WO2016007993A1 - Benzene sulfonamide-based inhibitors of sphingosine kinase - Google Patents

Abstract

A novel class of sphingosine kinase (SK) inhibitor compounds are disclosed which are useful in the treatment of cancer and other proliferative cell conditions. The compounds are benzene sulfonamides with a chemical structure according to formula (I).

Description

BENZENE SULFONAMIDE-BASED INHIBITORS OF SPHINGOSINE KINASE

TECHNICAL FIELD

[0001] The present invention relates to a novel class of sphingosine kinase (SK) inhibitors useful in the treatment of cancer.

PRIORITY DOCUMENT

[0002] The present application claims priority from Australian Provisional Patent Application No 2014902751 titled "Anti-cancer compounds" filed on 16 July 2014, the content of which is hereby incorporated by reference in its entirety.

BACKGROUND

[0003] In the last three decades, a number of 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). 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, however, 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.

[0004] The cellular levels of the sphingolipids are controlled by an array of bidirectional metabolic pathways that are subject to complex spatial and temporal regulation that controls flux in both directions (Pitson, 201 1; and Hannun and Obeid, 2008). Some of the most important regulators of this system are the sphingosine kinases (SKs), which, through their action of phosphorylating sphingosine to generate SIP, play a vital role in controlling the sphingolipid rheostat (Pitson, 201 1), and therefore, cell fate. Two SKs exist in mammals; SKI and SK2, which catalyse the same reaction and share a high degree of sequence similarity (Neubauer and Pitson, 2013). 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).

[0005] The 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. For example, 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). Similarly, 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).

[0006] Initial SK inhibitor development has utilised molecules derived from sphingosine including L- /Areo-dihydrosphingosine (DHS: safingol), N,N-dimethylsphingosine (DMS) and Ν,Ν,Ν- trimethylsphingosine (TMS). These compounds, however, have low specificity, with several important off-targets identified, such as protein kinase C, ceramide synthase and the 14-3-3 pro-survival protein (reviewed in Pitman and Pitson, 2010). Other inhibitors have been developed to selectively target the sphingosine-binding pockets of SKI and SK2 and have been successful in blocking cancer cell growth and reducing tumour burden in animal models (Paugh et al, 2008; Nagahashi et al, 2012; and Antoon et al, 2012).However, only one of these more selective inhibitors has reached clinical trials, with the SK2- selective inhibitor ABC294640 (ie 3-(4-chlorophenyl)-adamantane-l-carboxylic acid (pyridin-4- ylmethyl)amide)currently undergoing Phase I trials for treatment of advanced solid tumours.

[0007] Notably, some of the high affinity sphingosine-competitive SKI inhibitors recently developed have been controversial. That is, despite showing potent SKI inhibition in vitro and decreases in SIP in cells, these inhibitors failed to induce apoptosis or show anti-neoplastic properties in vivo (Kennedy et al, 2011 ; Kharel et al, 2011 ; and Rex et al, 2013). This has led to the groups that developed these reagents to reach the contentious conclusion that SK activity is not required for tumour cell viability (Rex et al, 2013), despite the large body of evidence to the contrary (reviewed in Heffeman-Stroud and Obeid, 2013; Pitman and Pitson, 2010; and Hannun and Obeid, 2008). Also, unlike other SK inhibitors or SK knockdown technology, where examined, these recent inhibitors failed to enhance cellular ceramide levels at concentrations where SKI was inhibited, suggesting that their similarity to sphingosine may result in non-specific effects on, for example, ceramide synthase. [0008] The present applicants have now identified a novel class of SK inhibitors through the use of a structure-based approach to target the ATP-binding pocket of SKI . This approach, it is believed, has exploited the known divergence of the SK ATP-binding site from other kinases (Pitson et al., 2002) thereby leading to a class of SK inhibitors (named benzene sulfonamide-based inhibitors) that show higher selectivity to SKI and SK2 over other kinases. In turn, this may mean that inhibitors of this class may overcome at least some of the off-target effects of sphingosine-like molecules (Pitman and Pitson, 2010; and Woodcock, 2006). Studies conducted on a representative compound of this class, 4-methyl-N- [2-[[2-[(4-ethylphenyl)sulfonylamino]phenyl]iminomethyl]phenyl]benzenesulfonamide (designated herein as MP-A08) with a panel of cancer cell lines in vitro as well as xenograft models of human cancer in vivo, revealed that this class of SK inhibitors possess a promising level of anti-neoplastic effects for use in the treatment of cancer.

SUMMARY

[0009] 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:

wherein,

X¹ is H or otherwise selected from the groups:

each of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³, R²⁴, R²⁵, R²⁶, R²⁷, R²⁸, R²⁹, R³⁰, R³¹ and R³²is independently selected from the group consisting of: H, halogen, OH, N0₂, CN, NH₂, optionally substituted C₁-C₁₂alkyl, optionally substituted C₂-C₁₂alkenyl, optionally substituted C₂-C₁₂alkynyl, optionally substituted C₂-C₁₂heteroalkyl, optionally substituted C₃- C₁₂cycloalkyl, optionally substituted C₂-C₁₂heterocycloalkyl, optionally substituted C₂- C₁₂heterocycloalkenyl, optionally substituted C₆-C₁₈ aryl, optionally substituted C₂-C₁₈heteroaryl, optionally substituted C₁-C₁₂alkyloxy, optionally substituted C₂-C_]2alkenyloxy, optionally substituted C₂- C₁₂alkynyloxy, optionally substituted C₂-C₁₂heteroalkyloxy, optionally substituted C₃-C₁₂cycloalkyloxy, optionally substituted C₃-C₁₂cycloalkenyloxy, optionally substituted C₁-C₁₂heterocycloalkyloxy, optionally substituted C₂-C₁₂heterocycloalkenyloxy, optionally substituted C₆-Ci₈aryloxy, optionally substituted C₂-C₁₈heteroaryloxy, optionally substituted C_rC₁₂alkylamino, SR³³, S0₃H, S0₂NH₂, S0₂R³³, SONH₂, SOR³³, COR³³, COOH, COOR³³, CONR³³R³⁴, NR³³COR³⁴, NR³³COOR³⁴, NR³³S0₂R³⁴, NR³³CONR³⁴R³⁵, NR³³R³⁴, and acyl, wherein R³³, R³⁴ and R³⁵ are each independently selected from the group consisting of H, C₁-C₁₂alkyl, Ci-Cnhaloalkyl, C₂-C₁₂alkenyl, C₂-C₁₂alkynyl, C₂-C₁₀heteroalkyl, optionally substituted C₃-C₁₂cycloalkyl, optionally substituted C₃-C₁₂cycloalkenyl, optionally substituted C₂-C₁₂heterocycloalkyl, optionally substituted C₂-C₁₂heterocycloalkenyl, optionally substituted C₆- C₁₈aryl, optionally substituted C₂-C₁₈heteroaryl, and acyl, or are such that any two or more of R³³, R³⁴ and R³⁵, when taken together with the atoms to which they are attached form a heterocyclic ring system with 3 to 12 ring atoms, or in the case of R¹⁵ and R¹⁶ and also R²⁷ and R²⁸may be collectively carbonyl (=0), or in the case of R²⁰ and R²¹ may be taken together with the carbon atoms to which R²⁰ and R²¹ are attached such that they form a (fused) C₂-C₈ cycloalkyl, C₂-C₈ heterocycloalkyl, aryl or any heteroaryl ring fused at the carbon atoms to which R²⁰ and R²¹ are attached; or a pharmaceutically acceptable salt or prodrug thereof.

[0010] In a second aspect, 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. [001 1] In a third aspect, 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.

[0012] In a fourth aspect, 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.

[0013] Further, in a fifth aspect, 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.

[0014] Still further, in a sixth aspect, 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.

