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WO2012131297A1 - Pyrido [3',2' :4,5] thieno [3, 2-d] pyrimidin- 4 - ylamine derivatives and their therapeutical use - Google Patents

Pyrido [3',2' :4,5] thieno [3, 2-d] pyrimidin- 4 - ylamine derivatives and their therapeutical use Download PDF

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Publication number
WO2012131297A1
WO2012131297A1 PCT/GB2012/000280 GB2012000280W WO2012131297A1 WO 2012131297 A1 WO2012131297 A1 WO 2012131297A1 GB 2012000280 W GB2012000280 W GB 2012000280W WO 2012131297 A1 WO2012131297 A1 WO 2012131297A1
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independently
compound according
present
nhr
compound
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French (fr)
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Jonathan Bayldon Baell
Ian Philip Street
Brad Edmund Sleebs
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention pertains generally to the field of therapeutic compounds, and more specifically to certain fused triaryl amine compounds (for convenience, collectively referred to herein as "FTA compounds"), which, inter alia, inhibit LIM kinase (LIMK) activity.
  • FAA compounds fused triaryl amine compounds
  • LIMK LIM kinase
  • the present invention also pertains to pharmaceutical compositions comprising such compounds, and the use of such compounds and compositions, both in vitro and in vivo, to inhibit LIMK activity, and in the treatment of diseases and conditions that are mediated by LIMK, that are ameliorated by the inhibition of LIMK activity, etc., including proliferative conditions such as cancer (e.g., breast cancer, prostate cancer, melanoma, glioma, etc.), as well as vasodilation (including, e.g., hypertension, angina, cerebral vasospasm, and ischemia following subarachnoid hemorrhage), neurodegenerative disorders, atherosclerosis, fibrosis, and inflammatory diseases (including, e.g., Crohn's disease and chronic obstructive pulmonary disease (COPD)), and glaucoma (also known as ocular hypertension).
  • cancer e.g., breast cancer, prostate cancer, melanoma, glioma, etc.
  • LIMK LIM kinase
  • Rho family of GTPases There are two members of the family, LIMK1 and LIMK2, both of which are directly involved in regulating multiple cellular processes via their ability to reorganise the actin-cytoskeleton network (see, e.g., Scott et al., 2007).
  • LIMKs share 50% homology (see, e.g., Mizuno et al., 1994; Nunoue et al., 1995). They are composed of two N-terminal LIM domains, each of which contains a double zinc-finger motif.
  • the LIM domains play an important role in regulating kinase activity (see, e.g., Nagata et al., 1999; Tomiyoshi et al., 2004) and may also contribute to LIMK function by mediating protein-to-protein and protein-to- DNA interactions (see, e.g., Hiraoka et al., 1996; Nishiya et al., 1998).
  • a proline/serine rich region then follows with the kinase domain of the protein being located at the extreme C-terminus.
  • the kinase domains of LIMK1 and LIMK2 are 70% identical and phosphorylation within the activation loop enhances their activity.
  • LIMK is activated via a number of signalling networks downstream of growth factors, integrins and cytokines (for a comprehensive review see, e.g., Scott et al., 2007).
  • Rho effector Rho kinase (ROCK) has been shown to phosphorylate LIMK on conserved threonine residues (Thr-508 on LIMK1 or Thr-505 LIMK2), modifications that are essential for kinase activation (see, e.g., Amano et al., 2001 ;
  • Pak1 see, e.g., Edwards et al., 1999
  • Pak4 see, e.g., Dan et al., 2001
  • MRCKa myotonic dystrophy kinase-related Cdc42-binding kinase
  • LIMK1 Transphosphorylation of LIMK1 , following association with HSP90, has been shown to increase the half life of the protein and increases its specific activity (see, e.g., Li et al., 2006). Autophosphorylation of LIMK also occurs, although the site of phosphorylation and functional consequence remains uncertain (see, e.g., Kobayashi et al., 2006; Proschel et al., 1995). Conversely, LIMK is subject to deactivation by the direct action of phosphatases.
  • Slingshotl (SSH1) binds directly to the kinase domain and dephosphorylates LIMK1 on Thr-508 and additional autophosphorylated serine residues resulting in decreased activity (see, e.g., Soosairajah et al., 2005).
  • cofilin 1 non-muscle cofilin
  • cofilin2 muscle cofilin
  • destrin also known as actin depolymerizing factor, ADF.
  • Unphosphorylated active cofilin binds to actin filaments in vivo, depolymerises filamentous actin (F-actin), and produces free barbed ends that serve to nucleate new actin filaments (see, e.g., Ghosh et al., 2004; Lorenz et al., 2004). Ultimately it is a balance between phosphorylated and
  • LIMK dephosphorylated cofilin that determines a cell's ability to reorganise the cytoskeleton, controlling cell shape and influencing its ability to move, invade or metastasise.
  • LIMK reorganises actin-cytoskeleton dynamics through direct phosphorylation of cofilin on serine-3 resulting in its inactivation and subsequent inhibition of its actin-severing activity (see, e.g., Maekawa et al., 1999; Sumi et al., 1999). Through this modulation of the actin- cytoskeleton, LIMK is implicated to play pivotal a role in several human diseases with inhibition via small molecule kinase inhibitors having potential utility.
  • LIMK1 has been found unregulated via chromosomal translocation in malignant melanoma cells (see, e.g., Okamoto et al., 2005), breast cancer tumours (see, e.g., Bagheri-Yarmand et al., 2006) and breast cancer cell lines (see, e.g., Yoshioka et al., 2003), in prostate tumours (see, e.g., Davila et al., 2003) and prostate cancer cell lines (see, e.g., Bagheri-Yarmand et al., 2006; Yoshioka et al., 2003).
  • LIMK expression levels have been linked to tumour cell growth. For example, reduction in LIMK1 caused a decrease in prostate cancer cell proliferation, arresting cells in G2/M (see, e.g., Davila et al., 2003). Similarly, lowering expression of LIMK2 in human fibrosarcoma cells limited their ability to form colonies in a long term growth assay (see, e.g., Suyama et al., 2004). LIMK has been reported to modify the p53 pathway (see, e.g., Freidman et al., 2002).
  • p53 is a potent tumour suppressor protein which is stabilised in response to cellular stress signals to induce cell-cycle arrest or apoptosis depending on signal strength and duration (see, e.g., Vousden et al., 2009).
  • LIMK regulates p53 signalling however remains unclear.
  • LIMK1 has been shown to increase the motility and invasive capacity of tumour cells in several different systems.
  • Overexpression of LIMK1 increased motility, invasiveness and metastatic ability of human breast cancer cells (see, e.g., Bagheri- Yarmand et al., 2006; Yoshioka et al., 2003) as well as increasing the invasive phenotype of benign prostate cells (see, e.g., Davila et al., 2003).
  • Glaucoma is one of the leading causes of irreversible blindness in the world. It is characterized by degeneration of the optic nerve and progressive visual field loss and is often associated with elevated intraocular pressure (IOP) (see, e.g., Ferrer, 2006).
  • IOP intraocular pressure
  • the actin cytoskeleton network is thought to be an important modulator of IOP and subsequently LIMK has been proposed to play a role in regulating IOP.
  • Downregulation of LIMK1 with short-interfering RNA in cultured corneal fibroblasts was shown to reduce actin polymerization, focal adhesion formation and delay cell migration. In an in vivo mouse model, deletion or downregulation of LIMK1 significantly reduced ocular inflammation (see, e.g., Gorovoy et al., 2008).
  • LIMK inhibitors have been proposed for the treatment of glaucoma (see, e.g., Hizaki et al., 2004; Harrison et al., 2009; Burgoon et al., 2009).
  • One aspect of the invention pertains to certain fused triaryl amine compounds
  • FTA compounds (for convenience, collectively referred to herein as “FTA compounds”), as described herein.
  • compositions e.g., a pharmaceutical composition
  • a composition comprising a FTA compound, as described herein, and a pharmaceutically acceptable carrier or diluent.
  • Another aspect of the invention pertains to method of preparing a composition (e.g., a pharmaceutical composition) comprising the step of admixing a FTA compound, as described herein, and a pharmaceutically acceptable carrier or diluent.
  • a composition e.g., a pharmaceutical composition
  • Another aspect of the present invention pertains to a method of inhibiting LIM kinase (LIMK) activity (e.g., LIMK1 activity and/or LIMK2 activity) in a cell, in vitro or in vivo, comprising contacting the cell with an effective amount of a FTA compound, as described herein.
  • LIMK LIM kinase
  • Another aspect of the present invention pertains to a method of regulating (e.g., inhibiting) cell proliferation (e.g., proliferation of a ce)) ⁇ , inhibiting cell cycle progression, promoting apoptosis, or a combination of one or more these, in vitro or in vivo, comprising contacting a cell with an effective amount of a FTA compound, as described herein.
  • Another aspect of the present invention pertains to a method of treatment comprising administering to a subject in need of treatment a therapeutically-effective amount of a FTA compound, as described herein, preferably in the form of a pharmaceutical composition.
  • Another aspect of the present invention pertains to a FTA compound as described herein for use in a method of treatment of the human or animal body by therapy.
  • Another aspect of the present invention pertains to use of a FTA compound, as described herein, in the manufacture of a medicament for use in treatment.
  • the treatment is treatment of a disease or condition that is mediated by LIM kinase (LIMK) (e.g., LIMK1 and/or LIMK2).
  • LIMK LIM kinase
  • the treatment is treatment of a disease or condition that is ameliorated by the inhibition of LIM kinase (LIMK) activity (e.g., LIMK1 activity and/or LIMK2 activity).
  • LIMK LIM kinase
  • the treatment is treatment of a proliferative condition. ln one embodiment, the treatment is treatment of cancer.
  • the treatment is treatment of cancer characterised by, or further characterised by, cancer cells which overexpress LlM kinase (LIMK) (e.g., LIMK1 and/or LIMK2).
  • LIMK LlM kinase
  • the treatment is treatment of solid tumour cancer. In one embodiment, the treatment is treatment of breast cancer, prostate cancer, melanoma, or glioma.
  • the treatment is treatment of vasodilation. In one embodiment, the treatment is treatment of hypertension, angina, cerebral vasospasm, or ischemia following subarachnoid hemorrhage.
  • the treatment is treatment of a neurodegenerative disorder. In one embodiment, the treatment is treatment of atherosclerosis.
  • the treatment is treatment of fibrosis.
  • the treatment is treatment of an inflammatory disease.
  • the treatment is treatment of Crohn's disease or chronic obstructive pulmonary disease (COPD).
  • COPD chronic obstructive pulmonary disease
  • the treatment is treatment of glaucoma (also known as ocular hypertension).
