WO2016007993A1 - Inhibiteurs de la sphingosine kinase à base de benzène-sulfonamides - Google Patents

Inhibiteurs de la sphingosine kinase à base de benzène-sulfonamides Download PDF

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WO2016007993A1
WO2016007993A1 PCT/AU2015/000414 AU2015000414W WO2016007993A1 WO 2016007993 A1 WO2016007993 A1 WO 2016007993A1 AU 2015000414 W AU2015000414 W AU 2015000414W WO 2016007993 A1 WO2016007993 A1 WO 2016007993A1
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group
optionally substituted
cells
cell
alkyl
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PCT/AU2015/000414
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English (en)
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Melissa Rose PITMAN
Stuart Maxwell Pitson
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University Of South Australia
Central Adelaide Local Health Network Inc.
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Priority claimed from AU2014902751A external-priority patent/AU2014902751A0/en
Application filed by University Of South Australia, Central Adelaide Local Health Network Inc. filed Critical University Of South Australia
Publication of WO2016007993A1 publication Critical patent/WO2016007993A1/fr

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    • 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
    • A61K31/18—Sulfonamides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a novel class of sphingosine kinase (SK) inhibitors useful in the treatment of cancer.
  • SK sphingosine kinase
  • sphingolipids including ceramide, sphingosine and sphingosine 1 -phosphate (SIP), have emerged as important signalling molecules for controlling a diverse array of important cell processes (Pitson, 201 1).
  • SIP in particular, has diverse cell signalling roles through its actions as both a ligand for a family of five S IP-specific G protein-coupled receptors (named SlPi.s) (Rosen et ah, 2009), as well as a modulator of a range of intracellular proteins (Alvarez et ah, 2010; Strub et ah, 2010; Chipuk et ah, 2012; Hait et ah, 2009; and Harikumar et ah, 2014).
  • SlPi.s S IP-specific G protein-coupled receptors
  • SIP receptor- mediated signalling most notably plays significant roles in immune cell trafficking and vascular integrity (Spiegel and Milstien, 201 1), while SIP in general confers pro-proliferative, pro-survival signalling (Hannun and Obeid, 2008).
  • Sphingosine and many ceramide species are pro-apoptotic, modulating the activity of a range of enzymes involved in the control of cell survival (Woodcock 2006, Hannun and Obeid 2008).Thus, the balance between the cellular levels of S IP and ceramide/sphingosine, the so-called “sphingolipid rheostat" (Cuvillier et ah, 1996), appears to be an important regulator of cell fate.
  • SKs sphingosine kinases
  • SKI and SK2 which catalyse the same reaction and share a high degree of sequence similarity
  • the two SKs share some redundant and related roles (Allende et ah, 2004; Mizugishi et ah, 2005; and Gao and Smith, 201 1), but also appear to possess some different functions, probably due to their different subcellular localisations, with SKI predominantly localised to the cytoplasm and SK2 mainly localised at the nucleus and other organelles (reviewed in Neubauer and Pitson, 2013).
  • SKs have been widely implicated in carcinogenesis. Indeed, SKI expression has been found to be elevated in a wide array of human solid cancers, with higher levels of SKI correlating with the severity of malignancy and shorter patient survival (Guan et al, 2011; Facchinetti et al, 2010; and Zhuge et al, 2011). Similarly, SK2 was recently found to be elevated in human non-small cell lung cancer, with high expression levels correlated with poor patient survival (Wang et al, 2014). Further, a large number of studies have shown that targeting SKs has considerable potential as an anti-cancer strategy.
  • RNAi-mediated knockdown or inhibition of SKI and SK2 has been widely demonstrated to induce apoptosis and enhance sensitivity to chemo- or radio-therapy of many different cancer cells (Gao and Smith 2011 ; Song et al, 2011 ; Akao et al, 2006; and Guillermet-Guibert et al, 2009).
  • genetic ablation of SKI and SK2 in mice has been found to reduce tumour growth in vivo in numerous cancer models (Kawamori et al, 2009; Weigert et al 2009; and Shirai et al, 2011). This body of evidence has secured the SKs as promising therapeutic targets in cancer and has driven drug development to target the enzymes in a range of cancer models (reviewed in Pitman and Pitson, 2010).
  • the present invention provides the use of a compound belonging to a novel class of sphingosine kinase (SK) inhibitors according to formula I for treating cancer or another proliferative cell condition in a subject:
  • X 1 is H or otherwise selected from the groups:
  • each of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 and R 32 is independently selected from the group consisting of: H, halogen, OH, N0 2 , CN, NH 2 , optionally substituted C 1 -C 12 alkyl, optionally substituted C 2 -C 12 alkenyl, optionally substituted C 2 -C 12 alkynyl, optionally substituted C 2 -C 12 heteroalkyl, optionally substituted C 3 - C 12 cycloalkyl, optionally substituted C 2 -C 12 heterocycloal
  • the present invention provides a method of treating cancer or another proliferative cell condition in a subject, the method comprising administering to said subject a therapeutically effective amount of a compound as defined in the first aspect or a pharmaceutically acceptable salt or prodrug thereof, optionally in combination with a pharmaceutically acceptable carrier, diluent and/or excipient.
  • a pharmaceutically acceptable carrier diluent and/or excipient.
  • the present invention provides the use of a compound as defined in the first aspect, or a pharmaceutically acceptable salt or prodrug thereof, in the manufacture of a medicament for treating cancer.
  • the present invention provides a method for modulating sphingolipid-mediated signalling in a cell, comprising introducing to said cell a therapeutically effective amount of a compound as defined in the first aspect or a pharmaceutically acceptable salt or prodrug thereof.
  • the present invention provides a compound as defined in the first aspect, or a pharmaceutically acceptable salt or prodrug thereof, in a substantially purified form.
  • the present invention provides a method of sensitising cancerous or other proliferative cells in a subject to chemo- and/or radio-therapy, comprising administering an effective amount of a compound as defined in the first aspect or a pharmaceutically acceptable salt or prodrug thereof.
  • Figure 1 provides graphical results showing the selectivity of an SK inhibitor compound according to the present invention (designated MP-A08) against human SKI, SK2, DAGK and CERK, and murine SKI and SK2. Data are represented as % activity compared to vehicle control. All data shown are mean ⁇ SD from four independent experiments;
  • Figure 2A-B provides Lineweaver-Burke plots showing inhibition kinetics of MP-A08 against recombinant SKI (A) and SK2 (B) with varying ATP concentration.
  • Data show MP-A08 employed at 50 mM ( ⁇ ) or 25 mM ( ⁇ ), or with vehicle control ( ⁇ ), and are mean ⁇ SD from four independent experiments.
  • C shows graphical results for the assessment of residual SK2 activity in the ATP-binding pocket mutants of residues predicted to be important for SK2 binding to MP-A08.
  • SK2 activities were determined after overexpression in HEK293T cells, and presented as % activity compared to wild-type SK2 (WT).
  • EV transfected cells show negligible contribution from endogenous SK to the activities displayed.
  • the lower panel shows similar expression levels of all SK2 variants, but all activities were adjusted for slight variations in expression.
  • D shows graphical results of the effect of MP-A08 on the activity of SK2 variants harbouring mutations in the ATP-binding site. All data shown are mean ⁇ SD from four independent experiments;
  • D Lysates from Jurkat or Bcl2-expressing Jurkat (Bcl2-Jurkat) cells treated with MP-A08 or vehicle control for 16 h were assessed for PARP cleavage by immunoblotting with PARP antibodies, with tubulin used as the loading control. Results are representative of two independent experiments.
  • Figure 5 provides results showing that MP-A08 reduces tumour burden in an A549 xenograft mouse model.
  • a Mice bearing A549 xenografts were treated i.p. with lOOmg/kg MP-A08 or vehicle control at the times indicated (arrows).
  • Figure 7 provides graphical results showing that MP- A08 induced caspase-dependent cell death in AML cell lines.