BRIEF DESCRIPTION OF FIGURES

[0015] 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;

[0016] 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). Empty vector (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;

[0017] Figure 3A shows the effect of MP-A08 on cellular S 1 P formation determined in Jurkat cells and presented as % vehicle control. Values are mean ± SD (n=4). Significance was determined by student t- test (*p< 0.05). B provides the results of mass spectrometric analysis of sphingolipids from Jurkat cells treated with MP-A08 (black bars) or vehicle control (grey bars) for 6 h (top) or 16 h (bottom).SlP levels in cell lysates were determined by SIP ELISA. Significance was determined by student t-test (*p< 0.05, **p< 0.01 and ***p< 0.001) (n=3). C shows results indicating that MP-A08 alters survival and proliferative signalling. Jurkat cells were treated with varying doses of MP-A08 (10-20μΜ) for 6 h. Lysates were resolved by SDS-PAGE and analysed by immunoblotting with antibodies against p- AKT(Ser473), p-ERKl/2, p-p38, and p-JNK (Cell Signaling Technology, Inc, Danvers, MA, United States of America). Actin was used as a loading control. Results are representative of two independent experiments;

[0018] Figure 4 A Jurkat cells were treated with vehicle, 10 or 15 μΜ MP-A08 for 5 h and then assessed for caspase 3 activity (mean ± SD, n=3; *p< 0.05 by Student's t-test). B Jurkat cells were treated with vehicle or MP-A08 (5-20 μΜ) for 6 h. Lysates were resolved by SDS-PAGE and analysed for caspase 3 and PARP cleavage by immunoblotting with antibodies against caspase 3 and PARP, with tubulin used as a loading control. C Jurkat (black bars) or Bcl2-expressing Jurkat cells (grey bars) were treated with MP- A08 (5-15 μΜ)θΓ vehicle control for 24 h, and then stained with Annexin V to assess apoptosis. Values are represented as % of no treatment control (mean ± SD, n=3; *p< 0.05 by Student's t-test). 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. E Jurkat cells were treated with MP-A08 or vehicle control for 5 h and stained with TMRE to test for changes in mitochondrial permeability (mean ± SD, n=3; *p< 0.05 by Student's t-test). F Mouse embryonic fibroblasts from wild- type (circles) or SK1/SK2 double knockout mice (squares) were treated with MP-A08 or vehicle control overnight. Cells were stained with DAPI and assessed for apoptosis by visualising nuclear condensation using confocal microscopy. Values are mean ± SD (n=3). G Growth of parental BJ1 (□) and transformed BJ7 (■) fibroblasts were determined by MTS assay with varying concentrations ( 1.25-50mM) of MP-A08 (48 h treatment). Values are displayed as % vehicle control, mean ± SD (n=3-4);

[0019] 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). Mean tumour volumes ± SEM are shown (n=8 per group).

Statistical analysis was performed by two-way ANOVA. B After two weeks of treatment with MP-A08 or vehicle control, tumours were excised and weighed (n=8 per group^/KO-OSby Student's t-test).C MP- A08 reduces tumour angiogenesis. Representative CD31 -stained tumour sections from MP-A08 or vehicle control-treated mice were analysed and CD31 positive vessels per field of view were quantitated (n=8 per group; */K0.05 by Student's t-test). D SIP/dhSIP levels in the tumour homogenates were determined by SIP ELISA. Values are represented as mean+SD (n=4-5),*/><0.05 by Student's t-test; [0020] Figure 6A Growth of glioblastoma cells was determined by MTS assay with vehicle control, doxorubicin alone (0.5 mM), MP-A08 treatment alone (ImM), or MP-A08 treatment (ImM) in combination with doxorubicin (0.5 mM) after 48 h treatment. Values are displayed as % vehicle control, mean ± SD (n=3). B provides results showing that MP-A08 reduces glioblastoma multiforme tumour growth in mice. Mice bearing U-87 xenografts were treated with i.p. with lOOmg/kg MP-A08 or vehicle control at the times indicated (arrows). Mean tumour volumes ± SEM are shown (n=8 per group).

Statistical analysis was performed by two-way ANOVA;

[0021 ] 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);

[0022] Figure 8 provides results showing that MP-A08(5uM) sensitised AML cell lines to killing by cytarabine (ΙΟηΜ) as measured by Annexin V positive cells. *Combinatorial Index < 1 indicating synergism;

[0023] Figure 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). *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;

[0024] Figure 10 provides graphical results demonstrating that MP-A08 chemosensitises AML leukaemic stem/progenitor cells to cytarabine; and

[0025] 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.

DETAILED DESCRIPTION

[0026] In a first aspect, the present invention provides the use of a compound according to formula I for treating cancer or another proliferative cell condition in a subject:

wherein,

X¹ is H or otherwise selected from the groups:

; wherein

each of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³, R²⁴, R²⁵, R²⁶, R²⁷, R²⁸, R²⁹, R³⁰, R³¹ and R³²is independently selected from the group consisting of: H, halogen, OH, N0₂, CN, NH₂, optionally substituted d-Cnalkyl, optionally substituted C₂-C₁₂alkenyl, optionally substituted C₂-C₁₂alkynyl, optionally substituted C₂-C₁₂heteroalkyl, optionally substituted C₃- C₁₂cycloalkyl, optionally substituted C₂-C₁₂heterocycloalkyl, optionally substituted C₂- C₁₂heterocycloalkenyl, optionally substituted C₆-C₁₈ aryl, optionally substituted C₂-Ci₈heteroaryl, optionally substituted C₁-C₁₂alkyloxy, optionally substituted C₂-C₁₂alkenyloxy, optionally substituted C₂- C₁₂alkynyloxy, optionally substituted C₂-C₁₂heteroalkyloxy, optionally substituted C₃-C₁₂cycloalkyloxy, optionally substituted C₃-C₁₂cycloalkenyloxy, optionally substituted CrC₁₂heterocycloalkyloxy, optionally substituted C₂-C₁₂heterocycloalkenyloxy, optionally substituted C₆-Ci_garyloxy, optionally substituted C₂-C₁₈heteroaryloxy, optionally substituted C₂-C₁₂alkylamino, SR³³, S0₃H, S0₂NH₂, S0₂R³³, SONH₂, SOR³³, COR³³, COOH, COOR³³, CONR³³R³⁴, NR³³COR³⁴, NR³³COOR³⁴, NR³³S0₂R³⁴, NR³³CONR³⁴R³⁵, NR³³R³⁴, and acyl, wherein R³³, R³⁴ and R³⁵ are each independently selected from the group consisting of H, C₁-C₁₂alkyl, Ci-Cnhaloatkyl, C₂-C₁₂alkenyl, C₂-C₁₂alkynyl, C₂-Cio heteroalkyl, optionally substituted C₃-C₁₂cycloalkyl, optionally substituted C₃-C_]2cycloalkenyl, optionally substituted C₂-C₁₂heterocycloalkyl, optionally substituted C₂-C₁₂heterocycloalkenyl, optionally substituted C₆- Cigaryl, optionally substituted C₂-Ci₈heteroaryl, and acyl, or are such that any two or more of R³³, R³⁴ and R³⁵, when taken together with the atoms to which they are attached form a heterocyclic ring system with 3 to 12 ring atoms, or in the case of R¹' and R¹⁶ and also R²⁷ and R²⁸ may be collectively carbonyl (=0), or in the case of R²⁰ and R²¹ may be taken together with the carbon atoms to which R²⁰ and R²¹ are attached such that they form a (fused) C₂-C₈ cycloalkyl, C₂-C₃ heterocycloalkyl, aryl or any heteroaryl ring fused at the carbon atoms to which R²⁰ and R²¹ are attached; or a pharmaceutically acceptable salt or prodrug thereof.

[0027] In a second aspect, 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.

[0028] In a third aspect, 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.

[0029] In a fourth aspect, 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.

[0030] Further, in a fifth aspect, 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.

[0031] Still further, in a sixth aspect, 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.

[0032] In this specification, a number of terms are used which are well known to those skilled in the art. Nevertheless, for the purposes of clarity, a number of these terms will be defined.

[0033] 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. In certain embodiments, the substituent groups are one or more groups independently selected from the group consisting of halogen, =0, =S, -CN, -N0₂, -CF₃, -OCF₃, alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, heteroarylalkyl, arylalkyl, cycloalkylalkenyl, heterocycloalkylalkenyl, arylalkenyl, heteroarylalkenyl, cycloalkylheteroalkyl, heterocycloalkylheteroalkyl, arylheteroalkyl, heteroarylheteroalkyl, hydroxy, hydroxyalkyl, alkyloxy, alkyloxyalkyl, alkyloxycycloalkyl, alkyloxyheterocycloalkyl, alkyloxyaryl, alkyloxyheteroaryl, alkyloxycarbonyl, alkylaminocarbonyl, alkenyloxy, alkynyloxy, cycloalkyloxy, cycloalkenyloxy, heterocycloalkyloxy, heterocycloalkenyloxy, aryloxy, phenoxy, benzyloxy, heteroaryloxy, arylalkyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, alkylsulfinyl, arylsulfinyl, aminosulfinylaminoalkyl, -C(=0)OH, -C(=0)R\ -C(=0)OR^a, C(=0)NR^aR^b, C(=NOH)R^a,