  • kits comprising (a) a FTA compound, as described herein, preferably provided as a pharmaceutical composition and in a suitable container and/or with suitable packaging; and (b) instructions for use, for example, written instructions on how to administer the compound.
  • Another aspect of the present invention pertains to a FTA compound obtainable by a method of synthesis as described herein, or a method comprising a method of synthesis as described herein.
  • Another aspect of the present invention pertains to a FTA compound obtained by a method of synthesis as described herein, or a method comprising a method of synthesis as described herein.
  • Another aspect of the present invention pertains to novel intermediates, as described herein, which are suitable for use in the methods of synthesis described herein.
  • Another aspect of the present invention pertains to the use of such novel intermediates, as described herein, in the methods of synthesis described herein.
  • the compounds are also characterized by (a) an amino group at the 4-position of the pyrimidine ring, (b) the lack of a substituent at the 2-position of the pyrimidine ring, and (c) the presence of a heteroatom (i.e., S, N, or O) at the 5-position in the central ring, for example, as shown below:
  • Some embodiments of the invention include the following:
  • -Y- is independently -S-, -NR Y -, or -0-;
  • -R Y is independently -H or saturated aliphatic C 1-6 alkyl
  • -R ze is independently -H, -R us , or -R ,
  • -R Z7 is independently -H, -R QS , or -R QL ;
  • -R Z8 is independently -H, -R QS , or -R QL ;
  • -R 29 is independently -H, -R QS , or -R QL ;
  • -R Z7 is independently -H, -R , or -R ;
  • -R ze is independently -H, -R QS , or -R QL ;
  • -R Z9 is independently -H, -R QS , or -R QL ;
  • each -R is independently -R CA , -R HA , -R cc , or -R ;
  • -R CA is independently phenyl or naphthyl, and is optionally substituted
  • -R HA is independently Cs-ia e eroaryl, and is optionally substituted;
  • -R cc is independently non-aromatic C 3 .7cycloalkyl or non-aromatic
  • -R HC is independently non-aromatic C 3 .7heterocyclyl, and is optionally substituted; and wherein: each - H 2 , -NHR S , -NR S 2
  • each -R s is independently saturated aliphatic Ci- 5 alkyl, phenyl,
  • each phenyl is optionally substituted with one or more groups selected from: -F, -CI, -Br, -I, -R ss , -CF 3 , -OH, -OR ss , and -OCF3, wherein each -R ss is independently saturated aliphatic
  • the compounds are optionally as defined herein, but with a proviso as defined in this section.
  • the compounds are optionally as defined herein, but without the proviso as defined in this section.
  • a reference to a particular group of compounds "without the recited proviso" is intended to be a reference to the compounds as defined, but wherein the definition no longer includes the indicated proviso.
  • the definition no longer includes the indicated proviso.
  • it is as if the indicated proviso has been deleted from the definition of compounds, and the definition has been expanded to encompass those compounds which otherwise would have been excluded by the indicated proviso.
  • -R 26 is independently -H, -R QS , or -R QL ;
  • -R Z7 is independently -H, -R QS , or -R QL ;
  • -R Z8 is independently -H, -R QS , or -R QL ;
  • -R Z9 is independently -H, -R QS , or -R QL ;
  • -R Z6 is independently -H or -R QS ;
  • -R Z7 is independently -R QL ;
  • -R Z8 is independently -H or -R QS ;
  • -R Z9 is independently -H or -R QS .
  • -R Z6 is independently -H
  • -R Z7 is independently -R QL ;
  • -R ze is independently -H
  • -R 29 is independently -H or -R QS .
  • -R Z6 is independently -H
  • -R ZT is independently -R QL ;
  • -R ze is independently -H
  • -R Z9 is independently -H. Examples of the previous embodiment include the following:
  • -R Z7 is independently -H, -R QS , or -R QL ;
  • -R ze is independently -H, -R QS , or -R QL ;
  • -R Z9 is independently -H, -R QS , or -R QL ;
  • -R Z7 is independently -R QL ;
  • -R Z8 is independently -H or -R QS ;
  • -R Z9 is independently -H or -R QS
  • -R Z7 is independently -R QL ;
  • -R Z8 is independently -H
  • -R Z9 is independently -H or -R QS .
  • -R Z7 is independently -R QL ;
  • -R ZB is independently -H
  • -R Z9 is independently -H.
  • a compound according to (1) selected from compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof:
  • -R ze is independently -H or -R QS ;
  • -R Z8 is independently -H or -R QS ;
  • -R Z9 is independently -H or -R QS .
  • a compound according to (1) selected from compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof, wherein -R Z9 is independently -H or -R QS :
  • each -R QS if present, is independently -F, -CI, -Br, -I, -R s , -CF 3 , -OH, -OR s , -OCF 3 , -NH 2 , -NHR S , -NR S 2 ,
  • each -R QS if present, is independently -F, -CI, -Br, -I, -R s , -CF 3 , -OH, -OR s , or -OCF 3 .
  • each -R s if present, is independently saturated aliphatic or phenyl, wherein said phenyl is optionally substituted with one or more groups selected from: -F, -CI, -Br, -I, -R sss , -CF 3 , -OH, -OR sss , or -OCF 3 , wherein each -R S5S is independently saturated aliphatic Ci. 4 alkyl.
  • each -R s is independently -Me, -Et, -nPr, -iPr, -nBu, -iBu, or -tBu.
  • each -R s is independently -Me.
  • each of -R , -R , -R , -R P5 , and -R is independently -H or a ring substituent; for example, wherein each ring substituent is independently -R x .
  • each of -R , -R , and -R P5 is independently -H or a ring substituent
  • each ring substituent is independently -R x .
  • each of -R P3 and -R w is independently -H or a ring substituent; for example, wherein each ring substituent is independently -R x
  • -R is independently a ring substituent; for example, wherein the ring substituent is independently -R x
  • -R is independently a ring substituent; for example, wherein the ring substituent is independently -R x .
  • each of -R P3 and -R P4 is independently a ring substituent; for example, wherein each ring substituent is independently -R*
  • each of -R and -R is independently -H or a ring substituent; for example, wherein each ring substituent is independently -R x .
  • each of -R P3 and -R P5 is independently a ring substituent; for example, wherein each ring substituent is independently -R x .
  • each of -R P2 and -R is independently a ring substituent; for example, wherein each ring substituent is independently -R x (58)
  • each of -R and -R is independently a ring substituent; for example, wherein each ring substituent is independently -R x .
  • -R is independently a ring substituent; for example, wherein the ring substituent is independently -R x .
  • each of -R , -R , and -R is independently a ring substituent; for example, wherein each ring substituent is independently -R x .
  • the Group -R' (62) A compound according to any one of (1) to (61), wherein -R , if present, or each -R HA , if present, is independently furanyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, oxazoiyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, pyridazinyl, indolyl, isoindolyl, indazolyl, benzofuranyl, isobenzofuranyl, benzothienyl, isobenzothienyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, benzoisoxazolyl, quinolinyl, isoquinolinyl, dihydroisoquinolinyl, cinnolinyl, or quinazolinyl; and is optionally substituted
  • two or more adjacent substituents -R X1 may together form -OCH 2 0-, -OCH 2 CH 2 0-, or -OCH 2 CH 2 CH 2 0-;
  • each -R ZL - is independently saturated aliphatic C- ⁇ alkylene
  • each -R NZ is independently azetidino, pyrrolidino, piperidino, piperazino, morpholino, azepino, or diazepino, and is optionally substituted with one or more substitutents selected from saturated aliphatic C ⁇ alkyl;
  • each -R z is independently saturated aliphatic C ⁇ alkyl, phenyl, benzyl, or
  • each pyrrolidinyl, piperidinyl, morpholinyl, piperizinyl, pyrrolidino, piperidino, morpholino, and piperizino is optionally substituted with one or more groups
  • each -R A1 is independently saturated aliphatic C 1-4 alkyl
  • each -R A2 is independently phenyl, C 5 . 6 heteroaryl, or non-aromatic
  • each -NR K3 R K4 is independently:
  • each -R A3 is independently saturated aliphatic C ⁇ alkyl, optionally substituted with one or more groups independently selected from -OH, -OR A ", -NH 2 , -NHR A4 , -NR M 2 , pyrrolidino, piperidino, morpholino, and piperizino;
  • each -R is independently saturated aliphatic C ⁇ alkyl; and wherein each -R KS is independently saturated aliphatic Ci. 4 alkyl, and is optionally substituted with one or more groups independently selected from: -F, -CI, -Br, -I, -CF 3 , -OH, -OR A5 , -OCF 3 , -NH 2 , -NHR A5 , and -NR A5 2 , wherein each -R AS is independently saturated aliphatic C ⁇ alkyl; and wherein each -R is independently saturated aliphatic C 1-4 alkyl, and is optionally substituted with one or more groups independently selected from: -F, -CI, -Br, -I, -CF 3l -OH, -OR A6 , -OCF 3 , -NH 2 , -NHR A6 , and -NR AS 2 , wherein each -R A6 is independently saturated aliphatic C
  • each -R X if present, is independently -R z , -F, -CI, -Br, -OH, or -OR z .
  • each -R NZ is independently pyrrolidino, piperidino, piperazino, or morpholino, and is optionally substituted with one or more substitutents selected from saturated aliphatic Ci. 4 alkyl.
  • each -R K if present, is independently -Me, -Et, -nPr, -iPr, -nBu, -iBu, -sBu, or -tBu.
  • each -R ⁇ if present, is independently -Me or -Et.
  • each -R A1 is independently -Me, -Et, -nPr, -iPr, -nBu, -iBu, -sBu, or -tBu.
  • the Group -R A2 (130) A compound according to any one of (98) to (129), wherein each -R* 2 , if present, is independently phenyl or C 5-6 heteroaryl.
  • each -NR K3 R 4 is independently pyrrolidine piperidino, morpholino, or piperizino.
  • each -R A3 is independently -Me, -Et, -nPr, -iPr, -nBu, -iBu, -sBu, or -tBu.
  • each -R A4 is independently -Me, -Et, -nPr, -iPr, -nBu, -iBu, -sBu, or -tBu.
  • the Group -R KS (140) A compound according to any one of (98) to (139), wherein each -R KS , if present, is independently saturated aliphatic d. 4 alkyl, and is optionally substituted with one or more groups independently selected from: -OH, -OR A5 , -NH 2 , -NHR AS , and -NR A5 2 .
  • each -R A5 is independently -Me, -Et, -nPr, -iPr, -nBu, -iBu, -sBu, or -tBu.
  • the Group -R K6 (145) A compound according to any one of (98) to (144), wherein each -R K6 , if present, is independently saturated aliphatic Ci. 4 alkyl, and is optionally substituted with one or more groups independently selected from: -OH, -OR A6 , -NH 2 , -NHR A6 , and -NR A6 2 .
