt thereof, and the HDAC inhibitor are administered in a therapeutically effective amount with respect to the individual components.
57. A method according to any of claims 49 to 55 wherein the compound of formula I or II, or pharmaceutically acceptable salt thereof, and the HDAC inhibitor are administered in a sub-therapeutically effective amount with respect to the individual components.
58. A method according to any of claims 49 to 57 wherein the HDAC inhibitor and the compound of formula I or II, or pharmaceutically acceptable salt thereof, are administered simultaneously.
59. A method according to any of claims 49 to 57 wherein the HDAC inhibitor and the compound of formula I or II, or pharmaceutically acceptable salt thereof, are administered sequentially or separately.
60. A method according to claim 59 wherein the HDAC inhibitor is administered sequentially or separately prior to the compound of formula I or II, or pharmaceutically acceptable salt thereof.
61. A method according to claim 59 wherein the compound of formula I or II, or pharmaceutically acceptable salt thereof, is administered sequentially or separately prior to the HDAC inhibitor.
62. A method according to any of claims 49 to 61 wherein the proliferative disorder is cancer.
63. A method according to claim 62 wherein the cancer is non-small cell lung cancer.
64. Use of a compound of formula I or II as defined in any of claims 1 to 29, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment of a proliferative disorder, wherein said treatment comprises simultaneously, sequentially or separately administering a HDAC inhibitor.
65. Use of a HDAC inhibitor in the preparation of a medicament for the treatment of a proliferative disorder, wherein said treatment comprises simultaneously, sequentially or separately administering to a subject a compound of formula I or II as defined in any of claims 1 to 29, or a pharmaceutically acceptable salt thereof.
66. Use of a compound of formula I or II as defined in any of claims 1 to 29, or a pharmaceutically acceptable salt thereof, and a HDAC inhibitor in the preparation of a medicament for treating a proliferative disorder.
67. Use of a HDAC inhibitor in the preparation of a medicament for the treatment of a proliferative disorder, wherein said medicament is for use in combination therapy with a compound of formula I or II as defined in any of claims 1 to 29, or a pharmaceutically acceptable salt thereof.
68. Use of a compound of formula I or II as defined in any of claims 1 to 29, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for the treatment of a proliferative disorder, wherein said medicament is for use in pretreatment therapy with a HDAC.
69. Use according to any of claims 64 to 68 wherein the HDAC inhibitor is selected from sodium butyrate, or a prodrug thereof, suberoylanilide hydroxamic acid (SAHA), sodium valproate and trichostatin A (TSA).
70. Use according to any of claims 64 to 69 wherein the HDAC inhibitor is sodium butyrate, or a prodrug thereof.
71. Use according to claim 70 wherein the prodrug is pivaloyloxymethyl butyrate.
72. Use according to any of claims 64 to 69 wherein the HDAC inhibitor is suberoylanilide hydroxamic acid (SAHA).
73. Use according to any of claims 61 to 69 wherein the HDAC inhibitor is trichostatin A (TSA).
74. Use according to any of claims claims 61 to 69 wherein the HDAC inhibitor is sodium valproate
75. Use according to any of claims 61 to 74 wherein the proliferative disorder is cancer.
76. Use according to claim 75 wherein the cancer is non-small cell lung cancer (NSCLC).
77. Use of a compound of formula I or II as defined in any of claims 1 to 29, or a pharmaceutically acceptable salt thereof, and a HDAC inhibitor selected from sodium butyrate, or a prodrug thereof, suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA), in the preparation of a medicament for the treatment of non- small cell lung cancer.
78. A kit of parts comprising:
(i) compound of formula I or II, as defined in any one of claims 1 to 25, or a pharmaceutically acceptable salt thereof, optionally admixed with a pharmaceutically acceptable diluent, excipient or carrier; and
(ii) a HDAC inhibitor, optionally admixed with a pharmaceutically acceptable diluent, excipient or carrier.
79. A combination, pharmaceutical composition, pharmaceutical product, method, use or kit of parts substantially as described herein.
EP06808516A 2005-11-11 2006-11-13 Combination of a cdk-inhibitor and a hdac-inhibitor Pending EP1951307A2 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GBGB0523040.4A GB0523040D0 (en) 2005-11-11 2005-11-11 Combination
GB0523160A GB0523160D0 (en) 2005-11-14 2005-11-14 Combination
PCT/GB2006/004224 WO2007054725A2 (en) 2005-11-11 2006-11-13 Combination of a cdk-inhibitor and a hdac-inhibitor