C(=NR^a)NR^bR^c, NR^aR^b, NR^aC(=0)R^b, NR^aC(=0)OR^b, NR^aC(=0)NR^bR^c, N R^aC(= N R^b) N R°R^d , NR^aS0₂R^b,-SR^a, S0₂NR^aR^b, -OR^a, OC(=0)NR^aR^b, OC(=0)R^a and acyl, wherein R^a, R^b, R^c and R^d are each independently selected from the group consisting of H, Ci-C^alkyl, C₁-C₁₂haloalkyl, C₂-C₁₂alkenyl, C₂-C₁₂alkynyl, C₂-C₁₀ heteroalkyl, C₃-C₁₂cycloalkyl, C₃-C ₁₂cycloalkenyl, C₂-C₁₂heterocycloalkyl, C₂- C₁₂heterocycloalkenyl, C₆-C₁₈aryl, C₂-C₁₈heteroaryl, and acyl, or any two or more of R^a, R^b, R^c and R^d, when taken together with the atoms to which they are attached form a heterocyclic ring system with 3 to 12 ring atoms. In certain embodiments, each optional substituent is independently selected from the group consisting of: halogen, =0, =S, -CN, -N0₂, -CF₃, -OCF₃, alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, hydroxy, hydroxyalkyl, alkyloxy, alkyloxyalkyl, alkyloxyaryl, alkyloxyheteroaryl, alkenyloxy, alkynyloxy, cycloalkyloxy, cycloalkenyloxy, heterocycloalkyloxy, heterocycloalkenyloxy, aryloxy, heteroaryloxy, arylalkyl, heteroarylalkyl, arylalkyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, aminoalkyl, -COOH, -SH and acyl. Examples of particularly suitable optional substituents include F, CI, Br, I, CH₃, CH₂CH₃, OH, OCH₃, CF₃, OCF₃, N0₂, NH₂ and CN. Alternatively, 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. Accordingly, the term "optionally substituted" includes a fused ring such as a cycloalkyl ring, a heterocycloalkyl ring, an aryl ring or a heteroaryl ring.

[0034] In the definitions of a number of the substituents provided below, it is stated that "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. Using the term "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.

[0035] "Acyl" means an R-C(=0)- group in which the R group may be an alkyl, cycloalkyl,

heterocycloalkyl, aryl or heteroaryl group as defined herein. Examples of 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.

[0036] "Acylamino" means an R-C(=0)-NH- group in which the R group may be an alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl group 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 nitrogen atom.

[0037] "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.

[0038] "Alkenyloxy" refers to an alkenyl-O- group in which alkenyl is as defined herein. Preferred alkenyloxy groups are C₂- C₆ 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.

[0039] "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 C₁-C₆ alkyl unless otherwise noted. Examples of suitable straight and branched C₁-C₆ 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.

[0040] "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)₂N- 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 C₁-C₆ 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. [0041] "Alkylaminocarbonyl" refers to a group of the formula (Alkyl)_x(H)_yNC(=0)- in which alkyl is as defined herein, x is 1 or 2, and the sum of X+Y =2. 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.

[0042] "Alkyloxy" refers to an alkyl-O- group in which alkyl is as defined herein. Preferably the alkyloxy is a C|-C_fialkyloxy. Examples include, but are not limited to, methoxy and ethoxy. The group may be a terminal group or a bridging group.

[0043] "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.

[0044] "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.

[0045] "Alkyloxycarbonyl" refers to an alkyl-0-C(=0)- group in which alkyl is as defined herein. The alkyl group is preferably a C₁-C₆ 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.

[0046] "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.

[0047] "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.

[0048] "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.

[0049] "Alkylsulfinyl" means an alkyl-S-(=0)- group in which alkyl is as defined herein.. The alkyl group is preferably a C₁-C₆ 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.

[0050] "Alkylsulfonyl" refers to an alkyl-S(=0)2- group in which alkyl is as defined above. The alkyl group is preferably a C_rC₆ 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.

[0051] "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.

[0052] "Alkynyloxy" refers to an alkynyl-O- group in which alkynyl is as defined herein. Preferred alkynyloxy groups are C₂-C₆ 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.

[0053] "Aminoalkyl" means an NH₂-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.

[0054] "Aminosulfonyl" means an NH₂-S(=0)₂- 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.

[0055] "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. Examples of aryl groups include phenyl, naphthyl and the like; (ii) an optionally substituted partially saturated bicyclic aromatic carbocyclic moiety in which a phenyl and a C₅. C₇ cycloalkyl or C₅-C₇ 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. Typically an aryl group is a C₆-Ci₈ aryl group.

[0056] "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. [0057] "Arylalkyl" means an aryl-alkyl- group in which the aryl and alkyl moieties are as defined herein. Preferred arylalkyl groups contain a C₁-C₆ alkyl group. Exemplary 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.

[0058] "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.

[0059] "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)₂N- 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.

[0060] "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.

[0061] "Aryloxy" refers to an aryl-O- group in which the aryl is as defined herein. Preferably, the aryloxy is a Ce-Cjgaryloxy, and more preferably a C₆-Ci₀aryloxy. 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.

[0062] "Arylsulfonyl" means an aryl-S(=0)2- group in which the aryl 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 sulfur atom.

[0063] 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.

[0064] "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. [0065] "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.

[0066] "Cycloalkylalkyl" means a cycloalkyl-alkyl- group in which the cycloalkyl and alkyl moieties are as defined herein. Exemplary monocycloalkylalkyl groups include cyclopropylmethyl,

cyclopentylmethyl, cyclohexylmethyl and cycloheptylmethyl. 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.

[0067] "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.

[0068] "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.

[0069] "Cycloalkyloxy" refers to a cycloalkyl-O- group in which cycloalkyl is as defined herein.

Preferably the cycloalkyloxy is a C₁-C₆ 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.

[0070] "Cycloalkenyloxy" refers to a cycloalkenyl-O- group in which the cycloalkenyl is as defined herein. Preferably the cycloalkenyloxy is a C₃-C₈ 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.

[0071 ] "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. Examples of haloalkyl include fluoromethyl, difluoromethyl and trifluoromethyl.

[0072] "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. [0073] "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.

[0074] "Halogen" refers to fluorine, chlorine, bromine or iodine.

[0075] "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 R¹ is selected from the group consisting of H, optionally substituted d-Cualkyl, optionally substituted Gs-Cucycloalkyl, optionally substituted C₆-C-igaryl, and optionally substituted C₂- Cisheteroaryl. Exemplary heteroalkyls include alkyl ethers, secondary and tertiary alkyl amines, amides, alkyl sulfides, and the like. Examples of heteroalkyl also include hydroxyC₁-C₆alkyl, d-Qalkyloxy Cr C₆alkyl, amino Cj-Qalkyl, C₁-C₆alkylaminoC₁-C₆alkyl, and di(C|-C₆alkyl)amino CrQalkyl. The group may be a terminal group or a bridging group.

[0076] "Heteroalkyloxy" refers to a heteroalkyl-O- group in which heteroalkyl is as defined herein. Preferably the heteroalkyloxy is a C2-C₆heteroalkyloxy. The group may be a terminal group or a bridging group.

[0077] "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. Examples of heteroaryl 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, oxazole, isooxazole,l,2,3-oxadiazole, 1 ,2,4-oxadiazole, 1,2,3-triazole, 1,2,4-triazole, 1 ,2,4-thiadiazole, 1,3,4-thiadiazole, furazane, phenoxazine, 2-, 3- or 4- pyridyl, 2-, 3-, 4-, 5-, or 8- quinolyl, 1-, 3-, 4-, or 5-isoquinolinyl 1-, 2-, or 3- indolyl, and 2-, or 3-thienyl. A heteroaryl group is typically a Q-C^ heteroaryl group. The group may be a terminal group or a bridging group.

[0078] "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. Exemplary

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. [0079] "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.

[0080] "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.

[0081] "Heteroaryloxy" refers to a heteroaryl-O- group in which the heteroaryl is as defined herein. Preferably, the heteroaryloxy is a d. Cis heteroaryloxy. 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.

[0082] "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. Examples of heterocyclic moieties include heterocycloalkyl, heterocycloalkenyl and heteroaryl.

[0083] "Heterocycloalkenyl" refers to a heterocycloalkyl group as defined herein but containing at least one double bond. A heterocycloalkenyl group typically is a C₂-C₁₂ heterocycloalkenyl group. The group may be a terminal group or a bridging group.

[0084] "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.

Examples of suitable heterocycloalkyl substituents include pyrrolidyl, tetrahydrofuryl,

tetrahydrothiofuranyl, piperidyl, piperazyl, tetrahydropyranyl, morphilino, 1,3-diazapane, 1,4-diazapane, 1,4- oxazepane and 1,4-oxathiapane. A heterocycloalkyl group typically is a C2-C12 heterocycloalkyl group. The group may be a terminal group or a bridging group.

[0085] "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.

[0086] "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.

[0087] "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.

[0088] "Heterocycloalkyloxy" refers to a heterocycloalkyl-O- group in which the heterocycloalkyl is as defined herein. Preferably the heterocycloalkyloxy is a C_r C₆heterocycloalkyloxy. 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.