  • each -R A6 is independently -Me, -Et, -nPr, -iPr, -nBu, -iBu, -sBu, or -tBu.
  • a compound according to (1) selected from compounds of the following formulae and pharmaceutically acceptable salts, hydrates, and solvates thereof:
  • a compound according to (1) selected from compounds of the following formulae and pharmaceutically acceptable salts, hydrates, and solvates thereof:
  • a compound according to (1) selected from compounds of the following formulae and pharmaceutically acceptable salts, hydrates, and solvates thereof:
  • a compound according to (1) selected from compounds of the following formulae and pharmaceutically acceptable salts, hydrates, and solvates thereof:
  • a compound according to (1) selected from compounds of the following formulae and pharmaceutically acceptable salts, hydrates, and solvates thereof:
  • a compound according to (1) selected from compounds of the following formulae and pharmaceutically acceptable salts, hydrates, and solvates thereof:
  • a compound according to (1) selected from compounds of the following formulae and pharmaceutically acceptable salts, hydrates, and solvates thereof:
  • a compound according to (1) selected from compounds of the following formulae and pharmaceutically acceptable salts, hydrates, and solvates thereof:
  • a compound according to (1) selected from compounds of the following formulae and pharmaceutically acceptable salts, hydrates, and solvates thereof:
  • the substantially purified form is at least 50% by weight, e.g., at least 60% by weight, e.g., at least 70% by weight, e.g., at least 80% by weight, e.g., at least 90%) by weight, e.g., at least 95% by weight, e.g., at least 97% by weight, e.g., at least 98% by weight, e.g., at least 99% by weight.
  • the substantially purified form refers to the compound in any
  • the substantially purified form refers to a mixture of stereoisomers, i.e., purified with respect to other compounds. In one embodiment, the substantially purified form refers to one
  • the substantially purified form refers to a mixture of enantiomers. In one embodiment, the substantially purified form refers to a equimolar mixture of enantiomers (i.e., a racemic mixture, a racemate). In one embodiment, the substantially purified form refers to one enantiomer, e.g., optically pure enantiomer.
  • the contaminants represent no more than 50% by weight, e.g., no more than 40% by weight, e.g., no more than 30% by weight, e.g., no more than 20% by weight, e.g., no more than 10% by weight, e.g., no more than 5% by weight, e.g., no more than 3% by weight, e.g., no more than 2% by weight, e.g., no more than 1% by weight.
  • the contaminants refer to other compounds, that is, other than stereoisomers or enantiomers. In one embodiment, the contaminants refer to other compounds and other stereoisomers. In one embodiment, the contaminants refer to other compounds and the other enantiomer.
  • the substantially purified form is at least 60% optically pure (i.e., 60% of the compound, on a molar basis, is the desired stereoisomer or enantiomer, and 40% is the undesired stereoisomer or enantiomer), e.g., at least 70% optically pure, e.g., at least 80% optically pure, e.g., at least 90% optically pure, e.g., at least 95% optically pure, e.g., at least 97% optically pure, e.g., at least 98% optically pure, e.g., at least 99% optically pure.
  • 60% optically pure i.e., 60% of the compound, on a molar basis, is the desired stereoisomer or enantiomer, and 40% is the undesired stereoisomer or enantiomer
  • at least 70% optically pure e.g., at least 80% optically pure, e.g., at least 90% optically pure, e
  • Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diastereoisomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and l-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; a- and ⁇ -forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as "isomers” (or "isomeric forms").
  • a reference to a class of structures may well include structurally isomeric forms falling within that class (e.g., d -7 alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).
  • d -7 alkyl includes n-propyl and iso-propyl
  • butyl includes n-, iso-, sec-, and tert-butyl
  • methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl
  • reference to a specifc group or substitution pattern is not intended to include other structural (or constitutional isomers) which differ with respect to the connections between atoms rather than by positions in space.
  • a reference to a methoxy group, -OCH 3 is not to be construed as a reference to its structural isomer, a
  • keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hydroxyazo, and nitro/aci-nitro.
  • keto enol enolate Note that specifically included in the term “isomer” are compounds with one or more isotopic substitutions.
  • H may be in any isotopic form, including 1 H, 2 H (D), and 3 H (T);
  • C may be in any isotopic form, including 12 C, 13 C, and 4 C;
  • O may be in any isotopic form, including 16 0 and 18 0; and the like.
  • a reference to a particular compound includes all such isomeric forms, including mixtures (e.g., racemic mixtures) thereof.
  • Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.
  • Salts It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the compound, for example, a pharmaceutically-acceptable salt.
  • a pharmaceutically-acceptable salt examples include pharmaceutically acceptable salts.
  • pharmaceutically acceptable salts are discussed in Berge ei a/., 1977, "Pharmaceutically Acceptable Salts," J. Pharm. Sci.. Vol. 66, pp. 1-19.
  • the compound is anionic, or has a functional group which may be anionic (e.g., -COOH may be -COO " )
  • a salt may be formed with a suitable cation.
  • suitable inorganic cations include, but are not limited to, alkali metal ions such as Na + and K + , alkaline earth cations such as Ca 2+ and Mg 2+ , and other cations such as Af 3 .
  • suitable organic cations include, but are not limited to, ammonium ion (i.e., NH ) and substituted ammonium ions (e.g., NH 3 R + , NH 2 R 2 + , NHR 3 ⁇ NR graffiti + ).
  • suitable substituted ammonium ions are those derived from:
  • ethylamine diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine.
  • An example of a common quaternary ammonium ion is N(CH 3 )4 +
  • a salt may be formed with a suitable anion.
  • suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.
  • Suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, trifluoroacetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric.
  • Suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose. Unless otherwise specified, a reference to a particular compound also includes salt forms thereof.
  • Solvates and Hydrates It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the compound.
  • solvate is used herein in the conventional sense to refer to a complex of solute (e.g., compound, salt of compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.
  • a reference to a particular compound also includes solvate and hydrate forms thereof.
  • chemically protected form is used herein in the conventional chemical sense and pertains to a compound in which one or more reactive functional groups are protected from undesirable chemical reactions under specified conditions (e.g., pH, temperature, radiation, solvent, and the like).
  • specified conditions e.g., pH, temperature, radiation, solvent, and the like.
  • well known chemical methods are employed to reversibly render unreactive a functional group, which otherwise would be reactive, under specified conditions.
  • one or more reactive functional groups are in the form of a protected or protecting group (also known as a masked or masking group or a blocked or blocking group).
  • a compound which has two nonequivalent reactive functional groups may be derivatized to render one of the functional groups "protected,” and therefore unreactive, under the specified conditions; so protected, the compound may be used as a reactant which has effectively only one reactive functional group.
  • the protected group may be "deprotected” to return it to its original functionality.
  • a hydroxy group may be protected as an ether (-OR) or an ester
  • the aldehyde or ketone group is readily regenerated, for example, by hydrolysis using water in the presence of acid.
  • an amine group may be protected, for example, as an amide (-NRCO-R) or a urethane (-NRCO-OR), for example, as: a methyl amide (-NHCO-CH 3 ); a benzyloxy amide (-NHCO-OCH 2 C 6 H 5 , -NH-Cbz); as a t-butoxy amide (-NHCO-OC(CH 3 ) 3 , -NH-Boc); a 2-biphenyl-2-propoxy amide (-NHCO-OC(CH 3 ) 2 C 6 H 4 C 6 H 5 , -NH-Bpoc), as a
  • 9-fluorenylmethoxy amide (-NH-Fmoc), as a 6-nitroveratryloxy amide (-NH-Nvoc), as a 2-trimethylsilylethyloxy amide (-NH-Teoc), as a 2,2,2-trichloroethyloxy amide (-NH-Troc), as an allyloxy amide (-NH-Alloc), as a 2-(phenylsulfonyl)ethyloxy amide (-NH-Psec); or, in suitable cases (e.g., cyclic amines), as a nitroxide radical (> ⁇ -0 ⁇ ).
  • a carboxylic acid group may be protected as an ester for example, as: an Ci. 7 alkyl ester (e.g., a methyl ester; a t-butyl ester); a C 1-7 haloalkyl ester (e.g., a
  • Ci. 7 trihaloalkyl ester a triC 1-7 alkylsilyl-C 1-7 alkyl ester; or a C 5-20 aryl-Ci. 7 alkyl ester (e.g., a benzyl ester; a nitrobenzyl ester); or as an amide, for example, as a methyl amide.
  • prodrug refers to a compound which yields the desired active compound in vivo. Typically, the prodrug is inactive, or less active than the desired active compound, but may provide advantageous handling, administration, or metabolic properties.
  • prodrugs are activated enzymatically to yield the active compound, or a compound which, upon further chemical reaction, yields the active compound (for example, as in antibody directed enzyme prodrug therapy (ADEPT), gene directed enzyme prodrug therapy (GDEPT), lipid directed enzyme prodrug therapy (LIDEPT), etc.).
  • the prodrug may be a sugar derivative or other glycoside conjugate, or may be an amino acid ester derivative.
  • compositions e.g., a pharmaceutical composition
  • a composition comprising a FTA compound, as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.
  • compositions e.g., a pharmaceutical composition
  • a composition comprising admixing a FTA compound, as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.
  • LIMK LIM kinase
  • LIMK LIM kinase
  • One aspect of the present invention pertains to a method of inhibiting LIM kinase (LIMK) activity (e.g., LIMK1 activity and/or LIMK2 activity), in vitro or in vivo, comprising contacting LIMK (e.g., LIMK1 and/or LIMK2) with an effective amount of a LIMK (e.g., LIMK1 and/or LIMK2) with an effective amount of a
  • LIMK LIM kinase
  • a method of inhibiting LIM kinase (LIMK) activity comprising contacting the cell with an effective amount of a FTA compound, as described herein.
  • LIMK activity e.g., LIMK1 activity and/or LIMK2 activity
  • Suitable assays for determining LI K activity inhibition are described herein and/or are known in the art.
  • the FTA compounds described herein e.g., (a) regulate (e.g., inhibit) cell proliferation; (b) inhibit cell cycle progression; (c) promote apoptosis; or (d) a combination of one or more of these.
  • One aspect of the present invention pertains to a method of regulating (e.g., inhibiting) cell proliferation (e.g., proliferation of a cell), inhibiting cell cycle progression, promoting apoptosis, or a combination of one or more these, in vitro or in vivo, comprising contacting a cell with an effective amount of a FTA compound, as described herein.
  • the method is a method of regulating (e.g., inhibiting) cell proliferation (e.g., proliferation of a cell), in vitro or in vivo, comprising contacting a cell with an effective amount of a FTA compound, as described herein.
  • the method is performed in vitro.