  • AML cell lines were incubated with increasing concentrations of MP-A08 and cell death assessed by Annexin V binding (A) and caspase 3 activity (B);
  • Figure 8 provides results showing that MP-A08(5uM) sensitised AML cell lines to killing by cytarabine ( ⁇ ) as measured by Annexin V positive cells. *Combinatorial Index ⁇ 1 indicating synergism;
  • FIG. 9 shows that MP-A08 induced dose-dependent cell death in primary AML blasts (A) and sensitised AML cell blasts to killing by cytarabine (luM), MP-A08(5uM) (B).
  • A primary AML blasts
  • B sensitised AML cell blasts to killing by cytarabine
  • B MP-A08(5uM)
  • *Combinatorial Index ⁇ 1 indicating synergism.
  • Normal healthy bone marrow derived CD34+ cells (C) were less sensitive indicating a potential therapeutic window. Cells were incubated with increasing concentrations of MP- A08 and cell death assessed by Annexin V binding;
  • Figure 10 provides graphical results demonstrating that MP-A08 chemosensitises AML leukaemic stem/progenitor cells to cytarabine;
  • Figure 11 provides results showing that MP-A08 reduces leukaemic burden of normal karyotype AML (A) and prolongs the survival (B). Mice were treated with MP-A08 (lOOmg/kg, daily i.p.) for 12 days.
  • the present invention provides the use of a compound according to formula I for treating cancer or another proliferative cell condition in a subject:
  • X 1 is H or otherwise selected from the groups:
  • each of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 and R 32 is independently selected from the group consisting of: H, halogen, OH, N0 2 , CN, NH 2 , optionally substituted d-Cnalkyl, optionally substituted C 2 -C 12 alkenyl, optionally substituted C 2 -C 12 alkynyl, optionally substituted C 2 -C 12 heteroalkyl, optionally substituted C 3 - C 12 cycloalkyl, optionally substituted C 2 -C 12 heterocycloalky
  • the present invention provides a method of treating cancer or another proliferative cell condition in a subject, the method comprising administering to said subject a therapeutically effective amount of a compound as defined in the first aspect or a pharmaceutically acceptable salt or prodrug thereof, optionally in combination with a pharmaceutically acceptable carrier, diluent and/or excipient.
  • the present invention provides the use of a compound as defined in the first aspect, or a pharmaceutically acceptable salt or prodrug thereof, in the manufacture of a medicament for treating cancer.
  • the present invention provides a method for modulating sphingolipid-mediated signalling in a cell, comprising introducing to said cell a therapeutically effective amount of a compound as defined in the first aspect or a pharmaceutically acceptable salt or prodrug thereof.
  • the present invention provides a compound as defined in the first aspect, or a pharmaceutically acceptable salt or prodrug thereof, in a substantially purified form.
  • the present invention provides a method of sensitising cancerous or other proliferative cells in a subject to chemo- and/or radio-therapy, comprising administering an effective amount of a compound as defined in the first aspect or a pharmaceutically acceptable salt or prodrug thereof.
  • the term "optionally substituted” as used throughout the specification denotes that the group may or may not be further substituted or fused (so as to form a condensed polycyclic system), with one or more non-hydrogen substituent groups.
  • optional substituents include F, CI, Br, I, CH 3 , CH 2 CH 3 , OH, OCH 3 , CF 3 , OCF 3 , N0 2 , NH 2 and CN.
  • two optional substituents on the same moiety when taken together may be joined to form a fused cyclic substituent attached to the moiety that is optionally substituted.
  • optionally substituted includes a fused ring such as a cycloalkyl ring, a heterocycloalkyl ring, an aryl ring or a heteroaryl ring.
  • the group may be a terminal group or a bridging group. This is intended to signify that the use of the term encompasses the situation where the group is a linker between two other portions of the molecule as well as where it is a terminal moiety.
  • alkyl as an example, some publications would use the term
  • alkylene for a bridging group and hence, in these other publications, there is a distinction between the terms “alkyl” (terminal group) and “alkylene” (bridging group). In the present specification, no such distinction is made and most groups may be either a bridging group or a terminal group.
  • heterocycloalkyl aryl or heteroaryl group as defined herein.
  • acyl include acetyl and benzoyl.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the carbonyl carbon.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the nitrogen atom.
  • alkenyl as a group or part of a group denotes an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and which may be straight or branched preferably having 2-12 carbon atoms, more preferably 2-10 carbon atoms, most preferably 2-6 carbon atoms, in the normal chain.
  • the group may contain a plurality of double bonds in the normal chain and the orientation about each is independently E or Z.
  • Exemplary alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl and nonenyl.
  • the group may be a terminal group or a bridging group.
  • alkenyloxy refers to an alkenyl-O- group in which alkenyl is as defined herein.
  • Preferred alkenyloxy groups are C 2 - C 6 alkenyloxy groups.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the oxygen atom.
  • Alkyl as a group or part of a group refers to a straight or branched aliphatic hydrocarbon group, preferably a Q-C- 12 alkyl, more preferably a Ci-Cio alkyl, most preferably C 1 -C 6 alkyl unless otherwise noted.
  • suitable straight and branched C 1 -C 6 alkyl substituents include methyl, ethyl, n- propyl, 2-propyl, n-butyl, sec-butyl, t-butyl, hexyl, and the like.
  • the group may be a terminal group or a bridging group.
  • Alkylamino includes both mono-alkylamino and dialkylamino, unless specified.
  • Mono- alkylamino means an alkyl-NH- group, in which alkyl is as defined herein.
  • Dialkylamino means a (alkyl) 2 N- group, in which each alkyl may be the same or different and are each as defined herein for alkyl.
  • the alkyl group is preferably a C 1 -C 6 alkyl group.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the nitrogen atom.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the carbonyl carbon.
  • Alkyloxy refers to an alkyl-O- group in which alkyl is as defined herein.
  • the alkyloxy is a C
  • the group may be a terminal group or a bridging group.
  • Alkyloxyalkyl refers to an alkyloxy-alkyl- group in which the alkyloxy and alkyl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the alkyl group.
  • Alkyloxyaryl refers to an alkyloxy-aryl- group in which the alkyloxy and aryl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the aryl group.
  • the alkyl group is preferably a C 1 -C 6 alkyl group. Examples include, but are not limited to, methoxycarbonyl and ethoxycarbonyl.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the carbonyl carbon.
  • Alkyloxycycloalkyl refers to an alkyloxy-cycloalkyl- group in which the alkyloxy and cycloalkyl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the cycloalkyl group.
  • Alkyloxyheteroaryl refers to an alkyloxy-heteroaryl- group in which the alkyloxy and heteroaryl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the heteroaryl group.
  • Alkyloxyheterocycloalkyl refers to an alkyloxy-heterocycloalkyl- group in which the alkyloxy and heterocycloalkyl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the heterocycloalkyl group.
  • the alkyl group is preferably a C 1 -C 6 alkyl group.
  • Exemplary alkylsulfinyl groups include, but not limited to, methylsulfmyl and ethylsulfinyl.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the sulfur atom.
  • the alkyl group is preferably a C r C 6 alkyl group. Examples include, but not limited to methylsulfonyl and ethylsulfonyl.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the sulfur atom.
  • Alkynyl as a group or part of a group means an aliphatic hydrocarbon group containing a carbon-carbon triple bond and which may be straight or branched preferably having from 2-12 carbon atoms, more preferably 2-10 carbon atoms, more preferably 2-6 carbon atoms in the normal chain.
  • Exemplary structures include, but are not limited to, ethynyl and propynyl.
  • the group may be a terminal group or a bridging group.
  • Alkynyloxy refers to an alkynyl-O- group in which alkynyl is as defined herein.
  • Preferred alkynyloxy groups are C 2 -C 6 alkynyloxy groups.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the oxygen atom.
  • Aminoalkyl means an NH 2 -alkyl- group in which the alkyl group is as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the alkyl group.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the sulfur atom.