[0089] "Heterocycloalkenyloxy" refers to a heterocycloalkenyl-O- group in which heterocycloalkenyl is as defined herein. Preferably the heterocycloalkenyloxy is a C_r C₆ 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.

[0090] "Hydroxyalkyl" 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_nH(2n+i. x)(OH)_x. In groups of this type, n is typically from 1 to 10, more preferably from 1 to 6, most preferably 1 to 3, and x is typically 1 to 6, more preferably 1 to 3.

[0091] "Sulfinyl" means an R-S(=0)- group in which the R group may be OH, alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl group 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 sulfur atom.

[0092] "Sulfinylamino" means an R-S(=0)-NH- group in which the R group may be OH, alkyl, cycloalkyl, heterocycloalkyl; aryl or heteroaryl group 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 nitrogen atom.

[0093] "Sulfonyl" means an R-S(=0)₂- group in which the R group may be OH, alkyl, cycloalkyl, heterocycloalkyl; aryl or heteroaryl group 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 sulfur atom. [0094] "Sulfonylamino" means an R-S(=0)₂-NH- 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.

[0095] 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.

[0096] Additionally, formula I is intended to cover, where applicable, solvated as well as unsolvated forms of the compounds. Thus, formula I includes compounds having the indicated structure, including the hydrated as well as the non-hydrated forms.

[0097] The term "pharmaceutically acceptable salt" as used herein 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.

[0098] In the case of compounds of formula I that are solid, it will be understood by those skilled in the art that the compounds (or pharmaceutically acceptable salts thereof) may exist in different crystalline or polymorphic forms, all of which are intended to be within the scope of the present invention.

[0099] "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). For example, 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. As another example, 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. Examples of ester prodrugs include those described by Leinweber FJ, 1987. Similarly, 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.

[00100] The term "therapeutically effective amount" or "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. Typically, a therapeutically effective amount is sufficient to palliate, ameliorate, stabilise, reverse, slow or delay the progression of the cancer disease or proliferative state. By way of example only, 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. However, notwithstanding the above, it will be understood by those skilled in the art that 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.

[00101] 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). As mentioned above, 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. As such, 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.

[00102] In some embodiments, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹are H.

[00103] In some embodiments, R³ is CH₃.

[00104] In some embodiments, R⁸ is NR³³S0₂R³⁴, wherein R³³ and R³⁴ are each independently selected from the group consisting of H, Cj-Cnalkyl, C₁-C₁₂haloalkyl, C₂-C₁₂alkenyl, C₂-C₁₂alkynyl, C₂-C₁₀ heteroalkyl, optionally substituted C₃-C₁₂cycloalkyl, optionally substituted C₃-C ₁₂cycloalkenyl, optionally substituted C₂-C₁₂heterocycloalkyl, optionally substituted C₂-C₁₂heterocycloalkenyl, optionally substituted C₆-C₁₈aryl, optionally substituted C₂-C₁₈heteroaryl, and acyl. However, preferably R³³ is H and R³⁴ is optionally substituted C₆ cycloalkyl.

100105] In some embodiments, X¹ is H. In such embodiments, it may be preferred that each of R¹ , R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ and R⁹is independently selected from H, CH₃ and NH₂. For example, one or both of R and R may beCH₃.

[00106] In some embodiments, X'is selected from the groups:

[00107] In some embodiments of group II, one or more of R¹⁰, R¹¹, R¹², R¹³ and R¹⁴ are H.

100108] In some embodiments of group II, one or more of R¹⁰, R¹ R¹², R¹³ and R¹⁴ are halogen.

100109] In some embodiments of group II, R¹² is selected from H, CH₃, OCH₃, N0₂, and N(CH₃)₂.

[001 10] In some embodiments of group II,R is N0₂ or NHS0₂R wherein R ' is selected from the group consisting of H, C_! -C₆alkyl, Ci -C₆haloalkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₂-Ci₀ heteroalkyl, optionally substituted C₃-C₆cycloalkyl, optionally substituted C₃-C₆cycloalkenyl, optionally substituted C₂-C₆heterocycloalkyl, optionally substituted C₂-C₆heterocycloalkenyl, optionally substituted C₆-C₁₈aryl, optionally substituted C₂-C₁₈heteroaryl, and acyl. In a preferred form of such embodiments, R¹⁸ is C₃- Qcycloalkyl optionally substituted with one or more alkyl groups (preferably CH₃).

001 1 1 ] In some embodiments of group III, one or more of R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹ and R²²are H.

[001 12] In some embodiments of group III, R¹⁵ and R¹⁶ are collectively carbonyl (=0). [00113] In some embodiments of group III, R²⁰ and R²¹ taken together with the carbon atoms to which R²⁰ and R²¹ are attached form a (fused) C₂-C₈cycloalkyl, C₂-C₈heterocycloalkyl, aryl or any heteroaryl ring fused at the carbon atoms to which R²⁰ and R²¹ are attached.

[00114] In some embodiments of group IV, one or more of R²³, R²⁴, R²⁵, R²⁶, R²⁷ and R²⁸ are H.

[00115] In some embodiments of group IV, R²⁴ is selected from H, halogen, OCH₃ and N0₂.

[00116] In some embodiments of group IV, each of R²³, R²⁵ and R²⁶ is independently selected from H, halogen and OCH₃.

[00117] In some embodiments of group IV, R²⁷ and R²⁸ are collectively carbonyl (=0). [00118] In some embodiments of group IV, R²⁶ is CH₂CH=CH₂.

[00119] In some embodiments of group V, R²⁹ is selected from C₃-C₅ heterocycloalkyl, C₃-C₅ heterocycloalkenyl and C$ heteroaryl, more preferably C₄-C₅ heterocycloalkyl comprising one or more heteroatoms selected from O, N and S.

[00120] In some embodiments of group VI, R³⁰ and R³¹ are collectively carbonyl (=0).

[00121] In some embodiments of group VI, R³² is selected from H and OH.

[00122] In some particularly preferred embodiments, X¹ is selected from the following:

0123] In specific embodiments, the compound is selected from:

(MP-A08)

(MP-A08.9) F F

(MP-A08.10)

(MP-A08.11)

(MP-A08.12)

(MP-A08.14)

(MP-A08.16)

(MP-A08.17)

[00124] 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.

[00125] Particularly preferred compounds of formula I may also include those compounds displaying inhibition of SKI characterised by an IC₅o value of less than 10, more preferably, less than 7 (as measured by, for example, SK assays as described herein). [00126] Particularly preferred compounds of formula I may also include those compounds displaying inhibition of SKI characterised by an IC₅₀ value that is at least a 3-fold improvement (ie increased inhibition) than the IC₅₀ value of MP-A08 (as measured by a suitable SK assays such as, for example, SK assays described herein).

[00127] Compounds of formula I (and pharmaceutically acceptable salts and prodrugs thereof)may be administered in combination with one or more additional agent(s) for the treatment of cancer. For example, 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) As such, the compounds of formula I may be used in combination with one or more of the following categories of anti-cancer agents:

• other anti-proliferative/antineoplastic drugs and combinations thereof, as used in medical

oncology, such as 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) and taxoids (eg taxol and taxotere; and 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;

• 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;

• inhibitors of growth factor function(eg the anti-erbB2 antibody trastuzumab (Herceptin™), 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-moφholinopropoxy)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)-quinazolin-4-amine (CI 1033), erbB2 tyrosine kinase inhibitors such as lapatinib); inhibitors of the hepatocyte growth factor family; inhibitors of the insulin growth factor family; inhibitors of the platelet-derived growth factor family such as imatinib and/or nilotinib (AMN107); inhibitors of serine/threonine kinases (eg Ras/Raf signalling inhibitors such as farnesyl transferase inhibitors, inhibitors of cell signalling through MEK and/or AKT kinases, c-kit inhibitors, abl kinase inhibitors, PI3 kinase inhibitors, Plt3 kinase inhibitors, CSF-1R kinase inhibitors, IGF receptor (insulin-like growth factor) kinase inhibitors; and cyclin dependent kinase inhibitors such as CDK2 and/or CDK9 inhibitors; and

• antiangiogenic agents such as those which inhibit the effects of vascular endothelial growth factor (eg the anti-vascular endothelial cell growth factor antibody bevacizumab (Avastin™) and the VEGF receptor tyrosine kinase inhibitor vandetanib (ZD6474)).

[00128] Where used in combination with other anti-cancer agents, 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)).

[00129] The present invention is typically applied to the treatment of cancer or another proliferative cell condition in a human subject. However, 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).