  • the method is performed in vivo.
  • the FTA compound is provided in the form of a pharmaceutically acceptable composition.
  • Any type of cell may be treated, including but not limited to, lung, gastrointestinal
  • a candidate compound regulates (e.g., inhibits) cell proliferation, etc.
  • assays which may conveniently be used to assess the activity offered by a particular compound are described herein.
  • a sample of cells e.g., from a tumour
  • a compound brought into contact with said cells, and the effect of the compound on those cells observed.
  • effect the morphological status of the cells (e.g., alive or dead, etc.) may be determined.
  • this may be used as a prognostic or diagnostic marker of the efficacy of the compound in methods of treating a patient carrying cells of the same cellular type.
  • Another aspect of the present invention pertains to a FTA compound, as described herein, for use in a method of treatment of the human or animal body by therapy.
  • Another aspect of the present invention pertains to use of a FTA compound, as described herein, in the manufacture of a medicament for use in treatment.
  • the medicament comprises the FTA compound.
  • Methods of Treatment pertains to a method of treatment comprising administering to a patient in need of treatment a therapeutically effective amount of a FTA compound, as described herein, preferably in the form of a pharmaceutical composition.
  • Conditions Treated - Conditions Mediated by LIM Kinase (LIMK) are described herein, preferably in the form of a pharmaceutical composition.
  • the treatment is treatment of a disease or condition that is mediated by LIM kinase (LIMK) (e.g., LIMK1 and/or LIMK2).
  • LIMK LIM kinase
  • the treatment is treatment of. a disease or condition that is ameliorated by the inhibition of LIM kinase (LIMK) activity (e.g., LIMK1 activity and/or LIMK2 activity).
  • LIMK LIM kinase
  • the treatment is treatment of: a proliferative condition.
  • proliferative condition pertains to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth.
  • the treatment is treatment of: a proliferative condition characterised by benign, pre-malignant, or malignant cellular proliferation, including but not limited to, neoplasms, hyperplasias, and tumours (e.g., histocytoma, glioma, astrocyoma, osteoma), cancers (see below), psoriasis, bone diseases, fibroproliferative disorders (e.g., of connective tissues), pulmonary fibrosis, atherosclerosis, smooth muscle cell proliferation in the blood vessels, such as stenosis or restenosis following angioplasty.
  • a proliferative condition characterised by benign, pre-malignant, or malignant cellular proliferation, including but not limited to, neoplasms, hyperplasias, and tumours (e.g., histo
  • the treatment is treatment of cancer.
  • the treatment is treatment of cancer characterised by, or further characterised by, cancer cells which overexpress LIM kinase (LIMK) (e.g., LIMK1 and/or LIMK2).
  • LIMK LIM kinase
  • the treatment is treatment of lung cancer, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, stomach cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, thyroid cancer, breast cancer, ovarian cancer, endometrial cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, renal cell carcinoma, bladder cancer, pancreatic cancer, brain cancer, glioma, sarcoma, osteosarcoma, bone cancer, nasopharyngeal cancer (e.g., head cancer, neck cancer), skin cancer, squamous cancer, Kaposi's sarcoma, melanoma, malignant melanoma, lymphoma, or leukemia.
  • lung cancer small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, stomach cancer, bowel cancer, colon cancer
  • rectal cancer colorectal cancer, thyroid cancer, breast cancer, ovarian cancer, endometrial cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, renal cell carcinoma, bladder cancer
  • the treatment is treatment of:
  • a carcinoma for example a carcinoma of the bladder, breast, colon (e.g., colorectal carcinomas such as colon adenocarcinoma and colon adenoma), kidney, epidermal, liver, lung (e.g., adenocarcinoma, small cell lung cancer and non-small cell lung carcinomas), oesophagus, gall bladder, ovary, pancreas (e.g., exocrine pancreatic carcinoma), stomach, cervix, thyroid, prostate, skin (e.g., squamous cell carcinoma); a hematopoietic tumour of lymphoid lineage, for example leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non- Hodgkin's lymphoma, hairy cell lymphoma, or Burkett's lymphoma;
  • hematopoietic tumor of myeloid lineage for example acute and chronic myelogenous leukemias, myelodysplastic syndrome, or promyelocytic leukemia;
  • tumour of mesenchymal origin for example fibrosarcoma or habdomyosarcoma
  • a tumor of the central or peripheral nervous system for example astrocytoma, neuroblastoma, glioma or schwannoma
  • melanoma melanoma; seminoma; teratocarcinoma; osteosarcoma; xenoderoma
  • pigmentoum pigmentoum
  • keratoctanthoma thyroid follicular cancer
  • Kaposi's sarcoma Kaposi's sarcoma
  • the treatment is treatment of solid tumour cancer. ln one embodiment, the treatment is treatment of breast cancer, prostate cancer, melanoma, or glioma. In one embodiment, the treatment is treatment of cancer metastasis.
  • the cancer is characterised by, or further characterised by, cancer stem cells.
  • the anti-cancer effect may arise through one or more mechanisms, including but not limited to, the regulation of cell proliferation, the inhibition of cell cycle progression, the inhibition of angiogenesis (the formation of new blood vessels), the inhibition of metastasis (the spread of a tumour from its origin), the inhibition of cell migration (the spread of cance cells to other parts of the body), the inhibition of invasion (the spread of tumour cells into neighbouring normal structures), or the promotion of apoptosis
  • the compounds of the present invention may be used in the treatment of the cancers described herein, independent of the mechanisms discussed herein. Conditions Treated - Additional Conditions
  • the treatment is treatment of vasodilation.
  • the treatment is treatment of hypertension, angina, cerebral vasospasm, or ischemia following subarachnoid hemorrhage.
  • the treatment is treatment of a neurodegenerative disorder.
  • the treatment is treatment of atherosclerosis.
  • the treatment is treatment of fibrosis.
  • the treatment is treatment of an inflammatory disease. In one embodiment, the treatment is treatment of Crohn's disease or chronic obstructive pulmonary disease (COPD).
  • COPD chronic obstructive pulmonary disease
  • the treatment is treatment of glaucoma (also known as ocular hypertension).
  • treatment refers generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, alleviatiation of symptoms of the condition, amelioration of the condition, and cure of the condition.
  • Treatment as a prophylactic measure i.e., prophylaxis
  • treatment is also included. For example, use with patients who have not yet developed the condition, but who are at risk of developing the condition, is encompassed by the term "treatment.”
  • treatment includes the prophylaxis of cancer, reducing the incidence of cancer, alleviating the symptoms of cancer, etc.
  • terapéuticaally-effective amount refers to that amount of a compound, or a material, composition or dosage form comprising a compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.
  • treatment includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously.
  • the compounds described herein may also be used in combination therapies, e.g., in conjunction with other agents, for example, cytotoxic agents, anticancer agents, etc.
  • treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g., drugs, antibodies (e.g., as in immunotherapy), prodrugs (e.g., as in photodynamic therapy, GDEPT, ADEPT, etc.); surgery; radiation therapy; photodynamic therapy; gene therapy; and controlled diets.
  • a compound as described herein may be beneficial to combine treatment with a compound as described herein with one or more other (e.g., 1 , 2, 3, 4) agents or therapies that regulates cell growth or survival or differentiation via a different mechanism, thus treating several characteristic features of cancer development.
  • one or more other agents or therapies that regulates cell growth or survival or differentiation via a different mechanism
  • One aspect of the present invention pertains to a compound as described herein, in combination with one or more additional therapeutic agents, as described below.
  • the particular combination would be at the discretion of the physician who would select dosages using his common general knowledge and dosing regimens known to a skilled practitioner.
  • the agents i.e., the compound described herein, plus one or more other agents
  • the agents can be administered at closely spaced intervals (e.g., over a period of 5-10 minutes) or at longer intervals (e.g., 1, 2, 3, 4 or more hours apart, or even longer periods apart where required), the precise dosage regimen being commensurate with the properties of the therapeutic agent(s).
  • agents i.e., the compound described here, plus one or more other agents
  • the agents may be formulated together in a single dosage form, or alternatively, the individual agents may be formulated separately and presented together in the form of a kit, optionally with instructions for their use.
  • the FTA compounds described herein may also be used as cell culture additives to inhibit (LIMK) activity (e.g., LIMK1 activity and/or LIMK2 activity), e.g., to inhibit cell proliferation, etc.
  • LIMK e.g., LIMK1 activity and/or LIMK2 activity
  • the FTA compounds described herein may also be used as part of an in vitro assay, for example, in order to determine whether a candidate host is likely to benefit from treatment with the compound in question.
  • the FTA compounds described herein may also be used as a standard, for example, in an assay, in order to identify other compounds, other LIMK activity inhibitors, other anti-proliferative agents, other anti-cancer agents, etc.
  • kits comprising (a) a FTA compound as described herein, or a composition comprising a FTA compound as described herein, e.g., preferably provided in a suitable container and/or with suitable packaging; and (b) instructions for use, e.g., written instructions on how to administer the compound or composition.
  • the written instructions may also include a list of indications for which the active ingredient is a suitable treatment.
  • the FTA compound or pharmaceutical composition comprising the FTA compound may be administered to a subject by any convenient route of administration, whether systemically/peripherally or topically (i.e., at the site of desired action).
  • Routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eyedrops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal,
  • the subject/patient may be a chordate, a vertebrate, a mammal, a placental mammal, a marsupial (e.g., kangaroo, wombat), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., a bird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., a cow), a primate, simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), an a rodent (e.
  • the subject/patient may be any of its forms of development, for example, a foetus.
  • the subject/patient is a human.
  • Formulations While it is possible for the FTA compound to be administered alone, it is preferable to present it as a pharmaceutical formulation (e.g., composition, preparation, medicament) comprising at least one FTA compound, as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents.
  • pharmaceutically acceptable carriers e.g., composition, preparation, medicament
  • diluents diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.
  • the formulation may further comprise other active agents, for example, other therapeutic or prophylactic agents.
  • the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition comprising admixing at least one FTA compound, as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, e.g., carriers, diluents, excipients, etc. If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the compound.
  • pharmaceutically acceptable pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit risk ratio.
  • Each carrier, diluent, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbook of Pharmaceutical Excipients, 5th edition, 2005.
  • the formulations may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the compound with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary.
  • carriers e.g., liquid carriers, finely divided solid carrier, etc.
  • the formulation may be prepared to provide for rapid or slow release; immediate, delayed, timed, or sustained release; or a combination thereof.
  • Formulations may suitably be in the form of liquids, solutions (e.g., aqueous, nonaqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, mouthwashes, drops, tablets (including, e.g., coated tablets), granules, powders, losenges, pastilles, capsules (including, e.g., hard and soft gelatin capsules), cachets, pills, ampoules, boluses, suppositories, pessaries, tinctures, gels, pastes, ointments, creams, lotions, oils, foams, sprays, mists, or aerosols.