  • Aryl as a group or part of a group denotes (i) an optionally substituted monocyclic, or fused polycyclic, aromatic carbocycle (ie a ring structure having ring atoms that are all carbon) preferably having from 5 to 12 atoms per ring.
  • aryl groups include phenyl, naphthyl and the like; (ii) an optionally substituted partially saturated bicyclic aromatic carbocyclic moiety in which a phenyl and a C 5 .
  • C 7 cycloalkyl or C 5 -C 7 cycloalkenyl group are fused together to form a cyclic structure, such as tetrahydronaphthyl, indenyl or indanyl.
  • the group may be a terminal group or a bridging group.
  • an aryl group is a C 6 -Ci 8 aryl group.
  • Arylalkenyl means an aryl-alkenyl- group in which the aryl and alkenyl are as defined herein.
  • Exemplary arylalkenyl groups include phenylallyl. The group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the alkenyl group.
  • Arylalkyl means an aryl-alkyl- group in which the aryl and alkyl moieties are as defined herein. Preferred arylalkyl groups contain a C 1 -C 6 alkyl group.
  • arylalkyl groups include benzyl, phenethyl, 1-naphthalenemethyl and 2-naphthalenemethyl.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the alkyl group.
  • Arylalkyloxy refers to an aryl-alkyl-O- group in which the alkyl and aryl are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the oxygen atom.
  • Arylamino includes both mono-arylamino and di-arylamino unless specified.
  • Mono-arylamino means a group of formula arylNH-, in which aryl is as defined herein, di-arylamino means a group of formula (aryl) 2 N- where each aryl may be the same or different and are each as defined herein for aryl.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the nitrogen atom.
  • Arylheteroalkyl means an aryl-heteroalkyl- group in which the aryl and heteroalkyl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the heteroalkyl group.
  • Aryloxy refers to an aryl-O- group in which the aryl is as defined herein.
  • the aryloxy is a Ce-Cjgaryloxy, and more preferably a C 6 -Ci 0 aryloxy.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the oxygen atom.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the sulfur atom.
  • a “bond” is a linkage between atoms in a compound or molecule.
  • the bond may be a single bond, a double bond, or a triple bond.
  • Cycloalkenyl means a non-aromatic monocyclic or multicyclic ring system containing at least one carbon-carbon double bond and preferably having from 5-10 carbon atoms per ring.
  • Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl and cycloheptenyl.
  • the cycloalkenyl group may be substituted by one or more substituent groups.
  • a cycloalkenyl group typically is a C3-Q2 alkenyl group. The group may be a terminal group or a bridging group.
  • Cycloalkyl refers to a saturated monocyclic or fused or spiro polycyclic, carbocycle preferably containing from 3 to 9 carbons per ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like, unless otherwise specified. It includes monocyclic systems such as cyclopropyl and cyclohexyl, bicyclic systems such as decalin, and polycyclic systems such as adamantane.
  • a cycloalkyl group typically is a C3-Q2 alkyl group. The group may be a terminal group or a bridging group.
  • Cycloalkylalkyl means a cycloalkyl-alkyl- group in which the cycloalkyl and alkyl moieties are as defined herein.
  • Exemplary monocycloalkylalkyl groups include cyclopropylmethyl,
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the alkyl group.
  • Cycloalkylalkenyl means a cycloalkyl-alkenyl- group in which the cycloalkyl and alkenyl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the alkenyl group.
  • Cycloalkylheteroalkyl means a cycloalkyl-heteroalkyl- group in which the cycloalkyl and heteroalkyl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the heteroalkyl group.
  • Cycloalkyloxy refers to a cycloalkyl-O- group in which cycloalkyl is as defined herein.
  • the cycloalkyloxy is a C 1 -C 6 cycloalkyloxy.
  • Examples include, but are not limited to, cyclopropanoxy and cyclobutanoxy.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the oxygen atom.
  • Cycloalkenyloxy refers to a cycloalkenyl-O- group in which the cycloalkenyl is as defined herein.
  • the cycloalkenyloxy is a C 3 -C 8 cycloalkenyloxy.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the oxygen atom.
  • Haloalkyl refers to an alkyl group as defined herein in which one or more of the hydrogen atoms has been replaced with a halogen atom selected from the group consisting of fluorine, chlorine, bromine and iodine.
  • halogen atom selected from the group consisting of fluorine, chlorine, bromine and iodine.
  • Examples of haloalkyl include fluoromethyl, difluoromethyl and trifluoromethyl.
  • Haloalkenyl refers to an alkenyl group as defined herein in which one or more of the hydrogen atoms has been replaced with a halogen atom independently selected from the group consisting of F, CI, Br and I.
  • Haloalkynyl refers to an alkynyl group as defined herein in which one or more of the hydrogen atoms has been replaced with a halogen atom independently selected from the group consisting of F, CI, Br and I.
  • Halogen refers to fluorine, chlorine, bromine or iodine.
  • Heteroalkyl refers to a straight- or branched-chain alkyl group preferably having from 2 to 12 carbons, more preferably 2 to 6 carbons in the chain, in which one or more of the carbon atoms (and any associated hydrogen atoms) are each independently replaced by a heteroatomic group selected from S, O, P and NR' where R 1 is selected from the group consisting of H, optionally substituted d-Cualkyl, optionally substituted Gs-Cucycloalkyl, optionally substituted C 6 -C-igaryl, and optionally substituted C 2 - Cisheteroaryl.
  • heteroalkyls include alkyl ethers, secondary and tertiary alkyl amines, amides, alkyl sulfides, and the like.
  • heteroalkyl also include hydroxyC 1 -C 6 alkyl, d-Qalkyloxy Cr C 6 alkyl, amino Cj-Qalkyl, C 1 -C 6 alkylaminoC 1 -C 6 alkyl, and di(C
  • the group may be a terminal group or a bridging group.
  • Heteroalkyloxy refers to a heteroalkyl-O- group in which heteroalkyl is as defined herein.
  • the heteroalkyloxy is a C2-C 6 heteroalkyloxy.
  • the group may be a terminal group or a bridging group.
  • Heteroaryl either alone or as part of a group refers to groups containing an aromatic ring (preferably a 5 or 6 membered aromatic ring) having one or more heteroatoms as ring atoms in the aromatic ring with the remainder of the ring atoms being carbon atoms. Suitable heteroatoms include nitrogen, oxygen and sulfur.
  • heteroaryl examples include thiophene, benzothiophene, benzofuran, benzimidazole, benzoxazole, benzothiazole, benzisothiazole, naphtho[2,3-b]thiophene, furan, isoindolizine, xantholene, phenoxatine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine,l ,3,5-triazene, tetrazole, indole, isoindole, 1 H-indazole, benzotriazole, purine, quinoline, isoquinoline, phthalazine, naphthyridine, quinoxaline, cinnoline, carbazole, phenanthridine, acridine, phenazine, thiazole, isothiazole, phenothiazine, oxazo
  • Heteroarylalkyl means a heteroaryl-alkyl group in which the heteroaryl and alkyl moieties are as defined herein. Preferred heteroarylalkyl groups contain a lower alkyl moiety.
  • heteroarylalkyl groups include pyridylmethyl.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the alkyl group.
  • Heteroarylalkenyl means a heteroaryl-alkenyl- group in which the heteroaryl and alkenyl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the alkenyl group.
  • Heteroarylheteroalkyl means a heteroaryl-heteroalkyl- group in which the heteroaryl and heteroalkyl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the heteroalkyl group.
  • Heteroaryloxy refers to a heteroaryl-O- group in which the heteroaryl is as defined herein.
  • the heteroaryloxy is a d.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the oxygen atom.
  • Heterocyclic refers to saturated, partially unsaturated or fully unsaturated monocyclic, bicyclic or polycyclic ring system containing at least one heteroatom selected from the group consisting of nitrogen, sulfur and oxygen as a ring atom.