[00130] 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 cancer, sarcomas (including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma), skin cancer (including melanoma, Kaposi's sarcoma, basocellular cancer, and squamous cell cancer), testicular cancer (including germinal tumours such as seminoma, non-seminoma teratomas, and choriocarcinomas), stromal tumours, germ cell tumours, thyroid cancer (including thyroid adenocarcinoma and medullar carcinoma), and renal cancer (including adenocarcinoma and Wilms¹ tumour).

[0013 ] ] In some embodiments of the fourth aspect, the method invlves modulating S 1 P-receptor mediated cell signalling and/or intracellular SIP cell signalling.

[00132] In some embodiments of the fifth aspect, 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.

[00133] In some embodiments of the sixth aspect, 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

[00127].

[00134] The invention is hereinafter described with reference to the following non-limiting examples and accompanying figures.

EXAMPLE(S)

Example 1 Identification of specific ATP-competitive inhibitors of sphingosine kinase

Materials and Methods

[00135] Prediction of SK structures by homology modelling

A multiple sequence alignment of human DAGKa (Genbank Accession No P23743.2), human DAGKz (NP_963290.1), human CERK (NP_073603.2), Escherichia coli YEGs (P76407.1), Staphylococcus aureus DgkB (PDB code 2QV7) and human SKla (Q9NYA1) was performed using ClustalW

(Thompson et ah, 1994). This alignment was used as input for Modeller 9v6 (Sali et al., 1995). DgkB (2QV7) and YegS (2JGR) structures were used as templates for homology modelling. Due to poor homology with the template structures, residues 1-12, 162-189 and 203-230 of human SKI were excluded from the SKI model. The model was optimised using 50 cycles of Refmac structure idealisation

(Collaborative Computational Project 4, 1994). The CCP4 software suite was used for model assessment and structural alignments using Procheck and Superpose modules, respectively (Collaborative

Computational Project 4, 1994). Figures were produced using the Pymol graphics program (DeLano, 2002). The structural model of human SK2 was produced as described above using the human SKI crystal structure (Z. Wang et al, 2013) bound with ADP (3VZD) as a template. Due to large insertions in the SK2 sequence (NP_001 191089), residues 1-134, 355-470 and 362-370 were excluded from the SK2 model.

[00136] In silico molecular screening

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. For in 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. Default high stringency docking parameters were used with a minimum anchor size of two atoms and scored with chemical matching. All compounds that docked at high stringency were assessed by score, rank and visual assessment. Candidates were chosen for biological testing and were sourced from the NCI/DTP Open Chemical Repository.

[00137] Chemical Synthesis

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:

2 3

Step 1. N-(2-aminophenyl)-4-methylbenzenesulfonamide (1)

To a solution of 1,2-phenilenediamine (25g, 0.23 mol) in pyridine (100 mL), tosyl chloride (47g, 0.23 mol) was added for 10 min. The reaction mixture was then stirred overnight and evaporated. The residue was suspended with cold water (350 mL) and filtered off. The precipitate was washed with an additional 100 ml of cold water, dried on air and recrystallised from 2-propanol (100 ml). The yield of compound 1: 52g (79%).

Step 2. Methyl 2-{[(4-methylphenyl)sulfonyl]amino}benzoate (2)

To a solution of methyl antranylate (25g, 0.17 mol) in pyridine (100 mL), tosyl chloride (32g, 0.18 mol) was added for 10 min. The reaction mixture was stirred overnight and was then evaporated. The residue was suspended with cold water (350 mL) and filtered off. The precipitate was washed with an additional 100 ml of cold water, dried on air and recrystallised from 2-propanol (50 ml). Yield of compound 2: 46 g (90%).

Step 3. N-(2-formylphenyl)-4-methylbenzenesulfonamide (3)

To a solution of compound 2 (46g, 0.15 mol) in anhydrous THF (500 mL), solution diisobutylaluminum hydride in THF (150 ml, 0.15 mol) was added dropwise for 30 min with cooling on water bath at RT. The reaction mixture was stirred when TLC showed an absence of starting material and was then evaporated. The residue was diluted with chloroform (500ml) and 5% HC1/H₂0 (500 ml), shaken, then water layer was removed and the organic layer washed by H₂0 (500ml) brine (200ml) dried by Na₂S0₄ and evaporated to give compound 3: 31 g (75%).

Step 4. 4-Methyl-N- {2-[(2- { [(4-methylphenyl)sulfonyl]amino } benzylidene)amino]

phenyl} benzenesulfonamide (4)

To compound l(26g, 0.1 mol) and compound 3(27.5g, 0.1 mol), absolute ethanol was added to the final volume of 200 mL. The reaction mixture was refluxed for 3 hours and cooled to 0°C. The precipitate was filtered, washed with diethyl ether to give compound 4 47 g (60%).

Those skilled in the art will recognise that this synthesis protocol may be readily adapted for the synthesis of other compounds according to formula I.

[00138] Construction of SK mutants

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.

[00139] Cell culture

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 )and 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). Jurkat (human T cell lymphoblast, ATCC#TIB-152) and 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.

[00140] SK1/SK2 double knockout mouse embryonic fibroblasts (MEFs) 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% C0₂. 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.

[00141] Assaying SK ATP-binding pocket mutants for activity

HEK293 cells were seeded in 6-well plates and were transiently transfected using Lipofectamine™ 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 Na₃V0₄, 10 mM NaF, 10 mM β-glycerophosphate, ImM dithiothreitol and protease inhibitor cocktail (F Hoffmann-La Roche Ltd, Basel, Switzerland). Cells were lysed by sonication and diluted in extraction buffer for assays (1 : 1000 final dilution). Expression levels of FLAG-tagged SKI/2 proteins were assessed by SDS-PAGE and anti-FLAG immunoblotting. SKI and SK2 activity was determined using O-erythro- sphingosine and [γ³²Ρ]ΑΤΡ as substrates, as described previously (Pitman et al, 2012a).

[00142] Generation of recombinant proteins in insect cells

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).

[00143] Purified recombinant human SK2 protein with 6xHis and 3xhemagglutinin (HA) tags was generated in Sf9 cells and purified as previously described(Roberts et al, 2004). [00144] Human ceramide kinase cDNA (Genbank Accession No NM_022766) was amplified from human placenta cDNA and cloned into pFastBacl following digestion with BamH/ and EcoRI. Human DAGK A cDNA (DAGKa; NM_201444) was PCR amplified from human foreskin fibroblast cDNA. 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 xlO⁹ 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. Cell pellets were resuspended in Buffer A (50 mM Tris-HCl buffer pH 7.6, 150 mM NaCl, 10% glycerol (w/v), 40 mM imidazole, and protease inhibitors (EDTA-free Complete™, F Hoffmann-La Roche Ltd). 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.

[00145] MP-A08 screening against recombinant kinases

SKI and SK2 activity was determined using D-ery/Aro-sphingosine (solubilised in fatty acid-free bovine serum albumin) and [γ³²Ρ]ΑΤΡ as substrates, as described previously (Pitman et al., 2012a). DAGK assays were performed using β-octylglucoside solubilised dioleoyl-s,n-glycerol (DAG) and

phosphatidylserine (PS), based on methods previously described (Walsh and Bell, 1986). CERK assays were performed as described previously (Wijesinghe et al., 2007). Cross-screening assays were carried out using 20μΜ ATP (0.5μα [γ³²Ρ]ΑΤΡ) 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. For the determination of kinetic constants, 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 50μΜ MP-A08. Kinetic constants were calculated using non-linear regression in GraphPad Prism 5 (GraphPad Software Inc, La Jolla, CA, United States of America).

[00146] Screening of MP-A08 for inhibitory activity against a panel of 140 protein kinases

encompassing the major enzymes within this protein family was performed at the International Centre for Kinase Profiling (University of Dundee, Dundee, Scotland) using methods previously described (Bain et al, 2007).

[00147] Sphingolipid analysis

SIP generation in cells was assessed in Jurkat cells suspended at 5x1 Or¹ cells in 1 ml RPMI containing 0.5% FBS were treated with 15 μΜ MP-A08 or vehicle control (0.06% (v/v) DMSO/0.54% (v/v) ethanol) for 4.5 h at 37°C and 5% C0₂. Cells were labelled with 0.5 μ£ϊ ¾-sphingosine

(PerkinElmer Inc, Waltham, MA, United States of America), incubated for 30 mins at 37°C and 5% C0₂, and then harvested by centrifugation at 1500 x g for 1 min, washed once with cold phosphate-buffered saline. Cells were lysed in 300 ml of acidified methanol (methanol :HC1, 100: 1 , by vol.), and SIP

(including ³H-S1P) extracted by the addition of 300 ml of chloroform, 300 ml of 2M KC1 and 30 μΐ of 3M NaOH. The samples were vortexed and then centrifuged for 13000 x g for 5 min to separate the chloroform and aqueous/methanol phases, which under these alkaline conditions contained the partitioned ³H-sphingosine and ³H-S1P, respectively. ³H-S1P in the upper aqueous/methanol phase was then determined by scintillation counting.