  • solutions e.g., aqueous, nonaqueous
  • suspensions e.g., aqueous, non-aqueous
  • emulsions
  • Formulations may suitably be provided as a patch, adhesive plaster, bandage, dressing, or the like which is impregnated with one or more compounds and optionally one or more other pharmaceutically acceptable ingredients, including, for example, penetration, permeation, and absorption enhancers. Formulations may also suitably be provided in the form of a depot or reservoir. The compound may be dissolved in, suspended in, or admixed with one or more other pharmaceutically acceptable ingredients. The compound may be presented in a liposome or other microparticulate which is designed to target the compound, for example, to blood components or one or more organs.
  • Formulations suitable for oral administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, tablets, granules, powders, capsules, cachets, pills, ampoules, boluses.
  • Formulations suitable for buccal administration include mouthwashes, losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs.
  • Losenges typically comprise the compound in a flavored basis, usually sucrose and acacia or tragacanth.
  • Pastilles typically comprise the compound in an inert matrix, such as gelatin and glycerin, or sucrose and acacia.
  • Mouthwashes typically comprise the compound in a suitable liquid carrier.
  • Formulations suitable for sublingual administration include tablets, losenges, pastilles, capsules, and pills.
  • Formulations suitable for oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil- in-water, water-in-oil), mouthwashes, losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs.
  • solutions e.g., aqueous, non-aqueous
  • suspensions e.g., aqueous, non-aqueous
  • emulsions e.g., oil- in-water, water-in-oil
  • mouthwashes e.g., gluges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs.
  • Formulations suitable for non-oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g. , oil-in-water, water-in-oil), suppositories, pessaries, gels, pastes, ointments, creams, lotions, oils, as well as patches, adhesive plasters, depots, and reservoirs.
  • solutions e.g., aqueous, non-aqueous
  • suspensions e.g., aqueous, non-aqueous
  • emulsions e.g. , oil-in-water, water-in-oil
  • suppositories e.g. , oil-in-water, water-in-oil
  • pessaries e.g., oil-in-water, water-in-oil
  • suppositories e
  • Formulations suitable for transdermal administration include gels, pastes, ointments, creams, lotions, and oils, as well as patches, adhesive plasters, bandages, dressings, depots, and reservoirs.
  • Tablets may be made by conventional means, e.g., compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the compound in a free-flowing form such as a powder or granules, optionally mixed with one or more binders (e.g., povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers or diluents (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica); disintegrants (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose); surface-active or dispersing or wetting agents (e.g., sodium lauryl sulfate); preservatives (e.g., ethy] p-hydroxybenzoate, prop
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the compound therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile.
  • Tablets may optionally be provided with a coating, for example, to affect release, for example an enteric coating, to provide release in parts of the gut other than the stomach.
  • Ointments are typically prepared from the compound and a paraffinic or a water-miscible ointment base.
  • Creams are typically prepared from the compound and an oil-in-water cream base.
  • the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1 ,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof.
  • the topical formulations may desirably include a compound which enhances absorption or penetration of the compound through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.
  • Emulsions are typically prepared from the compound and an oily phase, which may optionally comprise merely an emulsifier (otherwise known as an emulgent), or it may comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil.
  • an emulsifier also known as an emulgent
  • a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabiliser. It is also preferred to include both an oil and a fat.
  • the emulsifier(s) with or without stabilisers make up the so-called emulsifying wax
  • the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
  • Suitable emulgents and emulsion stabilisers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate.
  • suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the compound in most oils likely to be used in pharmaceutical emulsion formulations may be very low.
  • the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers.
  • Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
  • Formulations suitable for intranasal administration, where the carrier is a liquid include, for example, nasal spray, nasal drops, or by aerosol administration by nebuliser, include aqueous or oily solutions of the compound.
  • Formulations suitable for intranasal administration, where the carrier is a solid include, for example, those presented as a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
  • Formulations suitable for pulmonary administration include those presented as an aerosol spray from a pressurised pack, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichoro-tetrafluoroethane, carbon dioxide, or other suitable gases.
  • a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichoro-tetrafluoroethane, carbon dioxide, or other suitable gases.
  • Formulations suitable for ocular administration include eye drops wherein the compound is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the compound.
  • Formulations suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols, for example, cocoa butter or a salicylate; or as a solution or suspension for treatment by enema.
  • a suitable base comprising, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols, for example, cocoa butter or a salicylate; or as a solution or suspension for treatment by enema.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the compound, such carriers as are known in the art to be appropriate.
  • Formulations suitable for parenteral administration include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the compound is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate).
  • sterile liquids e.g., solutions, suspensions
  • Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient.
  • excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like.
  • suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection.
  • the concentration of the compound in the liquid is from about 1 ng/ml to about 10 g/ml, for example from about 10 ng/ml to about 1 g/ml.
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
  • FTA compounds, and compositions comprising the FTA compounds can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects.
  • the selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular FTA compound, the route of administration, the time of administration, the rate of excretion of the FTA compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health, and prior medical history of the patient.
  • the amount of FTA compound and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.
  • Administration can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.
  • a suitable dose of the FTA compound is in the range of about 10 pg to about 250 mg (more typically about 100 pg to about 25 mg) per kilogram body weight of the subject per day.
  • the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately.
  • N,N-dimethylformamide (0.025 mmol) in phosphorous oxychloride (6 mL) was allowed to stir at 90°C for 20 h.
  • the phosphorous oxychloride was removed in vacuo. Ice-water was added and the solution neutralized by portionwise addition of solid sodium hydrogen carbonate. The resulting precipitate was filtered off, washed with water and dried in a vacuum oven to give the chloro pyrimidine as a tanned solid (86%).
  • N,N-dimethylformamide (3 mL) containing under nitrogen was stirred at 0°C for 45 min and then warmed to 25°C for 2 h. After this time, water was added and a precipitate formed. This was filtered, washed with water and then oven dried to give the
  • N,N-dimethylformamide (3 mL) was allowed to stir for 1 h at room temperature. Ice-water was added to the reaction mixture. The precipitate that formed was filtered off, washing with water and dried in a vacuum oven to obtain a white solid (the solid was a mixture of acyclic and cyclic product). The solid was dissolved in N,N-dimethylformamide (3 mL) and potassium carbonate (0.7 mmol) was added. The mixture was allowed to stir at 50°C for 8 h. Ice-water was added to the reaction mixture. The precipitate that formed was filtered off, washing with water and dried in a vacuum oven to obtain the thieno pyridine as a white solid (68 %).
  • Morpholine (36.3 mmol) is added to a solution of 2-cyanothioacetamide (36.3 mmol) in ethanol (20 mL) and the solution stirred at 25°C for 5 min.
  • Ethyl acetoacetate (36.3 mmol) is added and the solution was then stirred at 25°C for 24 h.
  • Acetone was added to the solution and the precipitate that formed was filtered off washing with acetone to afford the pyridine as a white solid (61%).
  • the serine kinases LIMK-1 and LIMK-2 are kinases which have LIM domains or protein- motifs that are frequently found in cytosolic proteins which interact with the actin cytoskeleton involved in cell division and motility.
  • the purpose of the assay was to screen for inhibitors of LIMK-1 whereby kinase activity was measured by a luminescence ATP detection system in a single-point 384 well screening assay measuring ATP consumption by the kinase-substrate phosphorylation reaction.
  • the reaction measured was characterized by two steps as outlined below: APT/Mg ⁇
  • the first step LIMK-1 catalyses the phosphorylation of its substrate cofilin thereby consuming ATP.
  • the second step involves the detection of the remaining ATP using the Luminescence ATP detection system, Kinase-Glo® (Promega).
  • the firefly luciferase enzyme produces light by its reaction with its substrate D-Luciferin and ATP. The light which is produced as a result of the reaction is proportional to the amount of ATP that is present in the initial kinase reaction.
  • LimK-1 and cofilin were supplied by Ora Bernard, St Vincent's Medical Institute. ATP (99% purity) supplied by Sigma Aldrich whilst the Kinase-Glo detection kit was obtained from Promega Corp (Cat #V6714).
  • Common assay buffer reagents such as HEPES, KCI, MgCI 2 , MnCI 2 , NaF, NaVO, and BSA were purchased from Sigma Aldrich. Library compounds were pre-prepared in Metrical 50 ⁇ , V-bottom polypropylene plates. The assay was carried out in PerkinElmer Lifesciences 384 well ProxiplatesTM (white, shallow well of 28 ⁇ total volume, inverted chimney design) with Matrical deep well plates
  • MiniTrak IX PerkinElmer Lifesciences
  • Thermo Corporation was used to perform all other additions and measurements were detected using the Envision 2103 (PerkinElmer Lifesciences).
  • a MultiDrop reagent dispenser added 4.9 pL of LIMK-1 in kinase buffer to all wells except negative control wells. Those were filled with the same volume of assay buffer.
  • the plates were de-lidded and 10 pL Kinase-Glo reagent per well were added with a MultiDrop. The plates were lidded again and incubated for further 10 min in the dark to allow for stabilization of the luminescence signal. Afterwards, the plates were read in the EnVision 2103 reader using the program "Ultrasensitive Luminescence". Each well was read for 0.1 sec. Because the Kinase-Glo reagents were sensitive to light, all handlings were done under modest light conditions. 2.2.3. Data Analysis
  • x signal in samples after compound treatment.
  • ⁇ * mean signal of the blanks (no LI K-1).
  • Label 1 Ultra sensitive Luminescence.
  • Plate filter 384 wells, 16 * 24.
  • the substrate solution was prepared according to the supplier's manual, e.g., bringing the solutions to room temperature and reconstituting the lyophilized substrate solution. This was carried out in the dark due to the reagent's sensitivity to light.
  • the reconstituted substrate solution contained a mutant of firefly luciferase and its substrate D-Luciferin which were necessary for the detection of ATP levels after the kinase reaction. 3.3. Automation Protocol
  • This protocol describes a method to quantify the effect of inhibitors of LIMK1.
  • the 384 well format assay is based on the Transcreener FP technology to measure ADP formed during the reaction.
  • Triton X-100 Stored at room temperature.
  • LIMK1 0.8 ng/5 pL will give a final concentration of 1.9 nM during the kinase reaction.
  • Cofilin 1.9 pg/5 pL will give 10 ⁇ final concentration during the kinase reaction.
  • ATP 40 pM in dilution to give 20 pM during the kinase reaction.
  • the concentration of the ADP antibody is dependent on the ATP concentration present due to a limited selectivity.
  • ADP-antibody 28.1 pg/mL in the mix.