  • heterocyclic moieties include heterocycloalkyl, heterocycloalkenyl and heteroaryl.
  • Heterocycloalkenyl refers to a heterocycloalkyl group as defined herein but containing at least one double bond.
  • a heterocycloalkenyl group typically is a C 2 -C 12 heterocycloalkenyl group.
  • the group may be a terminal group or a bridging group.
  • Heterocycloalkyl refers to a saturated monocyclic, bicyclic, or polycyclic ring containing at least one heteroatom selected from nitrogen, sulfur, oxygen, preferably from 1 to 3 heteroatoms in at least one ring. Each ring is preferably from 3 to 10 membered, more preferably from 4 to 7 membered.
  • heterocycloalkyl substituents examples include pyrrolidyl, tetrahydrofuryl,
  • a heterocycloalkyl group typically is a C2-C12 heterocycloalkyl group.
  • the group may be a terminal group or a bridging group.
  • Heterocycloalkylalkyl refers to a heterocycloalkyl-alkyl- group in which the heterocycloalkyl and alkyl moieties are as defined herein.
  • exemplary heterocycloalkylalkyl groups include (2- tetrahydrofuryl)methyl and (2-tetrahydrothiofuranyl) methyl.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the alkyl group.
  • Heterocycloalkylalkenyl refers to a heterocycloalkyl-alkenyl- group in which the
  • heterocycloalkyl and alkenyl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the alkenyl group.
  • Heterocycloalkylheteroalkyl means a heterocycloalkyl-heteroalkyl- group in which the heterocycloalkyl and heteroalkyl moieties are as defined herein.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the heteroalkyl group.
  • Heterocycloalkyloxy refers to a heterocycloalkyl-O- group in which the heterocycloalkyl is as defined herein.
  • the heterocycloalkyloxy is a C r C 6 heterocycloalkyloxy.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the oxygen atom.
  • Heterocycloalkenyloxy refers to a heterocycloalkenyl-O- group in which heterocycloalkenyl is as defined herein.
  • the heterocycloalkenyloxy is a C r C 6 heterocycloalkenyloxy.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the oxygen atom.
  • Hydroalkyl refers to an alkyl group as defined herein in which one or more of the hydrogen atoms has been replaced with an OH group.
  • a hydroxyalkyl group typically has the formula C n H(2n+i. x)(OH) x .
  • n is typically from 1 to 10, more preferably from 1 to 6, most preferably 1 to 3
  • x is typically 1 to 6, more preferably 1 to 3.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the sulfur atom.
  • the group may be a terminal group or a bridging group. If the group is a terminal group, it is bonded to the remainder of the molecule through the nitrogen atom.
  • Some of the compounds of the disclosed embodiments may exist as single stereoisomers, racemates, and/or mixtures of enantiomers and/or diastereomers. All such single stereoisomers, racemates and mixtures thereof, are intended to be within the scope of the present invention.
  • the isomeric forms such as diastereomers, enantiomers, and geometrical isomers can be separated by physical and/or chemical methods known to those skilled in the art.
  • formula I is intended to cover, where applicable, solvated as well as unsolvated forms of the compounds.
  • formula I includes compounds having the indicated structure, including the hydrated as well as the non-hydrated forms.
  • pharmaceutically acceptable salt refers to salts that retain the desired biological activity of the compounds of formula I, and include pharmaceutically acceptable acid addition salts and base addition salts.
  • Suitable pharmaceutically acceptable acid addition salts of compounds of formula I may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, sulfuric and phosphoric acid.
  • Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, fumaric, maleic, alkyl sulfonic and arylsulfonic. Additional information on pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Co., Easton, PA 1995.
  • Prodrug means a compound that undergoes conversion to a compound of formula I within a biological system, usually by metabolic means (eg by hydrolysis, reduction or oxidation).
  • metabolic means eg by hydrolysis, reduction or oxidation.
  • an ester prodrug of a compound of formula I containing a hydroxyl group may be convertible by hydrolysis in vivo to the compound of formula I.
  • Suitable esters of compounds of formula I containing a hydroxyl group are, for example, acetates, citrates, lactates, tartrates, malonates, oxalates, salicylates, propionates, succinates, fumarates, maleates, methylene-bis-P-hydroxynaphthoates, gestisates, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p- toluenesulfonates, cyclohexylsulfamates and quinates.
  • an ester prodrug of a compound of formula I containing a carboxy group may be convertible by hydrolysis in vivo to the compound of formula I.
  • ester prodrugs include those described by Leinweber FJ, 1987.
  • an acyl prodrug of a compound of formula I containing an amino group may be convertible by hydrolysis in vivo to the compound of formula I. Examples of prodrugs for these and other functional groups, including amines, are provided in Prodrugs: challenges and rewards, Valentino J Stella (ed), Springer, 2007.
  • a therapeutically effective amount is an amount sufficient to effect beneficial or desired clinical results.
  • a therapeutically effective amount can be administered in one or more administrations.
  • a therapeutically effective amount is sufficient to palliate, ameliorate, stabilise, reverse, slow or delay the progression of the cancer disease or proliferative state.
  • a therapeutically effective amount of a compound of formula 1, or a pharmaceutically acceptable salt or prodrug thereof may comprise between about 0.1 and about 250 mg/kg body weight per day, more preferably between about 0.1 and about 100 mg/kg body weight per day and, still more preferably between about 0.1 and about 25 mg/kg body weight per day.
  • the therapeutically effective amount may vary and depend upon a variety of factors including the activity of the particular compound (or salt or prodrug thereof), the metabolic stability and length of action of the particular compound (or salt or prodrug thereof), the age, body weight, sex, health, route and time of administration, rate of excretion of the particular compound (or salt or prodrug thereof), and the severity of the cancer or another proliferative cell condition to be treated.
  • Compounds of formula (I), and pharmaceutically acceptable salts and prodrugs thereof, are capable of inhibiting sphingosine kinases (SKs), and may show higher selectivity (to inhibit) SKI and SK2 over other kinases such as the related human lipid kinases, diacylglycerol kinase (DAGK) and ceramide kinase (CERK).
  • SKI and SK2 act by phosphorylating sphingosine to generate S IP, which has diverse cell-signalling roles and, in general, confers pro-proliferative, pro- survival cell signalling.
  • the compounds of formula (I), and pharmaceutically acceptable salts and prodrugs thereof are capable of modulating sphingolipid-mediated cell signalling (eg SIP cell signalling) and, therefore, have utility in both in vitro and in vivo applications (eg in vitro cell-based assays) and as the basis of a therapeutic method of treating cancer or another proliferative cell condition in a subject.
  • sphingolipid-mediated cell signalling eg SIP cell signalling
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 are H.
  • R 3 is CH 3 .
  • R 8 is NR 33 S0 2 R 34 , wherein R 33 and R 34 are each independently selected from the group consisting of H, Cj-Cnalkyl, C 1 -C 12 haloalkyl, C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, C 2 -C 10 heteroalkyl, optionally substituted C 3 -C 12 cycloalkyl, optionally substituted C 3 -C 12 cycloalkenyl, optionally substituted C 2 -C 12 heterocycloalkyl, optionally substituted C 2 -C 12 heterocycloalkenyl, optionally substituted C 6 -C 18 aryl, optionally substituted C 2 -C 18 heteroaryl, and acyl.
  • R 33 is H and R 34 is optionally substituted C 6 cycloalkyl.
  • X 1 is H.
  • each of R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 and R 9 is independently selected from H, CH 3 and NH 2 .
  • R and R may beCH 3 .
  • X' is selected from the groups:
  • one or more of R 10 , R 11 , R 12 , R 13 and R 14 are H.
  • R 10 , R 1 R 12 , R 13 and R 14 are halogen.
  • R 12 is selected from H, CH 3 , OCH 3 , N0 2 , and N(CH 3 ) 2 .