[00148] Sphingolipid mass spectrometric analyses analysis was performed on5 x 10⁶Jurkat cells treated in the same manner as above for 6 h or 16 h with either 15 μΜ 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).

[00149] Assessment of apoptosis. cell viability, and proliferation

Jurkat cells (5 x 10⁵ cells/ml) were treated with vehicle (70% PEG, 0.28% v/v final) or MP-A08 (5-15 μΜ) 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.

[00150] For population cell growth assays, 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).

[00151 ] Colony formation in soft agar

Assays were performed as previously described (Xia et al., 2000) with the following modifications. Cells were prepared in growth media containing 0.33% DMEM-low-melting point agarose (Sigma-Aldrich Inc, St Louis, MO, United States of America) with either MP-A08 or vehicle control. This was overlaid onto 0.5% DMEM-low-melting agarose gel. After 14-21 days, the cells were analysed by light microscopy and the number of colonies was quantified using ImageJ software (Schneider et ah, 2012).

[00152] Toxicity study of MP-A08 in NOD/SCID mice

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).

[00153] A549 xenograft model

A549 cells (5 x 10⁶) 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 70mm³, 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. For immunohistological analysis of vascularisation, 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) at 0.2 μg/ml was incubated overnight at 4°C followed by a 35 min incubation with 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.

[00154] Cross-screening of the SK inhibitory activity of compounds similar to MP-A08

A search for compounds similar to MP-A08 was performed in the Pubchem database

(https://pubchem.ncbi.nlm.nih.gov). Examples of the identified compounds were subsequently sourced from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute (Bethesda, MD, United States of America). The compounds were tested for their ability to inhibit recombinant SKI activity in the manner described above for MP-A08. [00155] Effects on growth of human glioblastoma multiforme xenografts in mice

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- 100mm³. Mice were then treated with MP-A08 at 100 mg/kg, six times a week for two weeks.

[00156] Effects on progression of human acute myeloid leukaemia

Acute myeloid leukaemia (AML) is a heterogeneous disorder arising from the transformation of normal hematopoietic stem and progenitor cells (HSPC) to leukaemic stem and progenitor cells (LSPC) followed by clonal expansion and the accumulation of undifferentiated myeloid blasts in the bone marrow and peripheral blood. Using RT-qPCR assays to quantify levels of SKI mRNA in a small set of primary AML patients (n=56), it was found that in 32 patients that completed standard induction chemotherapy, high SKI mRNA levels correlated with poor patient outcome (data not shown).To examine the effect of inhibiting SKI and SK2 on cell viability in vitro, AML cell lines (ie MOLM13, MV41 1, TF1, UT7, THP1 and ME1) representing various AML subtypes were treated with MP-A08.

[00157] Xeno-transplantation models are widely accepted within the field for the in vivo analysis of therapies that target the clinically relevant LSPC population. In this study, 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.

Results

[00158] Characterisation and validation of the predicted ATP pocket of SKI

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). In the model, residues from all five regions highly conserved in all SKs (designated motifs 1- 5) were involved in forming the ATP-binding pocket of SKI, with the motif comprising residues 79-86 (motif 3) forming the centre of the pocket. To identify the residues that are most important for ATP binding, ATP was computationally docked into the predicted SKI ATP-binding pocket. The docked ATP was predicted to form hydrogen bonds with multiple residues in motifs 1 to 4 (Asn22, Thr54, Gly80, Asp81, Gly82, Leu83, Glu86 and Serl 12). Additionally, 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). [00159] To validate the modelling and ATP docking, 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. Mutation of Asp341 and Glu343 abolished SKI activity, consistent with their proposed role in the coordination of the magnesium ion cofactor. Together, these findings supported the accuracy of the model. Notably, the recent availability of the SKI crystal structure (Z. Wang et al, 2013) also enabled a retrospective assessment of the validity of the model. Structural alignment of the ATP-binding pocket residues of the SKI ATP-binding site model with those of the ADP-bound SKI structure (3VZD;Z. Wang et al, 2013) showed very close alignment (RMSD =1.1366 A),further confirming the validity of the model. Interestingly, the main differences between the model and the SKI structure were in Argl 85 and Argl91, which were excluded from the model because the sequence identity in that region was below the required threshold for homology modelling (<30%). These residues contribute to binding of the b-phosphate of ADP in the crystal structure and were confirmed to contribute to ATP binding as alanine mutations of Argl85 and Argl91 substantially reduced SKI activity.

[00160] 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.

Physical screening of the top candidate compounds was then performed to examine the ability of the compounds to inhibit the activity of purified recombinant SKI in vitro. This identified the "hit molecule"4-methyl-N-[2-[[2-[(4- methylphenyl)sulfonylamino]phenyl]iminomethyl]phenyl]benzenesulfonamide (MP-A08) (Figure 2A) as an inhibitor of SKI :

This compound contains two benzenesulfonamide groups joined by a central benzylidene-aniline group.

[00161] 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. To confirm this orientation of MP-A08 binding, its ability to inhibit the ATP- binding pocket mutants of SKI was assessed. 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. Interestingly, the R24A mutation did not affect SKI activity, but reduced MP-A08 inhibition, which was in agreement with the docking. These findings confirmed that 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.

[00162] To assess its selectivity, MP-A08 was tested against purified recombinant human SKI and SK2, with both enzymes being similarly inhibited. Notably, unlike some other recently developed SK inhibitors (Gustin et al., 2013; and Rex et al., 2013), comparable inhibition of murine SKI and SK2 was also observed (Figure 1). In addition, the inhibitory activity of MP-A08 against the related human lipid kinases, diacylglycerol kinase (DAGK) and ceramide kinase (CERK) was assessed (Figure 1); it was found that MP-A08 showed no inhibition of human DAGK, and only weakly inhibited CERK. To further assess specificity, 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).

Table 1 In vitro screening for the effects of MP-A08 against a panel of protein kinases

[00163] Inhibition kinetics confirmed that MP-A08 was an ATP-competitive inhibitor of human SKI and SK2 (Figure 2 A and 2B). Somewhat surprisingly, MP-A08 was a higher affinity inhibitor of SK2 than SKI, with K, values of 6.9 ± 0.8 mM and 27 ± 3 mM, respectively. In order to analyse these differences in binding to SKI and SK2, a homology model of SK2 was produced using the recent SKI crystal structure and the MP-A08 docked into the predicted SK2 ATP-binding pocket. Comparison of the SKI crystal structure and the SK2 model revealed a highly conserved ATP pocket with the exception of the substitutions at SK2^Phe154 /SKl^and SK2^A"^nl87/SKl^A,g57. In the SKI structure, Arg24 and Thr54 appear to coordinate one of the central phenyl rings of MP-A08 and shield the amine groups from the basic side-chains of Arg24 and Arg57. The second central phenyl ring likely shields the internal amine group from the Argl85 and Argl91 side-chains, pointing the sulfonyl groups towards Asn22, Ser79 and Leu83 in SKI . 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. Compared to the SKI pocket, the central phenyl rings are shifted towards the bottom of the SK2 pocket, coordinated by Arg315 and Arg321. The sulfonyl groups are tethered via hydrogen bonding to Asnl52 and Ser242 side-chains. To validate the SK2 model, alanine mutants of the SK2 ATP pocket predicted to contribute to MP-A08 binding were tested. As was found with the corresponding residues in SKI , SK2 activity was significantly reduced with alanine mutations at Asnl52, Thrl84, Ser242, Arg315 and Arg321 (Figure 2C). Conversely, the F154A mutant displayed a two-fold increase in SK2 activity (Figure 2C), which is likely to be due to the removal of the bulky, hydrophobic side-chain. Next, SK2 mutants were assessed for MP-A08 binding. In agreement with the docking, it was found that alanine mutations at Asnl52 and Thrl84gave some reduction in SK2 inhibition by MP-A08, while mutations at Arg315 and Arg321 resulted in much less inhibition of SK2 by this inhibitor (Figure 2D). As expected, alanine mutation at Phel54 had only a minor effect on SK2 inhibition by MP-A08, as the side-chain is not predicted to directly interact with MP-A08 (Figure 2D). Surprisingly, mutation at Ser242 did not affect MP-A08 binding in SK2 despite the predicted hydrogen bonding interaction of its side-chain hydroxyl. It appears that for SK2, Arg315 and Arg321 tether MP- A08 in the pocket without the involvement of Ser242, whilst in SKI, Serl 12, R185 and R191 all contribute to MP-A08 binding.