  • Minitrak program for pin tool transfer is optimized to pick up the dilution out of 7.5 pL of DMSO, therefore 6 is usually 7.5 ul. Choose a to achieve any desired dilution factor of
  • Min/trak program 11_pt_titration_P30 the Min/trak program 11_pt_titration_P30.
  • MultiDrop prior to all additions flush the MultiDrop cassette with assay buffer containing ovalbumine to minimize loss of the enzyme/ substrate in the tubings.
  • excitation filter 620/40 nm the excitation filter 620/40 nm.
  • Plate layout 384 wells, 16 * 24.
  • Reading mode Fluorescence polarization.
  • 1X Kinase Buffer A 50 mM HEPES at pH 7.5, 10 mM gCI 2> 1 m EGTA, 0.01 % Brij-35.
  • the antibody tube was centrifuged at approximately 10,000 x g for 0 minutes and the desired volume aspirated from the top of the solution to eliminate spurious data points that can arise on occasion due to any particulates in the product.
  • a working solution of tracer 178 was prepared at 3x the final desired concentration in 1 X Buffer A.
  • 5 pL of kinase/antibody solution was added to all test compound wells and positive control wells.
  • An antibody solution containing no UMK2 kinase was added to the final two columns to give negative control wells.
  • 5 uL of Tracer 78 was added to all wells of the plate and then incubated at room temperature for 60 minutes. Following the incubation period the plate was read on a Tecan Ultra plate reader where two sequential measurements were measured: one at 620 nm for the antibody/donor emission, and the other at 665 nm for the acceptor/tracer emission. The emission ratio is calculated by the ratio of the two fluorescence intensities (acceptor/donor).
  • Measurement 1 Excitation Filter 337 nm, Emission Filter 620 nm, Mirror Dichroic2 (e.g., Fl 96), Lag time 100 ps, Integration time 200 ps, optimal Gain, optimal Z-position.
  • Mirror Dichroic2 e.g., Fl 96
  • Measurement 2 Excitation Filter 337 nm, Emission Filter 665 nm, Mirror Dichroic2 (e.g., Fl 96), Lag time 100 ps, Integration time 200 ps, optimal Gain, optimal Z-position.
  • Mirror Dichroic2 e.g., Fl 96
  • the following compounds were found to have
  • Biological Data - Assay 2 - LI K1 (Kinase Domain) Enzyme Binding Assay 2 The following compounds were examined using Assay 2: AA-001 , AA-002, AA-003, AA-005, AA-006, AA-007, AA-008, AA-009, AA-010, AA-0 1 , AA-012, AA-013, AA-014, AA-015, AA-016, AA-017, AA-0 8, AA-0 9, AA-020, AA-021 , AA-023, AA-028, AA-029, AA-032, AA-033, AA-036, AA-037, AA-038, AA-040, AB-001 , AB-002, AB-003, AB-004, AB-005, AB-006, AB-007, AB-009, AB-010, AB-011 , AB-012, AB-013, AB
  • the IC50 for AA-040 was 0.202 ⁇ .

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Abstract

The present invention pertains generally to the field of therapeutic compounds, and more specifically to certain fused triaryl amine compounds of the following formula (for convenience, collectively referred to herein as "FTA compounds"), which, inter alia, inhibit LIM kinase (LIMK) activity. The present invention also pertains to pharmaceutical compositions comprising such compounds, and the use of such compounds and compositions, both in vitro and in vivo, to inhibit LIMK activity, and in the treatment of diseases and conditions that are mediated by LIMK, that are ameliorated by the inhibition of LIMK activity, etc., including proliferative conditions such as cancer (e.g., breast cancer, prostate cancer, melanoma, glioma, etc.), as well as vasodilation (including, e.g., hypertension, angina, cerebral vasospasm, and ischemia following subarachnoid hemorrhage), neurodegenerative disorders, atherosclerosis, fibrosis, and inflammatory diseases (including, e.g., Crohn's disease and chronic obstructive pulmonary disease (COPD)), and glaucoma (also known as ocular hypertension).

Description

PYRIDO [3' ,2" :4,5] THIENO [3, 2-D] PYRIMIDIN- 4 - YLAMINE
DERIVATIVES AND THEIR THERAPEUTICAL USE
RELATED APPLICATION This application is related to United States (US) provisional patent application number 61/468,136 filed 28 March 2011, the contents of which are incorporated herein by reference in their entirety.
TECHNICAL FIELD
The present invention pertains generally to the field of therapeutic compounds, and more specifically to certain fused triaryl amine compounds (for convenience, collectively referred to herein as "FTA compounds"), which, inter alia, inhibit LIM kinase (LIMK) activity. The present invention also pertains to pharmaceutical compositions comprising such compounds, and the use of such compounds and compositions, both in vitro and in vivo, to inhibit LIMK activity, and in the treatment of diseases and conditions that are mediated by LIMK, that are ameliorated by the inhibition of LIMK activity, etc., including proliferative conditions such as cancer (e.g., breast cancer, prostate cancer, melanoma, glioma, etc.), as well as vasodilation (including, e.g., hypertension, angina, cerebral vasospasm, and ischemia following subarachnoid hemorrhage), neurodegenerative disorders, atherosclerosis, fibrosis, and inflammatory diseases (including, e.g., Crohn's disease and chronic obstructive pulmonary disease (COPD)), and glaucoma (also known as ocular hypertension). BACKGROUND
A number of patents and publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.
Throughout this specification, including the claims which follow, unless the context requires otherwise, the word "comprise," and variations such as "comprises" and "comprising," will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like. Ranges are often expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent "about," it will be understood that the particular value forms another embodiment.
This disclosure includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
LIMK
The LIM kinase (LIMK) family of serine/threonine protein kinases sit at the centre of multiple signalling pathways downstream of the Rho family of GTPases. There are two members of the family, LIMK1 and LIMK2, both of which are directly involved in regulating multiple cellular processes via their ability to reorganise the actin-cytoskeleton network (see, e.g., Scott et al., 2007). Overall, the LIMKs share 50% homology (see, e.g., Mizuno et al., 1994; Nunoue et al., 1995). They are composed of two N-terminal LIM domains, each of which contains a double zinc-finger motif. The LIM domains play an important role in regulating kinase activity (see, e.g., Nagata et al., 1999; Tomiyoshi et al., 2004) and may also contribute to LIMK function by mediating protein-to-protein and protein-to- DNA interactions (see, e.g., Hiraoka et al., 1996; Nishiya et al., 1998). Next lies a PDZ domain which also influences protein-protein interactions but additionally contains two nuclear export signals (see, e.g., Yang et al., 1998; Yang et al., 1999) which together with nuclear localisation signals (see, e.g., Goyal et al., 2006) most likely regulate
nuclear/cytoplasmic shuttling. A proline/serine rich region then follows with the kinase domain of the protein being located at the extreme C-terminus. The kinase domains of LIMK1 and LIMK2 are 70% identical and phosphorylation within the activation loop enhances their activity.
LIMK is activated via a number of signalling networks downstream of growth factors, integrins and cytokines (for a comprehensive review see, e.g., Scott et al., 2007).
Specifically, the Rho effector, Rho kinase (ROCK) has been shown to phosphorylate LIMK on conserved threonine residues (Thr-508 on LIMK1 or Thr-505 LIMK2), modifications that are essential for kinase activation (see, e.g., Amano et al., 2001 ;
Ohashi et al., 2000; Sumi et al., 2001a), Pak1 (see, e.g., Edwards et al., 1999), Pak4 (see, e.g., Dan et al., 2001), and the myotonic dystrophy kinase-related Cdc42-binding kinase (MRCKa) have also been shown to phosphorylate LIMK1 with MRCKa also phosphorylating LIMK2 (see, e.g., Sumi et al., 2001b). Transphosphorylation of LIMK1 , following association with HSP90, has been shown to increase the half life of the protein and increases its specific activity (see, e.g., Li et al., 2006). Autophosphorylation of LIMK also occurs, although the site of phosphorylation and functional consequence remains uncertain (see, e.g., Kobayashi et al., 2006; Proschel et al., 1995). Conversely, LIMK is subject to deactivation by the direct action of phosphatases. Slingshotl (SSH1) binds directly to the kinase domain and dephosphorylates LIMK1 on Thr-508 and additional autophosphorylated serine residues resulting in decreased activity (see, e.g., Soosairajah et al., 2005).
Downstream of LIMK there are relatively few substrates known. The best characterised are the cofilin family of proteins, cofilin 1 (non-muscle cofilin), cofilin2 (muscle cofilin) and destrin (also known as actin depolymerizing factor, ADF). Unphosphorylated active cofilin binds to actin filaments in vivo, depolymerises filamentous actin (F-actin), and produces free barbed ends that serve to nucleate new actin filaments (see, e.g., Ghosh et al., 2004; Lorenz et al., 2004). Ultimately it is a balance between phosphorylated and
dephosphorylated cofilin that determines a cell's ability to reorganise the cytoskeleton, controlling cell shape and influencing its ability to move, invade or metastasise. LIMK reorganises actin-cytoskeleton dynamics through direct phosphorylation of cofilin on serine-3 resulting in its inactivation and subsequent inhibition of its actin-severing activity (see, e.g., Maekawa et al., 1999; Sumi et al., 1999). Through this modulation of the actin- cytoskeleton, LIMK is implicated to play pivotal a role in several human diseases with inhibition via small molecule kinase inhibitors having potential utility.
LIMK and Cancer
Enhanced LIMK activity has been associated with tumour types. LIMK1 has been found unregulated via chromosomal translocation in malignant melanoma cells (see, e.g., Okamoto et al., 2005), breast cancer tumours (see, e.g., Bagheri-Yarmand et al., 2006) and breast cancer cell lines (see, e.g., Yoshioka et al., 2003), in prostate tumours (see, e.g., Davila et al., 2003) and prostate cancer cell lines (see, e.g., Bagheri-Yarmand et al., 2006; Yoshioka et al., 2003). In line with increased LIMK activity, co-ordinately increased phosphorylated cofilin has also been found in several studies (see, e.g., Davila et al., 2003; Wang et al., 2004; Wang et al., 2005).
LIMK expression levels have been linked to tumour cell growth. For example, reduction in LIMK1 caused a decrease in prostate cancer cell proliferation, arresting cells in G2/M (see, e.g., Davila et al., 2003). Similarly, lowering expression of LIMK2 in human fibrosarcoma cells limited their ability to form colonies in a long term growth assay (see, e.g., Suyama et al., 2004). LIMK has been reported to modify the p53 pathway (see, e.g., Freidman et al., 2002). p53 is a potent tumour suppressor protein which is stabilised in response to cellular stress signals to induce cell-cycle arrest or apoptosis depending on signal strength and duration (see, e.g., Vousden et al., 2009). The exact mechanism by which LIMK regulates p53 signalling however remains unclear.