  • R is N0 2 or NHS0 2 R wherein R ' is selected from the group consisting of H, C ! -C 6 alkyl, Ci -C 6 haloalkyl, C 2 -C 6 alkenyl, C 2 -C 6 alkynyl, C 2 -Ci 0 heteroalkyl, optionally substituted C 3 -C 6 cycloalkyl, optionally substituted C 3 -C 6 cycloalkenyl, optionally substituted C 2 -C 6 heterocycloalkyl, optionally substituted C 2 -C 6 heterocycloalkenyl, optionally substituted C 6 -C 18 aryl, optionally substituted C 2 -C 18 heteroaryl, and acyl.
  • R 18 is C 3 - Qcycloalkyl optionally substituted with one or more alkyl groups (preferably CH 3 ).
  • R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , R 21 and R 22 are H.
  • R 20 and R 21 taken together with the carbon atoms to which R 20 and R 21 are attached form a (fused) C 2 -C 8 cycloalkyl, C 2 -C 8 heterocycloalkyl, aryl or any heteroaryl ring fused at the carbon atoms to which R 20 and R 21 are attached.
  • R 23 , R 24 , R 25 , R 26 , R 27 and R 28 are H.
  • R 24 is selected from H, halogen, OCH 3 and N0 2 .
  • each of R 23 , R 25 and R 26 is independently selected from H, halogen and OCH 3 .
  • R 29 is selected from C 3 -C 5 heterocycloalkyl, C 3 -C 5 heterocycloalkenyl and C$ heteroaryl, more preferably C 4 -C 5 heterocycloalkyl comprising one or more heteroatoms selected from O, N and S.
  • R 32 is selected from H and OH.
  • X 1 is selected from the following:
  • the compound is selected from:
  • Particularly preferred compounds of formula I include those designated as MP-A08.9, MP- A08.17, MP-A08.23, MP-A08.24, MP-A08.26 and MP-A08.27.
  • Particularly preferred compounds of formula I may also include those compounds displaying inhibition of SKI characterised by an IC 5 o value of less than 10, more preferably, less than 7 (as measured by, for example, SK assays as described herein).
  • Particularly preferred compounds of formula I may also include those compounds displaying inhibition of SKI characterised by an IC 50 value that is at least a 3-fold improvement (ie increased inhibition) than the IC 50 value of MP-A08 (as measured by a suitable SK assays such as, for example, SK assays described herein).
  • Compounds of formula I may be administered in combination with one or more additional agent(s) for the treatment of cancer.
  • the compounds may be used in combination with other anti-cancer agents in order to inhibit more than one cancer signalling pathway simultaneously so as to make cancer cells more susceptible to anti-cancer therapies (eg treatments with other anti-cancer agents, chemotherapy, radiotherapy or a combination thereof)
  • the compounds of formula I may be used in combination with one or more of the following categories of anti-cancer agents:
  • alkylating agents eg cis-platin, oxaliplatin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulphan, temozolamide and nitrosoureas
  • alkylating agents eg cis-platin, oxaliplatin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulphan, temozolamide and nitrosoureas
  • antimetabolites eg gemcitabine, methotrexate, cytarabine and hydroxyurea
  • antitumour antibiotics eg anthracyclines such as bleomycin, doxorubicin, daunomycin and epirubicin
  • antimitotic agents eg vinca alkaloids such as vincristine and vinblastine
  • taxoids eg taxol and taxotere
  • topoisomerase inhibitors eg epipodophyllotoxins such as etoposide, topotecan and camptothecin
  • cytostatic agents such as antiestrogens (eg tamoxifen), antiandrogens (eg bicalutamide, flutamide and cyproterone acetate), LHRH antagonists or LHRH agonists (eg goserelin, leuprorelin and buserelin), progestogens (eg megestrol acetate), aromatase inhibitors (eg anastrozole, letrozole, vorazole and exemestane) and inhibitors of 5a-reductase such as finasteride;
  • antiestrogens eg tamoxifen
  • antiandrogens eg bicalutamide, flutamide and cyproterone acetate
  • LHRH antagonists or LHRH agonists eg goserelin, leuprorelin and buserelin
  • progestogens eg megestrol acetate
  • aromatase inhibitors e
  • anti-invasion agents eg c-Src kinase family inhibitors such as 4-(6-chloro-2,3- methylenedioxyanilino)-7-[2-(4-methylpiperazin-l-yl)ethoxy]-5-tetrahydropyran-4- yloxyquinazoline (AZD0530; International Patent Publication No WO 01/94341), N-(2-chloro-6- methylphenyl)-2- ⁇ 6-[4-(2-hydroxyethyl)piperazin-l -yl]-2-methylpyrimidin-4-ylamino ⁇ thiazole- 5-carboxamide (dasatinib) and bosutinib (SKI-606), and metalloproteinase inhibitors such as marimastat, and inhibitors of urokinase plasminogen activator receptor function;
  • anti-invasion agents eg c-Src kinase family inhibitors such as 4-(
  • inhibitors of growth factor function eg the anti-erbB2 antibody trastuzumab (HerceptinTM), the anti-EGFR antibody panitumumab, and the anti-erbB 1 antibody cetuximab (Erbitux, C225); such inhibitors also include tyrosine kinase inhibitors, for example inhibitors of the epidermal growth factor family (eg EGFR family tyrosine kinase inhibitors such as N-(3-chloro-4-fluorophenyl)-7- methoxy-6-(3-mo ⁇ holinopropoxy)quinazolin-4-amine (gefitinib, ZD 1839), N-(3-ethynylphenyl)- 6,7-bis(2-methoxyethoxy)quinazolin-4-amine (erlotinib, OSI-774) and 6-acrylamido-N-(3-chloro- 4-fluorophenyl)-7-(3-morpholinopropoxy
  • antiangiogenic agents such as those which inhibit the effects of vascular endothelial growth factor (eg the anti-vascular endothelial cell growth factor antibody bevacizumab (AvastinTM) and the VEGF receptor tyrosine kinase inhibitor vandetanib (ZD6474)).
  • vascular endothelial growth factor eg the anti-vascular endothelial cell growth factor antibody bevacizumab (AvastinTM) and the VEGF receptor tyrosine kinase inhibitor vandetanib (ZD6474)
  • the compound of formula I and the other anti-cancer agent can be administered in the same pharmaceutical composition or in separate pharmaceutical compositions. If administered in separate pharmaceutical compositions, the compound of formula I and the other anti-cancer agent may be administered simultaneously or sequentially in any order (eg within seconds or minutes or even hours (eg 2 to 48 hours)).
  • the present invention is typically applied to the treatment of cancer or another proliferative cell condition in a human subject.
  • the subject may also be selected from, for example, livestock animals (eg cows, horses, pigs, sheep and goats), companion animals (eg dogs and cats) and exotic animals (eg non-human primates, tigers, elephants etc).
  • Cancers and other proliferative cell conditions that may be treated in accordance with the present invention include biliary tract cancer, brain cancer (including glioblastomas and
  • medulloblastomas breast cancer, cervical cancer; choriocarcinoma, colonic cancer, endometrial cancer, oesophageal cancer, gastric cancer, haematological neoplasms (including acute lymphocytic leukaemia (ALL), chronic lymphocytic leukaemia (CLL), chronic myeloid leukaemia (CML),acute myeloid leukaemia(AML), multiple myeloma, AIDS-associated leukaemias and adult T-cell leukaemia lymphoma, intraepithelial neoplasms (including Bowen's disease and Paget's disease), liver cancer, lung cancer, lymphomas (including Hodgkin's disease and lymphocytic lymphomas, neuroblastomas, oral cancer (including squamous cell carcinoma), ovarian cancer (including those arising from epithelial cells, stromal cells, germ cells, and mesenchymal cells), pancreatic cancer, prostate cancer, colorectal
  • the method invlves modulating S 1 P-receptor mediated cell signalling and/or intracellular SIP cell signalling.