[00164] MP-A08 inhibits SIP production in cells and increases pro-apoptotic sphingolipids

To test whether MP-A08 is a cell permeable SK inhibitor, its ability to block SIP generation in cells was examined. Treatment of Jurkat cells with 15 mM MP-A08 significantly reduced cellular SIP generation (Figure 3A), confirming this compound is cell permeable and able to block SK activity in cells. In order to assess the impact of MP- AOS on other cellular sphingolipids, the sphingolipid profile of MP-A08- treated Jurkat cells was analysed (Figure 3B). Significant increases in the levels of dihydrosphingosine, dihydroceramide and all ceramides examined were observed at both 6 and 16 h after cell exposure to MP- A08. Indeed, cells treated with MP-A08 for 6 h displayed significant increases in dihydrosphingosine and all ceramide species compared to vehicle-treated cells, but most notably in C 18-, C20- and C20: 1- ceramide, which displayed 3.7, 3.5 and 5.8-foldincreases, respectively (Figure 3B). At 16 h after MP-A08 treatment, the levels of CI 8-, C20- and C20: 1 -ceramides remained significantly elevated compared to vehicle controls (3.4, 2.8, 3.9-fold, respectively), while levels of C14- and C 18: 1 -ceramide,

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.

[00165] MP-A08 induces mitochondrial-mediated apoptosis

As 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).

[00166] Previous studies have demonstrated that knockdown or inhibition of SKs induces apoptosis in many cell types (Taha et al., 2006; Gao and Smith, 2011 ; and Pitman and Pitson, 2010). Using Jurkat T cell leukaemia cells that have been shown to undergo mitochondrial-mediated apoptosis when treated with the dual SK inhibitor, DMS (Cuvillier et al. 2000), the effects of MP-A08 on induction of apoptosis via an array of apoptosis markers (ie caspase-3 cleavage and activation, PARP cleavage, annexin V staining, and mitochondrial permeability transition by TMRE staining) was investigated (Figure 4A-E). Treatment of cells with 10 and 15 uM MP-A08 caused a dose-dependent induction of caspase 3 activity (Figure 4A). This was confirmed by immunoblotting for caspase 3 and PARP cleavage products, which demonstrated a dose-dependent activation of caspase 3 and inactivation of PARP with MP-A08 treatment (Figure 4B). Consistent with this, MP-A08 also induced cell surface expression of annexin V in a dose- dependent manner (Figure 4C). Further, to verify that MP-A08 was inducing mitochondrial-mediated apoptosis, Jurkat cells overexpressing the anti-apoptotic Bcl2 protein were treated with MP-A08 and assessed for apoptosis. Bcl2 overexpression blocked both MP-A08-induced annexin V staining and PARP cleavage compared to the parental cells (Figure 4C and 4D). MP-A08 treatment of cells also reduced TMRE staining in a dose-dependent manner, indicating mitochondrial membrane permeabilisation consistent with induction of mitochondrial-mediated apoptosis (Figure 4E). [00167] To validate the SKs as the targets of MP-A08-induced apoptosis, the sensitivity of SK 1/SK2 double knockout (dKO) mouse embryonic fibroblasts (MEFs) to MP-A08 treatment was assessed (Figure 4F). Unlike wild-type MEFs, which showed a dose-dependent increase in apoptosis with MP-A08 treatment, dKO MEFs had an elevated basal level of apoptosis that was unaffected by MP-A08 treatment (Figure 4F). These results indicate that MP-A08 induced-apoptosis is mediated by inhibition of the SKs. In order to test the sensitivity of non-cancerous cells compared to transformed/cancerous cells, MP-A08 was tested on fibroblast cell lines BJ1 (non-transformed) and BJ7 (transformed/cancerous). While the transformed BJ7 cells were sensitive to dose-dependent growth inhibition by MP-A08, the untransformed BJ1 cells were not (Figure 4G), indicating that cancer cell lines are more sensitive to sphingosine kinase inhibition by MP-A08.

[00168] MP-A08 blocks survival and neoplastic growth of cancer cell lines

Due to the ability of MP-A08 to induce apoptosis in Jurkat cells, its effects on the growth of a range of solid and blood cancer cell lines were tested. Amongst 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-Ras, the telomerase catalytic subunit and the simian virus 40 large and small T antigens (Hahn et al., 1999). These cell lines, along with Jurkat T cell lymphoma cells, were treated with varying doses of MP-A08 to determine the median effective concentration (EC5₀) to block cell growth (Table 2). Growth of all the cell lines was blocked by MP-A08, but with varying sensitivities. MP-A08 was effective at inhibiting growth of U87 and LNCaP prostate cells with EC5₀ values below 5mM concentration (Table 2). In addition, MP-A08 was effective at inhibiting K-562, H929, Jurkat, and MDA-MB-468 cells at concentrations below lOmM (Table 2). With EC5₀ values ranging from 10-20mM, MP-A08 was also effective at growth inhibition of OVCAR-3, SK- Mel-28, MV41 1, MOLM13, BJ7, MCF-7 and MDA-MB-231 cells, while A549 cells were less sensitive (Table 2). An assessment was then made of the EC50 of MP-A08 required to block neoplastic, anchorage- independent growth of a subset of the solid tumour cell lines in in vitro colony formation assays, as a better indicator of anti-neoplasticactivity. Again, MP-A08 blocked neoplastic growth of all four cell lines tested (Table 2). Table 2

Cancer cell lines were treated at a range of inhibitor compounds and growth was measured by MTS proliferation assay after hours. Effective concentrations were determined using non-linear regression in GraphPad Prism, where the EC50 is the concentration of inhibitor required to kill 50% of the cells. Values are n=3 ± stdev of triplicate measurements. ND = not determined.

[00169] MP-A08 suppresses the growth of human lung tumour xenografts in mice

Prior to examining the anti-neoplastic effects of MP-A08 in vivo, the maximal tolerated dose of MP-A08 in NOD/SCID mice was established. No adverse side effects were observed following daily i.p.

administration of 50, 75 or lOOmg/kg MP-A08 to mice for 14 days. Indeed, no significant changes were observed in body weight, white cells, blood haemoglobin, or platelet numbers in these mice after 14 days of MP-A08 administration. The in vivo efficacy of MP-A08 to attenuate growth of A549 human lung adenocarcinoma xenografts in mice was then examined. Subcutaneous xenografts of A549 cells were established in NOD/SCID mice and grown to a volume of 50- 100mm³ before mice were treated with MP- A08 at lOOmg/kg, six times a week for two weeks. MP-A08 significantly reduced tumour volume and weight (Figure 5A and 5B). 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.

[00170] Cross-screening of the SK inhibitory activity of compounds similar to MP- AOS

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). Eight compounds, MP-A08.9, 8.16, 8.17, 8.21, 8.23, 8.24, 8.26 and 8.27 displayed improved inhibition of SKI compared to the parental MP-A08 (Table 3). In a subsequent cross-screen of the compounds against recombinant SK2, improved or comparable inhibition was demonstrated by MP-A08.9, 8.16, 8.17, 8.23 and 8.24, but not by the compounds MP-A08.21, 8.26 and 8.27) (Table 3). The compounds were tested against SKI at varying doses to assess dose-dependent inhibition and their IC₅₀ values (Table 4). All of the tested compounds displayed dose-dependent inhibition of SKI. MPA-08.9, 8.17, 8.23, and 8.27 displayed an approximate 3-fold improvement in IC₅o value compared to MP-A08.

[00171] All eight compounds with improved inhi bition of SK 1 were then tested in Jurkat T-cell lymphoma cancer cells to measure their effect on cell growth. MP-A08 was found to inhibit cell growth with an effective concentration (EC₅o) of 7.8mM. The most potent compound in this cell-based assay was MP-A08.27 with ~ 5-fold improvement in growth inhibition compared to MP-A08 (Table 4). Compounds MP-A08.9, 8.17, 8.23 and 8.26 demonstrated approximately 2-3-fold increases in growth inhibition compared to MP-A08, while MP-A08.21 and MP-A08.24 showed similar growth inhibition to MP-A08. Surprisingly, MP-A08.16 did not demonstrate any growth inhibition, possibly due to low cell

permeability. While not wishing to be bound by theory, it is considered that increases in potency in the cells compared to the in vitro IC50 determinations are likely due to inhibition of SK2 in the cell-based assays.