Consistent with LIMKs ability to reorganise the actin-cytoskeleton network, the most convincing role for LIMK in cancer is to modulate tumour cell invasion and metastasis. Metastasis occurs through a series of complex biological events which together allow tumour cells to detach and extravasate from their site of origin and repopulate at a distal site within the body (see, e.g., Klein, 2009). Currently there are no therapeutic strategies to control metastasis although it is estimated to account for over 90% of all human deaths from cancer (see, e.g., Sporn, 1996).
Overexpression of LIMK has been shown to increase the motility and invasive capacity of tumour cells in several different systems. Overexpression of LIMK1 increased motility, invasiveness and metastatic ability of human breast cancer cells (see, e.g., Bagheri- Yarmand et al., 2006; Yoshioka et al., 2003) as well as increasing the invasive phenotype of benign prostate cells (see, e.g., Davila et al., 2003).
Importantly, it has been demonstrated in several studies that interfering with LIMK function reduces tumour cell invasion. Silencing of LIMK expression with anti-sense oligonuceotides in metastatic prostate cells resulted in decreased cell invasion (see, e.g., Davila et al., 2003), and ribozyme-mediated knockdown of LIMK2 inhibited the motility of metastatic fibrosarcoma cells (see, e.g., Suyama et al., 2004). Furthermore, expression of dominant-negative LIMK1 resulted in decreased invasion of metastatic breast cancer cells in vitro and in an in vivo model of breast cancer metastasis caused a decrease in the number osteolytic lesions formed after tumour cells were injected into nude mice (see, e.g., Yoshioka et al., 2003). Collectively these studies suggest that targeting LIMK may be a viable therapeutic strategy for preventing or controlling metastatic disease.
LIMK and Glaucoma
Glaucoma is one of the leading causes of irreversible blindness in the world. It is characterized by degeneration of the optic nerve and progressive visual field loss and is often associated with elevated intraocular pressure (IOP) (see, e.g., Ferrer, 2006). The actin cytoskeleton network is thought to be an important modulator of IOP and subsequently LIMK has been proposed to play a role in regulating IOP. Downregulation of LIMK1 with short-interfering RNA in cultured corneal fibroblasts was shown to reduce actin polymerization, focal adhesion formation and delay cell migration. In an in vivo mouse model, deletion or downregulation of LIMK1 significantly reduced ocular inflammation (see, e.g., Gorovoy et al., 2008). Accordingly, LIMK inhibitors have been proposed for the treatment of glaucoma (see, e.g., Hizaki et al., 2004; Harrison et al., 2009; Burgoon et al., 2009). SUMMARY OF THE INVENTION
One aspect of the invention pertains to certain fused triaryl amine compounds
(for convenience, collectively referred to herein as "FTA compounds"), as described herein.
Another aspect of the invention pertains to a composition (e.g., a pharmaceutical composition) comprising a FTA compound, as described herein, and a pharmaceutically acceptable carrier or diluent.
Another aspect of the invention pertains to method of preparing a composition (e.g., a pharmaceutical composition) comprising the step of admixing a FTA compound, as described herein, and a pharmaceutically acceptable carrier or diluent. Another aspect of the present invention pertains to a method of inhibiting LIM kinase (LIMK) activity (e.g., LIMK1 activity and/or LIMK2 activity) in a cell, in vitro or in vivo, comprising contacting the cell with an effective amount of a FTA compound, as described herein. Another aspect of the present invention pertains to a method of regulating (e.g., inhibiting) cell proliferation (e.g., proliferation of a ce))}, inhibiting cell cycle progression, promoting apoptosis, or a combination of one or more these, in vitro or in vivo, comprising contacting a cell with an effective amount of a FTA compound, as described herein. Another aspect of the present invention pertains to a method of treatment comprising administering to a subject in need of treatment a therapeutically-effective amount of a FTA compound, as described herein, preferably in the form of a pharmaceutical composition. Another aspect of the present invention pertains to a FTA compound as described herein for use in a method of treatment of the human or animal body by therapy.
Another aspect of the present invention pertains to use of a FTA compound, as described herein, in the manufacture of a medicament for use in treatment.
In one embodiment, the treatment is treatment of a disease or condition that is mediated by LIM kinase (LIMK) (e.g., LIMK1 and/or LIMK2).
In one embodiment, the treatment is treatment of a disease or condition that is ameliorated by the inhibition of LIM kinase (LIMK) activity (e.g., LIMK1 activity and/or LIMK2 activity).
In one embodiment, the treatment is treatment of a proliferative condition. ln one embodiment, the treatment is treatment of cancer.
In one embodiment, the treatment is treatment of cancer characterised by, or further characterised by, cancer cells which overexpress LlM kinase (LIMK) (e.g., LIMK1 and/or LIMK2).
In one embodiment, the treatment is treatment of solid tumour cancer. In one embodiment, the treatment is treatment of breast cancer, prostate cancer, melanoma, or glioma.
In one embodiment, the treatment is treatment of vasodilation. In one embodiment, the treatment is treatment of hypertension, angina, cerebral vasospasm, or ischemia following subarachnoid hemorrhage.
In one embodiment, the treatment is treatment of a neurodegenerative disorder. In one embodiment, the treatment is treatment of atherosclerosis.
In one embodiment, the treatment is treatment of fibrosis.
In one embodiment, the treatment is treatment of an inflammatory disease.
In one embodiment, the treatment is treatment of Crohn's disease or chronic obstructive pulmonary disease (COPD).
In one embodiment, the treatment is treatment of glaucoma (also known as ocular hypertension).
Another aspect of the present invention pertains to a kit comprising (a) a FTA compound, as described herein, preferably provided as a pharmaceutical composition and in a suitable container and/or with suitable packaging; and (b) instructions for use, for example, written instructions on how to administer the compound.
Another aspect of the present invention pertains to a FTA compound obtainable by a method of synthesis as described herein, or a method comprising a method of synthesis as described herein.
Another aspect of the present invention pertains to a FTA compound obtained by a method of synthesis as described herein, or a method comprising a method of synthesis as described herein. Another aspect of the present invention pertains to novel intermediates, as described herein, which are suitable for use in the methods of synthesis described herein.
Another aspect of the present invention pertains to the use of such novel intermediates, as described herein, in the methods of synthesis described herein.
As will be appreciated by one of skill in the art, features and preferred embodiments of one aspect of the invention will also pertain to other aspect of the invention.
DETAILED DESCRIPTION OF THE INVENTION
Compounds The present invention relates to certain compounds which are structurally related to the following compounds:
Figure imgf000009_0001
The compounds are also characterized by (a) an amino group at the 4-position of the pyrimidine ring, (b) the lack of a substituent at the 2-position of the pyrimidine ring, and (c) the presence of a heteroatom (i.e., S, N, or O) at the 5-position in the central ring, for example, as shown below:
Figure imgf000010_0001
Thus, one aspect of the present invention pertains to compounds selected from compounds of the following formulae, and salts, hydrates, and solvates thereof (e.g. pharmaceutically acceptable salts, hydrates, and solvates thereof), wherein -Y-, -X= -R28, and -R29 are as defined herein (for convenience, collectively referred to herein "fused triaryl amine compounds" or "FTA compounds"):
Figure imgf000010_0002
Some embodiments of the invention include the following:
(1) A compound selected from compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000011_0001
wherein:
-X= is independently -CR - or -N=;
-Y- is independently -S-, -NRY-, or -0-;
-RY is independently -H or saturated aliphatic C1-6alkyl; and wherein, if -X= is -CR26=, then:
-Rze is independently -H, -Rus, or -R ,
-RZ7 is independently -H, -RQS, or -RQL;
-RZ8 is independently -H, -RQS, or -RQL; and
-R29 is independently -H, -RQS, or -RQL;
with the proviso that at least one of -RZ6, -RZ7, -RZ8, and -RZ9 is -R' and wherein, i -X= is -N=, then:
-RZ7 is independently -H, -R , or -R ;
-Rze is independently -H, -RQS, or -RQL; and
-RZ9 is independently -H, -RQS, or -RQL;
with the proviso that at least one of -R27, -R28, and -RZ9 is -R( wherein: each -R is independently -RCA, -RHA, -Rcc, or -R ;
wherein:
-RCA is independently phenyl or naphthyl, and is optionally substituted;
-RHA is independently Cs-ia e eroaryl, and is optionally substituted;
-Rcc is independently non-aromatic C3.7cycloalkyl or non-aromatic
C3.7cycloalkenyl, and is optionally substituted;
-RHC is independently non-aromatic C3.7heterocyclyl, and is optionally substituted; and wherein: each - H2, -NHRS, -NRS2
-C(=0)NHRs,
Figure imgf000012_0001
wherein each -Rs is independently saturated aliphatic Ci-5alkyl, phenyl,
-CH2-phenyl, -CH2CH2-phenyl, or -CH^CH-phenyl, wherein each phenyl is optionally substituted with one or more groups selected from: -F, -CI, -Br, -I, -Rss, -CF3, -OH, -ORss, and -OCF3, wherein each -Rss is independently saturated aliphatic
Figure imgf000012_0002
Optional Provisos
In one or more aspects of the present invention (e.g., compounds, compositions, methods of treatment, compounds for use in therapy, use of compounds in the manufacture of a medicament, etc.), the compounds are optionally as defined herein, but with a proviso as defined in this section.
In one or more aspects of the present invention (e.g., compounds for use in therapy, use of compounds in the manufacture of a medicament, methods, methods of treatment, etc.), the compounds are optionally as defined herein, but without the proviso as defined in this section.
For example, a reference to a particular group of compounds "without the recited proviso" (e.g., for use in therapy) is intended to be a reference to the compounds as defined, but wherein the definition no longer includes the indicated proviso. In such cases, it is as if the indicated proviso has been deleted from the definition of compounds, and the definition has been expanded to encompass those compounds which otherwise would have been excluded by the indicated proviso. (2) A compound according to (1), with the proviso that the compound is not a compound selected from: compounds PP-001 to PP-034, and pharmaceutically acceptable salts, hydrates, and solvates thereof.
Code Reg No Structure
PP-001 340816-57-1
Figure imgf000013_0001
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0001
Figure imgf000017_0001
Figure imgf000018_0001
The Group -Y-
(3) A compound according to (1) or (2), wherein -Y- is independently -S- or -0-. (4) A compound according to (1) or (2), wherein -Y- is independently -S- or -NRY-.
(5) A compound according to (1) or (2), wherein -Y- is independently -S-.
(6) A compound according to (1) or (2), wherein -Y- is independently -0-.