  • the compound in a substantially purified form is other than a compound designated herein as MP-A08 or MP-A08.1 to MP-A08.29.
  • the method may comprise administering the compound to the subject prior to, or simultaneously with, the chemo- and/or radio-therapy.
  • the chemotherapy may be, for example, treatment with any of the anti-cancer agents mentioned above at paragraph
  • the SKI structural model and DgkB crystal structure were prepared for docking using the DockPrep module in Chimera (Pettersen et al., 2004). Docking was performed using DOCK6 (Shoichet et al., 1992: and Ewing and Kuntz, 1996) and docking parameters optimised using comparisons of the structure of DgkB co-crystallised with ADP and with the DgkB structure docked with ADP.
  • DOCK6 Shownazeet et al., 1992: and Ewing and Kuntz, 1996) and docking parameters optimised using comparisons of the structure of DgkB co-crystallised with ADP and with the DgkB structure docked with ADP.
  • silico screening a virtual library of 120,000 compounds was generated from databases (Sigma- Aldrich, Calbiochem, and the National Cancer Institute Chemical and Natural Products libraries). The docking was carried out in two stages: initial low stringency screening, and then high stringency screening.
  • the compound designated MP-A08 (4-methyl-N- ⁇ 2-[(2- ⁇ [(4-methylphenyl)sulfonyl]amino ⁇ benzylidene) amino]phenyl ⁇ benzenesulfonamide was synthesised, purified and identity-verified by ChemBridge Inc (San Diego, CA, United States of America) with >95% purity.
  • the synthesis protocol was as follows:
  • FLAG-tagged human SKI and SK2 constructs in pCDNA3 vector were as previously described (Pitson et al., 2000; and Roberts et al., 2004).
  • Quikchange® PCR mutagenesis was carried out with forward and reverse mutagenic oligonucleotides. DNA sequencing verified the integrity and orientation of all cDNAs.
  • HEK293 human embryonic kidney, ATCC# CRL-1573
  • A549 human lung adenocarcinoma, ECACC# 86012804
  • transformed human foreskin fibroblast BJ7 Hahn et al., 1999
  • MCF7 human mammary adenocarcinoma, ECACC# 86012803
  • MDA-MB-231 human mammary adenocarcinoma, ATCC# HTB-26 cells were cultured in Dulbecco's modified Eagle's medium (GIBCO, Invitrogen Corporation, Carlsbad, CA, United States of America), containing 10% foetal bovine serum (Bovagen, Seguin, TX, United States of America), 2 mM glutamine, 0.2% (w/v) sodium bicarbonate, 1 mM HEPES, penicillin (1.2 mg/ml) and streptomycin (1.6 mg/ml).
  • Dulbecco's modified Eagle's medium GIBCO, Invit
  • Jurkat human T cell lymphoblast, ATCC#TIB-152
  • Jurkat-Bcl2 cells were cultured in suspension in RPMI medium containing 10% foetal bovine serum (Bovagen Biologicals Pty Ltd, East Keilor, VIC, Australia), 2 mM glutamine, 0.2% (w/v) sodium bicarbonate, 1 mM HEPES, penicillin (1.2 mg/ml) and streptomycin (1.6 mg/ml). All cells were grown at 37°C, 5% CO2 in a humidified incubator.
  • SK1/SK2 double knockout mouse embryonic fibroblasts were generated from timed matings of female SK1 + 7SK2 ' ' mice with a male SKI " 7SK2 ⁇ " mice.
  • Fibroblasts from 1 1.5 day post coitum embryos were isolated and cultured in DMEM containing 10% bovine calf serum (Bovogen), penicillin ( 1.2 mg/ml) and streptomycin (1.6 mg/ml) at 37°C in a humidified atmosphere with 10% C0 2 .
  • Cells were genotyped to identify cultures with SK1/SK2 double knockout genotype. Wild-type MEFs were generated from 14.5 day post coitum embryos and cultured as described above.
  • HEK293 cells were seeded in 6-well plates and were transiently transfected using LipofectamineTM 2000 Transfection Reagent (Invitrogen) according to the manufacturer's protocol. Cells were harvested 24 h post-transfection, and cell pellets were resuspended in 50 mM Tris-HCl buffer (pH 7.4) containing 150mM NaCl, 10% glycerol, ImM EDTA, 0.05% Triton X-100 (excluded for SK2 samples), 2mM Na 3 V0 4 , 10 mM NaF, 10 mM ⁇ -glycerophosphate, ImM dithiothreitol and protease inhibitor cocktail (F Hoffmann-La Roche Ltd, Basel, Switzerland).
  • LipofectamineTM 2000 Transfection Reagent Invitrogen
  • Baculovirus SKI expression constructs encoding for human SKla with a C-terminal TEV-cleavable 6xHis tag was generated by PCR with oligonucleotide primers using pcDNA3-SKl (Pitson et al., 2000) as a template. The resultant product was cloned into pFastBacl (Invitrogen) by digestion with EcoRI. Recombinant human SKI protein was expressed using the baculovirus expression system in Sf9 cells and purified as previously described (Pitson et al., 2002).
  • the PCR product was digested with Hindlll and cloned into pFastBacl . Sequencing verified the orientation and integrity of all the cloned cDNAs. Recombinant bacmids and baculoviruses were produced according to the manufacturer's protocols. To generate CERK and DAGKa proteins Sf9 cells (2 xlO 9 cells) were infected with the recombinant baculovirus (MOI 5-10) for 96 h. Infected cells were harvested and snap frozen and stored at -80°C until required.
  • Triton X-100 was added to a final concentration of 1% (v/v) and the cell suspension incubated on ice for 30 mins. The lysate was clarified by centrifugation at 50,000 x g for 20 minutes at 4°C.
  • the resulting lysate was incubated with 1.5 ml Buffer A-equilibrated Nickel-NTA sepharose (GE Healthcare, Fairfield, CT, United States of America) and incubated at 4°C for 30 minutes with shaking.
  • the Nickel-NTA was packed into a column and washed with 5 column volumes of Buffer A.
  • the protein was eluted with Buffer A containing 0.2 M imidazole.
  • DAGK assays were performed using ⁇ -octylglucoside solubilised dioleoyl-s,n-glycerol (DAG) and
  • phosphatidylserine PS
  • CERK assays were performed as described previously (Wijesinghe et al., 2007).
  • Cross-screening assays were carried out using 20 ⁇ ATP (0.5 ⁇ [ ⁇ 32 ⁇ ] ⁇ ) and contained either MP-A08 (0.1 mg/ml) or vehicle control (1% DMSO/ 9% (v/v) ethanol). Data are represented at % activity compared to vehicle control.
  • SK assays were carried out as described above with 7.8-500 mM ATP and 100 mM sphingosine and treated with either vehicle (0.25% DMSO/ 2.25% (v/v) ethanol), 25 or 50 ⁇ MP-A08.
  • Kinetic constants were calculated using non-linear regression in GraphPad Prism 5 (GraphPad Software Inc, La Jolla, CA, United States of America).
  • Sphingolipid mass spectrometric analyses analysis was performed on5 x 10 6 Jurkat cells treated in the same manner as above for 6 h or 16 h with either 15 ⁇ MP-A08 or vehicle control. Cells were then pelleted, lyophilised and subjected to mass spectrometric analyses of sphingosine, ceramides and their dihydro species using methods described previously (Bielawski et al. 2006).
  • Jurkat cells (5 x 10 5 cells/ml) were treated with vehicle (70% PEG, 0.28% v/v final) or MP-A08 (5-15 ⁇ ) for 5 or 24 h in RPMI medium containing 0.5% FBS. Analysis of TMRE staining and caspase 3 activity were carried out as described previously (Woodcock et al., 2010). Annexin V staining was carried out according to manufacturer's instructions (Annexin- V-Fluos, F Hoffmann-La Roche) and analysed by flow cytometry as described previously (Woodcock et al, 2010). Jurkat cells stably expressing human Bcl-2awere generated by lentiviral transduction.