[00172] In summary, six SK inhibitors with improved properties were discovered from screening MP- A08 analogues. The six compounds MP-A08.9, 8.17, 8.23, 8.24, 8.26 and 8.27 displayed both improved inhibition of recombinant SKI and improved growth inhibition in cancer cells compared to MP-A08. In addition, these analogue compounds are both smaller in molecular weight and are predicted to have greater solubility(as predicted from lower LogP values) than MP-A08. Table 3 Screening of compounds structurally similar to MP-A08

Compounds were resuspended in DMSO to 25mM. Compounds were screened at 250mM against recombinant purified human SKI and SK2. ND = not determined. Table 4 In vitro assessment and growth inhibition with MP-A08 analogues in Jurkat T

Lymphoma cells

Recombinant SKI was treated with varying doses of MP-A08 to determine the median inhibition concentration (IC5₀) to block enzyme activity in a 96-well plate format assay. Jurkat T cell lymphocytes were treated with a range of inhibitor compound concentrations and growth was measured by MTS proliferation assay after 48 hours. Effective concentrations were determined using non-linear regression in GraphPad Prism, where the EC₅₀ is the concentration of inhibitor required to kill 50% of the cells. Values are n=3 ± SD of triplicate measurements

[00173] MP-A08 suppresses the growth of human glioblastoma multiforme xenografts in mice

To test the application of MP-A08 in a combinational therapy in solid cancer, 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).

Therefore, 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.

[00174] Effects on progression of human acute myeloid leukaemia

MP-A08 induced dose-dependent induction of caspase-dependent apoptosis in all cell lines examined (Figure 7). Importantly, 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. Next, 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). Importantly, 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 EC₅₀ values varied.

[00175] MP-A08 reduces leukaemic burden and prolongs survival in vivo

In the xeno-transplantation model, it was shown that intra-peritoneal (i.p.) administration of MP-A08 for two weeks after establishment of engrafted AML cells, resulted in a significant reduction in tumour burden of normal karyotype AML (Figure 1 1A). Further, MP-A08 significantly prolonged the survival of mice engrafted with normal karyotype AML (Figure 1 IB). Immuno-histochemical examination of the bone marrow revealed MP-A08 significantly reduced the proportion of engrafted human leukaemic cells therefore facilitating the re-establishment of normal host mouse hematopoiesis (data not shown).

Discussion

[00176] 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. An investigation with the representative compound 4-methyl-N-[2-[[2-[(4-methylphenyl) sulfonylamino]phenyl]iminomethyl]phenyl]benzenesulfonamide (MP-A08) has shown that these inhibitors are active in vivo; with MP-A08 able to inhibit the growth of human lung adenocarcinoma xenograft tumours in mice via a mechanism that involved both induction of tumour cell apoptosis and reduction in tumour angiogenesis. MP-A08 also significantly reduced tumour burden in xenograft models of glioblastoma and AML. Also, MP-A08 sensitised cancer cell lines to chemotherapeutic agents, displaying synergistic cell death. Therefore, this novel class of SK inhibitors, that targets the ATP- binding pocket of SK enzymes, shows considerable promise for further development in cancer therapy. [00177] Throughout the specification and the claims that follow, unless the context requires otherwise, the words "comprise" and "include" and variations such as "comprising" and "including" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[00178] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement of any form of suggestion that such prior art forms part of the common general knowledge.

[00179] It will be appreciated by those skilled in the art that the invention is not restricted in its use to the particular application described. Neither is the present invention restricted in its preferred embodiment with regard to the particular elements and/or features described or depicted herein. It will be appreciated that the invention is not limited to the embodiment or embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the invention as set forth and defined by the following claims.

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Claims

1. The use of a compound according to formula I for treating cancer or another proliferative cell condition in a subject:

wherein,

X¹ is H or is otherwise selected from the groups:

each of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²¹, R²², R²³, R²⁴, R²⁵, R²⁶, R²⁷, R²⁸, R²⁹, R³⁰, R³¹ and R³²is independently selected from the group consisting of: H, halogen, OH, N0₂, CN, NH₂, optionally substituted C₁-C₁₂alkyl, optionally substituted C₂-C₁₂alkenyl, optionally substituted C₂-C₁₂alkynyl, optionally substituted C₂- C₁₂heteroalkyl, optionally substituted C₃-C₁₂cycloalkyl, optionally substituted C₂-C₁₂heterocycloalkyl, optionally substituted C₂-C₁₂heterocycloalkenyl, optionally substituted C₆-C|₈ aryl, optionally substituted C₂-C₁₈heteroaryl, optionally substituted Ci-C|₂alkyloxy, optionally substituted C₂- C₁₂alkenyloxy, optionally substituted C₂-C₁₂alkynyloxy, optionally substituted C₂-C₎₂heteroalkyloxy, optionally substituted C₃-C₁₂cycloalkyloxy, optionally substituted C₃-C₁₂cycloalkenyloxy, optionally substituted CrCnheterocycloalkyloxy, optionally substituted C₂-C₁₂heterocycloalkenyloxy, optionally substituted C₆-Ci₈aryloxy, optionally substituted C₂-C₁₈heteroaryloxy, optionally substituted C _1- C₁₂alkylamino, SR³³, S0₃H, S0₂NH₂, S0₂R³³, SONH₂, SOR³³, COR³³, COOH, COOR³³, CONR³³R³⁴, NR³³COR³⁴, NR³³COOR³⁴, NR³³S0₂R³⁴, NR³³CONR³⁴R³⁵, NR³³R³⁴, and acyl, wherein R³³, R³⁴ and R³⁵ are each independently selected from the group consisting of H, C₁-C₁₂alkyl, C₁-C₁₂haloalkyl, C₂- C₁₂alkenyl, C₂-C_]2alkynyl, C₂-Cio heteroalkyl, optionally substituted C₃-C₁₂cycloalkyl, optionally substituted C₃-C₁₂cycloalkenyl, optionally substituted C₂-C₍₂heterocycloalkyl, optionally substituted C₂-C₁₂heterocycloalkenyl, optionally substituted C₆-C₁₈aryl, optionally substituted C₂-Ci₈heteroaryl, and acyl, or are such that any two or more of R³³, R³⁴ and R³⁵, when taken together with the atoms to which they are attached form a heterocyclic ring system with 3 to 12 ring atoms, or in the case of R¹⁵ and R¹⁶ and also R²⁷ and R²⁸ may be collectively carbonyl (=0), or in the case of R²⁰ and R²¹ may be taken together with the carbon atoms to which R²⁰ and R²¹ are attached such that they form a (fused) C₂-C₈ cycloalkyl, C₂-C₈heterocycloalkyl, aryl or any heteroaryl ring fused at the carbon atoms to which R²⁰ and R²¹ are attached; or a pharmaceutically acceptable salt or prodrug thereof.

2. The use of claim 1 , wherein R³ is CH₃.

3. The use of claim 1 or 2, wherein X¹ comprises a group according to (II).

4. The use of claim 3, wherein one or more of R¹⁰, R¹ R¹², R¹³ and R¹⁴ are halogen.

5. The use of claim 3, wherein R¹² is selected from H, CH₃, OCH₃, N0₂, and N(CH₃)₂.

6. The use of claim 3, wherein R¹⁴ is N0₂ or NHS0₂R³², wherein R³² is selected from the group consisting of H, C₁-C₆alkyl, C₁-C₆haloalkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₂-Ci₀ heteroalkyl, optionally substituted C₃-C₆cycloalkyl, optionally substituted C₃-C₆cycloalkenyl, optionally substituted C₂-C₆heterocycloalkyl, optionally substituted C₂-C₆heterocycloalkenyl, optionally substituted C₆-C₁₈aryl, optionally substituted C₂-Ci₈heteroaryl, and acyl.

7. The use of claim 1 or 2, wherein X¹ comprises a group according to (III).

8. The use of claim 7, wherein R¹⁵ and R¹⁶ are collectively carbonyl (=0).

9. The use of claim 7, wherein R²⁰ and R²¹ taken together with the carbon atoms to which R²⁰ and R²¹ are attached form a (fused) C₂-C₈cycloalkyl, C₂-C₈heterocycloalkyl, aryl or any heteroaryl ring fused at the carbon atoms to which R²⁰ and R²¹ are attached.

10. The use of claim 1 or 2, wherein X¹ comprises a group according to (IV).

1 1. The use of claim 10, wherein R²⁶ is selected from H and (Χ¾.

12. The use of claim 10, wherein R²⁷ and R²⁸ are collectively carbonyl (=0).

13. The use of claim 1 , wherein the compound is selected from:

14. 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 any one of claims 1 to 13or a pharmaceutically acceptable salt or prodrug thereof, optionally in combination with a pharmaceutically acceptable carrier, diluent and/or excipient.

15. The use of a compound as defined in any one of claims 1 to 13, or a pharmaceutically acceptable salt or prodrug thereof, in the manufacture of a medicament for treating cancer.

16. 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 any one of claims 1 to 13or a pharmaceutically acceptable salt or prodrug thereof.

17. A compound as defined in any one of claims 1 to 13, or a pharmaceutically acceptable salt or prodrug thereof, in a substantially purified form.

18. 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 any one of claims 1 to 13 or a pharmaceutically acceptable salt or prodrug thereof.