Publications (1)

Publication Number Publication Date
EP1951307A2 true EP1951307A2 (en) 2008-08-06

Family

ID=37603117

Family Applications (1)

Application Number Title Priority Date Filing Date
EP06808516A Pending EP1951307A2 (en) 2005-11-11 2006-11-13 Combination of a cdk-inhibitor and a hdac-inhibitor

Country Status (2)

Country Link
EP (1) EP1951307A2 (en)
WO (1) WO2007054725A2 (en)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9636298B2 (en) * 2014-01-17 2017-05-02 Methylgene Inc. Prodrugs of compounds that enhance antifungal activity and compositions of said prodrugs
WO2017020065A1 (en) * 2015-08-04 2017-02-09 University Of South Australia N-(pyridin-2-yl)-4-(thiazol-5-yl)pyrimidin-2-amine derivatives as therapeutic compounds
EP4114375A1 (en) * 2020-03-02 2023-01-11 Sironax Ltd Ferroptosis inhibitors-diarylamine para-acetamides

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2001287157A1 (en) * 2000-09-12 2002-03-26 Virginia Commonwealth University Promotion of adoptosis in cancer cells by co-administration of cyclin dependent kinase inhibitors and cellular differentiation agents
GB0219054D0 (en) * 2002-08-15 2002-09-25 Cyclacel Ltd New purine derivatives
EP1648887A1 (en) * 2003-07-30 2006-04-26 Cyclacel Limited Pyridinylamino-pyrimidine derivatives as protein kinase inhibitors
BRPI0415759A (en) * 2003-10-21 2006-12-19 Cyclacel Ltd pyrimidin-4-yl-3,4-thione compounds and their use in therapy

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2007054725A2 *

Also Published As

Publication number Publication date
WO2007054725A2 (en) 2007-05-18
WO2007054725A3 (en) 2007-08-09

Similar Documents

Publication Publication Date Title
JP7058345B2 (en) Combination products of Bcl-2 inhibitors and MDM2 inhibitors and their use in the prevention and / or treatment of diseases
JP5478262B2 (en) Anticancer drug
US20170157134A1 (en) Combination therapy
JP7114478B2 (en) Combination drugs for the treatment of cancer
WO2020103921A1 (en) Pharmaceutical composition and use thereof
JP2016503003A (en) Pyrimido [4,5-B] quinoline-4,5 (3H, 10H) -dione as a nonsense mutation inhibitor
JP2005200419A (en) Method for treating cancer
WO2012104823A2 (en) Pyridopyrimidinone compounds in the treatment of neurodegenerative diseases
US20130196938A1 (en) Combination comprising cndac (2&#39;-cyano-2&#39;-deoxy-n4-palmitoyl-1-beta-d-arabinofuranosyl-cytosine) and a cytotoxic agent
ES2385850T3 (en) Radiotherapy enhancer
US20090306098A1 (en) Combination of roscovitine and a hdca inhibitor to treat proliferative diseases
WO2020103922A1 (en) Novel pharmaceutical composition and use thereof
EP1951307A2 (en) Combination of a cdk-inhibitor and a hdac-inhibitor
US9872874B2 (en) Dosage regimen for sapacitabine and seliciclib
JP2015510945A (en) Methods of treating cancer using aurora kinase inhibitors
KR20080044277A (en) Combination of organic compounds
WO2018186366A1 (en) Novel cyclin-dependent kinase 8 and/or 19 inhibitor
AU2015324072B2 (en) A compound for anti-cancer therapy that acts by targeting GOF mutant P53 and stimulates P73
US20050164976A1 (en) Combination of a cdk inhibitor and 5-fu for the tratment of cancer
CN113004207B (en) Compound of PLK4 small molecule inhibitor and application thereof
US20230404973A1 (en) New carbazole derivatives sensitizing cells to anti-cancer agents
US20180303797A1 (en) Chalcone Compounds
WO2017208019A1 (en) Dosing regimen for sapacitabin and seliciclib
JP2013166703A (en) Antitumor agent containing indole compound

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20080331

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI SK TR

DAX Request for extension of the european patent (deleted)
D18D Application deemed to be withdrawn (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20090603