  • the lentiviral construct contained the cDNA for Bcl-2a upstream of an IRES followed by cDNA of the mIL-2Ra chain. After transduction, cells were sorted for mIL-2Ra expression using flow cytometry to enrich for Bcl-2a expression.
  • adherent cancer cell lines (A549, BJ7, MCF7 and MDA-MB- 231) were seeded into 48-well plates (20,000 cells per well) 8 h prior to treatment. Cells were then treated with MP-A08 or vehicle control (10% DMSO/90% ethanol) in DMEM containing 0.5% FBS, 1.2 mg/ml penicillin, 1.6 mg/ml streptomycin, and lmM HEPES. After 48h, relative viable cell numbers were determined using the MTS assay (Promega Corporation, Madison, WI, United States of America) according to the manufacturer's protocol. Apoptosis in MEFs was assessed by DAPI staining as previously described (Pitson et al., 2005).
  • MP-A08 dissolved in 70% (v/v) polyethylene glycol 400 (PEG 400), was administered at 50, 75 and lOOmg/kg by intraperitoneal (i.p.) injection daily for 2 weeks, as was vehicle control. Mice were weighed daily and murine cell blood counts were determined after two weeks of treatment by a SYSMEX XE 2100 hematology analyser (Sysmex Corporation, Kobe, Japan).
  • A549 cells (5 x 10 6 ) were subcutaneously injected into the flanks of 6-8 week old female NOD/SCID mice. Tumour development was assessed daily and measured by calliper. Four weeks post-engraftment, when tumour sizes reached approximately 70mm 3 , MP-A08 was administered at lOOmg/kg six days a week for 2 weeks, with daily measurement of tumours. Tumours were then excised, fixed in 10% formalin, paraffin embedded and sectioned. Apoptosis was then examined by TUNEL staining using the Fluorescein in situ cell death detection kit (F Hoffmann-La Roche) according the manufacturer's instructions.
  • F Hoffmann-La Roche Fluorescein in situ cell death detection kit
  • tumour tissue sections underwent a citrate buffer antigen retrieval process followed by blocking with 10% serum/PBS at room temperature for 60 min.
  • Affinity purified goat polyclonal antibody to CD31/PECAM-l sc-1506; Santa Cruz Biotechnology Inc, Santa Cruz, CA, United States of America
  • biotinylated rabbit anti-goat antibody (1 :500; Abeam) at room temperature.
  • Sections were then incubated with VECTASTAIN Elite ABC Reagent (Vector Laboratories Inc, Burlingame, CA, United States of America) at room temperature for 30 min, followed by peroxidise substrate solution.
  • Sections were counterstained with Mayer's haematoxylin, mounted using DPX and visualised on an Olympus BX45 microscope equipped with an XC10 camera. A single image was collected using the 10X objective which covered 30-60% of the total area of the section, and CD31- immunoreactive vessels were enumerated by a person blinded to the identity of the samples.
  • Glioblastoma multiforme is the most common and most aggressive malignant primary brain tumour in humans. Recent studies have implicated SKI and SIP in progression of glioblastoma multiforme tumours (Abuhusain et al., 2013).
  • In vivo efficacy of MP-A08 to attenuate growth of U-87 human glioblastoma multiforme (ATCC#HTB-14) was investigated using subcutaneous xenografts of U-87 cells established in NOD/SCID mice and grown to a volume of 50- 100mm 3 . Mice were then treated with MP-A08 at 100 mg/kg, six times a week for two weeks.
  • AML Acute myeloid leukaemia
  • HSPC normal hematopoietic stem and progenitor cells
  • LSPC leukaemic stem and progenitor cells
  • AML cell lines ie MOLM13, MV41 1, TF1, UT7, THP1 and ME1 representing various AML subtypes were treated with MP-A08.
  • Xeno-transplantation models are widely accepted within the field for the in vivo analysis of therapies that target the clinically relevant LSPC population.
  • a xeno-transplantation model of primary human AML was used wherein cells were transplanted into sub-lethally irradiated immunocompromised mice and allowed to grow and establish disease.
  • Homology modelling was used to predict the structure of the ATP-binding pocket of SKI using the solved structures of two bacterial lipid kinases, DgkB (Miller et al., 2008) and YegS (Bakali et al., 2007) that, while possessing little overall sequence similarity to SKI , do show some sequence similarity with residues proposed to contribute to ATP binding in SKI (Pitman et a/., 2013; Pitson, 201 1; and Pitson et al., 2002).
  • Asp341 and Glu343 from motif 5 were predicted to coordinate a magnesium ion, as inferred from similarity to the DgkB structure (Miller et al., 2008).
  • alanine mutagenesis was carried out to predict the residues that contribute to the ATP-binding pocket. Mutagenesis of all the SKI residues predicted to form hydrogen-bonds with ATP either abolished or substantially reduced enzyme activity, while mutation of other residues in this region that had not been not predicted to directly interact with ATP (Arg24, Glu55 and Ser79), generally had less effect.
  • Virtual screening identifies a novel SK-selective inhibitor
  • the validated model of the ATP-binding pocket of SKI was then used in a virtual screen to identify novel inhibitors of SKI.
  • Libraries were docked into the ATP-binding pockets of the SKI model and DgkB structure using a two-step screening approach.
  • Candidate compounds were chosen that displayed preferential docking scores and orientation for the ATP-binding pocket of SKI over that of DgkB.
  • This compound contains two benzenesulfonamide groups joined by a central benzylidene-aniline group.
  • MP-A08 docked into the ATP-binding pocket of SKI in close association with conserved motifs 1-3, and was predicted to form close associations with Asn22, Arg24, Thr54, Ser79, Gly80, Asp81, Gly82, Leu83 and Serl 12.
  • MP-A08 inhibition of SKI was reduced by around three- fold by the T54A, L83A,R185A and SI 12A mutations and approximately two-fold by the S79A, R24A and R191 A mutations.
  • the R24A mutation did not affect SKI activity, but reduced MP-A08 inhibition, which was in agreement with the docking.
  • MP-A08 is an inhibitor targeting the ATP-binding pocket of SKI . Together with the docking, it also suggests that a subset of the residues that bind the adenosine component of ATP, namely Arg24, Thr54, Glu55 and Leu83, accommodate the amine group from the benzene sulfonamide and the central imine in MPA-08. Additionally, the polar side-chains of Ser79 and Serl 12 and the positively charged Argl85 and Argl91, which coordinate the negatively charged phosphates of ATP, accommodate the bulky phenyl rings and sulfonyl group in MPA-08.
  • MP-A08 was also tested against a panel of 140 human protein kinases. Consistent with the structural divergence of the ATP-binding pocket of SKI from that of the protein kinases, initial screening showed that very few protein kinases were affected by 25uM MP-A08, and those that were, only displayed modest inhibition (Table 1). Moreover, when very high concentrations of MP-A08 (250uM) were tested against the seven protein kinases that were inhibited by more than 30% in the initial screen, six of these failed to show a dose-dependent trend. Only testis-specific serine kinase 1 (TSSK1) was modestly inhibited at this very high concentration of MP-A08 (Table 1).
  • TSSK1 testis-specific serine kinase 1
  • One methyl substituted ring points out towards the outside of the pocket and the other is orientated towards the internal SKl SelU2 side-chain.
  • the predicted orientation of MP-A08 in the ATP- binding pocket of SK2 is altered due to the substituted residues at Phel 54 (SKl ⁇ 824 ) and Asnl87 (SKI'* 1857 ).
  • the bulky aromatic side-chain of Phel54 and the smaller basic side-chain of Asnl87 alter both the size and charge of the of the ATP pocket in SK2.
  • the terminal methylphenyl rings point towards the side-chain of Thrl84 and the backbone of Phel54.
  • MP-A08 inhibits SIP production in cells and increases pro-apoptotic sphingolipids
  • dihydrosphingosine and sphingosine were elevated further compared to the 6 h treatment. Consistent with previous reports of sphingolipid modulation in cells with SKI and SK2 knockdown (Taha et al., 2006; and Gao and Smith, 2011), these results demonstrated that SK inhibition by MP-A08 significantly decreased cellular SIP generation, and increased upstream sphingosine/dihydrosphingosine and ceramides/dihydroceramides.
  • MP-A08 induces mitochondrial-mediated apoptosis
  • MP-A08 treatment was found to increase levels of pro-apoptotic sphingosine and ceramides, the effects of MP-A08 on signalling pathways associated with survival and proliferation was assessed.
  • SIP is known to regulate the Akt and MAPK pathways via signalling through the SIP G-protein coupled receptors and via unknown intracellular pathways (Cuvillier et al., 1996).
  • MP-A08 treatment caused a dose-dependent loss in activation of the pro-survival and pro-proliferative Akt and ERK1/2 pathways, and induction of the apoptosis-associated p38 and JNK pathways (Figure 3C).
  • MP-A08 blocks survival and neoplastic growth of cancer cell lines
  • the cell lines were seven from the NCI60 human tumour cell line panel (A549 lung adenocarcinoma cells, and MCF-7 (estrogen receptor positive) and MDA-MB-231 (triple-negative) breast adenocarcinoma cells), U251 glioblastoma cells, SK-Mel-28 melanoma cells, OVCAR-3 ovarian cancer cells and K-562 chronic myeloid leukaemia (CML) cells), as well as MV411 and MOLM13 acute myeloid leukaemia (AML) cells, H929 myeloma cells, LNCaP prostate cancer cells, MDA-MB-468 breast adenocarcinoma, U87 glioblastoma cells and BJ7 human foreskin fibroblasts transformed via the expression of V12-
  • NCI60 human tumour cell line panel A549 lung adenocarcinoma cells, and MCF-7 (estrogen receptor positive) and MDA-MB-231 (trip
  • MP-A08 suppresses the growth of human lung tumour xenografts in mice
  • MP-A08 treated tumours contained significantly lower levels of S IP compared to the vehicle control tumours confirming inhibition of sphingosine kinases in vivo. Further, MP-A08 treated tumours exhibited significantly higher levels of apoptotic cell death compared to the vehicle control as indicated by TUNEL staining. In addition, MP-A08 treated tumours showed reduced vasculature as signified by a reduction in CD31 positive blood vessels ( Figure 5C). This data therefore indicates that MP-A08 acts as an anti-cancer agent by both inducing cell death and blocking angiogenesis.
  • MP-A08.1-MP-A08.28 Twenty-eight structural analogues of MP-A08 (designated herein as MP-A08.1-MP-A08.28) were tested in biological assays against recombinant sphingosine kinase 1 (SKI) to screen the compounds for their ability to inhibit SKI activity. Sixteen of the compounds (MP-A08.1-8.8, 8.13, 8.15, 8.18-8.20, 8.22, 8.25 and 8.28) showed reduced ability to inhibit SKI compared to the parent MP-A08 compound (Table 3). Four compounds (MP-A08.10-8.12, and 8.14) showed similar inhibition levels to MP-A08 (Table 3).
  • Recombinant SKI was treated with varying doses of MP-A08 to determine the median inhibition concentration (IC5 0 ) to block enzyme activity in a 96-well plate format assay.
  • IC5 0 median inhibition concentration
  • MP-A08 suppresses the growth of human glioblastoma multiforme xenografts in mice
  • glioblastoma cell lines were treated with sub-optimal concentrations of MP-A08, the chemotherapeutic agent doxorubicin or the combination, and measured the effect on growth.
  • Treatment of U251 and U87 glioblastoma cells with very low concentrations of MP-A08 or doxorubicin had no effect on cell growth; however, the combined treatment demonstrated significant growth inhibition, indicating a synergistic action (Figure 6A).
  • MP-A08 can be used to enhance chemotherapeutic sensitivity in glioblastoma. Similar chemosensitising effects through blocking sphingosine kinases have been observed with other inhibitors (Gao and Smith 2011 ; Song et al, 2011 ; Akao et al, 2006; and Guillermet-Guibert et al, 2009).Results of experiments conducted to assess the effect of MP-A08 on human glioblastoma multiforme U-87 cells are provided in Figure 6B. It was found that MP-A08 significantly reduced tumour growth.
  • MP-A08 induced dose-dependent induction of caspase-dependent apoptosis in all cell lines examined (Figure 7).
  • combinational treatment of MP-A08 with the chemotherapeutic agent cytarabine resulted in synergistic cell death ( Figure 8) indicating combinational treatment of existing induction chemotherapeutics with MP-A08 may have significant clinical utility.
  • primary AML blasts from a number of different patients spanning various cytogenetic subgroups were treated with MP-A08 and as seen in the AML cell lines, MP-A08 induced apoptosis dose-dependant apoptosis (Figure 9A) and sensitised blasts to killing by the chemotherapeutic cytarabine ( Figure 9B).
  • MP-A08 had no effect on normal haematopoietic stem/progenitor cells (CD34+) derived from the bone marrow of healthy donors except at very high concentrations (>50mM, Figure 9C). This indicates the existence of a useful therapeutic window. Further, examination of AML LSPCs from a number of patients indicated that all were sensitive to MP-A08 and sensitised LSPCs to killing by the chemotherapeutic cytarabine ( Figure 10), although the EC 50 values varied.
  • MP-A08 reduces leukaemic burden and prolongs survival in vivo
  • a novel class of SK inhibitors has been identified that show higher selectivity to SKI and SK2 over other kinases. These inhibitors are cell permeable and are capable of altering the balance of the sphingolipid rheostat away from anti-apoptotic, pro-proliferative S IP, towards pro-apoptotic sphingosine and ceramide.
  • MP-A08 4-methyl-N-[2-[[2-[(4-methylphenyl) sulfonylamino]phenyl]iminomethyl]phenyl]benzenesulfonamide
  • Facchinetti MM et al, Cells Tissues Organs 192(5):314-324 (2010).

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Abstract

La présente invention concerne une nouvelle classe de composés inhibiteurs de la sphingosine kinase (SK) qui sont utiles dans le traitement du cancer et d'autres affections liées à la prolifération cellulaire. Les composés sont des benzène-sulfonamides avec une structure chimique répondant à la formule (I).
PCT/AU2015/000414 2014-07-16 2015-07-16 Inhibiteurs de la sphingosine kinase à base de benzène-sulfonamides WO2016007993A1 (fr)

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Cited By (2)

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WO2018237296A1 (fr) * 2017-06-23 2018-12-27 City Of Hope Inhibiteurs parg et procédé d'utilisation de ceux-ci
CN113999148A (zh) * 2021-11-11 2022-02-01 浙江省中医药研究院 一种n-(4-(取代磺酰基氨基)苯基)磺酰胺类化合物及其应用

Citations (2)

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WO2007050673A2 (fr) * 2005-10-25 2007-05-03 University Of Florida Inhibiteurs des kinases dependantes des cyclines

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GARNOVSKII, DA ET AL.: "Electrochemical synthesis of adducts of 2-aminopyridine or methanol in metal chelates of a N,N,N-tridentate Schiff base ligand. X-ray crystal structures of the Ni(II) and Zn(II) derivatives.", POLYHEDRON, vol. 22, 2003, pages 1335 - 1340 *
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018237296A1 (fr) * 2017-06-23 2018-12-27 City Of Hope Inhibiteurs parg et procédé d'utilisation de ceux-ci
US11858879B2 (en) 2017-06-23 2024-01-02 City Of Hope PARG inhibitors and method of use thereof
CN113999148A (zh) * 2021-11-11 2022-02-01 浙江省中医药研究院 一种n-(4-(取代磺酰基氨基)苯基)磺酰胺类化合物及其应用

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