(7) A compound according to (1) or (2), wherein -Y- is independently -NRY-. The Group -X=
(8) A compound according to any one of (1) to (7), wherein -X= is independently -CR26=.
(9) A compound according to any one of (1) to (7), wherein -X= is independently -N=.
Combinations of the Group -Y- and the Group -X=
(10) A compound according to (1), wherein the compound is selected from compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000019_0001
(11) A compound according to (1), wherein the compound is selected from compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000019_0002
(12) A compound according to (1), wherein the compound is selected from compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000020_0001
(13) A compound according to (1) or (2), wherein the compound is selected from compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000020_0002
(14) A compound according to (1), wherein the compound is selected from compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000020_0003
(15) A compound according to (1), wherein the compound is selected from compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000020_0004
The Group -RY
(16) A compound according to any one of (1) to (15), wherein -RY, if present, is independently -H, -Me, -Et, -nPr, -iPr, -nBu, -iBu, -sBu, or -tBu.
(17) A compound according to any one of (1) to (15), wherein -RY, if present, is independently -H, -Me, or -Et. (18) A compound according to any one of (1) to (15), wherein -RY, if present, is independently -H or -Me. (19) A compound according to any one of (1) to (15), wherein -RY, if present, is independently -Me.
(20) A compound according to any one of (1) to (15), wherein -RY, if present, is independently -H.
The Groups -RZ6, -RZ7, -RZ8, and -R >Z9
(21 ) A compound according to any one of (1) to (20), wherein, if -X= is -CR'
-R26 is independently -H, -RQS, or -RQL;
-RZ7 is independently -H, -RQS, or -RQL;
-RZ8 is independently -H, -RQS, or -RQL; and
-RZ9 is independently -H, -RQS, or -RQL;
with the proviso that exactly one of -R26, -RZ7, -R28, and -RZ9 is -RQL. (22) A compound according to any one of (1) to (20), wherein, if -X= is -CRZ =, then:
-RZ6 is independently -H or -RQS;
-RZ7 is independently -RQL;
-RZ8 is independently -H or -RQS; and
-RZ9 is independently -H or -RQS.
(23) A compound according to any one of (1) to (20), wherein, if -X
-RZ6 is independently -H;
-RZ7 is independently -RQL;
-Rze is independently -H; and
-R29 is independently -H or -RQS.
Examples of the previous embodiment include the following:
Figure imgf000021_0001
(24) A compound according to any one of (1) to (20), wherein, if -X= is -CR then:
-RZ6 is independently -H;
-RZT is independently -RQL;
-Rze is independently -H; and
-RZ9 is independently -H. Examples of the previous embodiment include the following:
Figure imgf000022_0001
(25) A compound according to any one of (1) to (20), wherein, if -X= is -N=, then:
-RZ7 is independently -H, -RQS, or -RQL;
-Rze is independently -H, -RQS, or -RQL; and
-RZ9 is independently -H, -RQS, or -RQL;
with the proviso that exactly one of -R27, -Rze, and -R29 is -RQL;
(26) A compound according to any one of (1) to (20), wherein, if -X= is -N=, then:
-RZ7 is independently -RQL;
-RZ8 is independently -H or -RQS; and
-RZ9 is independently -H or -RQS
(27) A compound according to any one of (1) to (20), wherein, if -X= is -N=, then:
-RZ7 is independently -RQL;
-RZ8 is independently -H; and
-RZ9 is independently -H or -RQS.
Examples of the previous embodiment include the following:
Figure imgf000022_0002
(28) A compound according to any one of (1) to (20), wherein, if -X
-RZ7 is independently -RQL;
-RZB is independently -H; and
-RZ9 is independently -H.
Examples of the previous embodiment include the following:
Figure imgf000022_0003
Some Preferred Combinations
(29) A compound according to (1), selected from compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000023_0001
wherein:
-Rze is independently -H or -RQS;
-RZ8 is independently -H or -RQS; and
-RZ9 is independently -H or -RQS.
(30) A compound according to (1), selected from compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof, wherein -RZ9 is independently -H or -RQS:
Figure imgf000023_0002
(31) A compound according to (1), selected from compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000023_0003
(32) A compound according to (1), selected from compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof, wherein -RZ9 is independently -H or -RQS:
Figure imgf000023_0004
(32) A compound according to (1) or (2), selected from compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000024_0001
(33) A compound according to (1 ), selected from compounds of the following formula, and pharmaceutically acceptable sal thereof:
Figure imgf000024_0002
The Group -R
(34) A compound according to any one of (1) to (33), wherein each -RQS, if present, is independently -F, -CI, -Br, -I, -Rs, -CF3, -OH, -ORs, -OCF3, -NH2, -NHRS, -NRS 2,
-NHC(=0)Rs, -NHC(=0)ORs, -C(=0)OH, -C(=0)ORs, -C(=0)NH2, -C(=0)NHRs, or -C(=0)NRs 2.
(35) A compound according to any one of (1) to (33), wherein each -RQS, if present, is independently -F, -CI, -Br, -I, -Rs, -CF3, -OH, -ORs, or -OCF3.
(36) A compound according to any one of (1) to (33), wherein each -RQS, if present, is independently -Rs.
(37) A compound according to any one of (1) to (33), wherein each -Rs, if present, is independently saturated aliphatic
Figure imgf000024_0003
or phenyl, wherein said phenyl is optionally substituted with one or more groups selected from: -F, -CI, -Br, -I, -Rsss, -CF3, -OH, -ORsss, or -OCF3, wherein each -RS5S is independently saturated aliphatic Ci.4alkyl.
(38) A compound according to any one of (1) to (33), wherein each -Rs, if present, is independently saturated aliphatic
Figure imgf000024_0004
or phenyl, (39) A compound according to any one of (1) to (33), wherein each -Rs, if present, is independently saturated aliphatic C alkyl.
(40) A compound according to any one of (1) to (33), wherein each -Rs, if present, is independently -Me, -Et, -nPr, -iPr, -nBu, -iBu, or -tBu. (41) A compound according to any one of (1) to (33), wherein each -Rs, if present, is independently -Me.
The Group -R1
(42) A compound according to any one of (1) to (41), wherein -RQ , or each -R , is independently -RCA or -RHA.
(42) A compound according to any one of (1) to (41), wherein -RQL, or each -RQL, is independently -RCA.
(44) A compound according to any one of (1) to (41), wherein -RQL, or each -RQL, is independently -Rm. (45) A compound according to any one of (1) to (41), wherein -RQL, or each -RQL, is independently -Rcc
(46) A compound according to any one of (1) to (41), wherein -RQL, or each -RQL, is independently -RHC.
The Group -R'
(47) A compound according to any one of (1) to (46), wherein -R , if present, or each -RCA, if present, is independently phenyl; and is optionally substituted, for example, with one or more groups -R .
(48) A compound according to any one of (1) to (46), wherein -RCA, if present, or each -RCA, if present, is independently naphthyl; and is optionally substituted, for example, with one or more groups
The Group -R . Phenyl
(49) A compound according to any one of (1) to (46), wherein -R , if present, or each -RCA, if present, is independently:
Figure imgf000025_0001
wherein each of -R , -R , -R , -RP5, and -R is independently -H or a ring substituent; for example, wherein each ring substituent is independently -Rx. (50) A compound according to any one of (1) to (46), wherein -RCA, if present, or each -RCA, if present, is independently:
Figure imgf000026_0001
wherein each of -R , -R , and -RP5 is independently -H or a ring substituent;
for example, wherein each ring substituent is independently -Rx.
(51) A compound according to any one of (1) to (46), wherein -RCA, if present, or each -RCA, if present, is independently:
Figure imgf000026_0002
wherein each of -RP3 and -Rw is independently -H or a ring substituent; for example, wherein each ring substituent is independently -Rx
(52) A compound according to any one of (1) to (46), wherein -RCA, if present, or each -RCA, if present, is independently:
Figure imgf000026_0003
wherein -R is independently a ring substituent; for example, wherein the ring substituent is independently -Rx
(53) A compound according to any one of (1) to (46), wherein -RCA, if present, or each -RCA, if present, is independently:
Figure imgf000026_0004
wherein -R is independently a ring substituent; for example, wherein the ring substituent is independently -Rx. (54) A compound according to any one of (1) to (46), wherein -RCA, if present, or each -RCA, if present, is independently:
Figure imgf000027_0001
wherein each of -RP3 and -RP4 is independently a ring substituent; for example, wherein each ring substituent is independently -R*
(55) A compound according to any one of (1) to (46), wherein -RCA, if present, or each -RCA, if present, is independently:
Figure imgf000027_0002
wherein each of -R and -R is independently -H or a ring substituent; for example, wherein each ring substituent is independently -Rx.
(56) A compound according to any one of (1) to (46), wherein -RCA, if present, or each -RCA, if present, is independently:
Figure imgf000027_0003
wherein each of -RP3 and -RP5 is independently a ring substituent; for example, wherein each ring substituent is independently -Rx.
(57) A compound according to any one of (1) to (46), wherein -R , if present, or each -RCA, if present, is independently:
Figure imgf000027_0004
wherein each of -RP2 and -R is independently a ring substituent; for example, wherein each ring substituent is independently -Rx (58) A compound according to any one of (1) to (46), wherein -R , if present, or each -RCA, if present, is independently:
Figure imgf000028_0001
wherein each of -R and -R is independently a ring substituent; for example, wherein each ring substituent is independently -Rx.
(59) A compound according to any one of (1) to (46), wherein -RCA, if present, or each -RCA, if present, is independently:
Figure imgf000028_0002
wherein -R is independently a ring substituent; for example, wherein the ring substituent is independently -Rx.
(60) A compound according to any one of (1) to (46), wherein -RCA, if present, or each -RCA, if present, is independently:
Figure imgf000028_0003
wherein each of -R , -R , and -R is independently a ring substituent; for example, wherein each ring substituent is independently -Rx.
(61) A compound according to any one of (1) to (46), wherein -RCA, if present, or each -RCA, if present, is independently:
Figure imgf000028_0004
The Group -R' (62) A compound according to any one of (1) to (61), wherein -R , if present, or each -RHA, if present, is independently furanyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, oxazoiyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, pyridazinyl, indolyl, isoindolyl, indazolyl, benzofuranyl, isobenzofuranyl, benzothienyl, isobenzothienyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, benzoisoxazolyl, quinolinyl, isoquinolinyl, dihydroisoquinolinyl, cinnolinyl, or quinazolinyl; and is optionally substituted, for example, with one or more groups -Rx.
(63) A compound according to any one of (1) to (61), wherein -RHA, if present, or each -RHA, if present, is independently furanyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, pyridazinyl, and indolyl; and is optionally substituted, for example, with one or more groups -Rx.
(64) A compound according to any one of (1) to (61), wherein -RHA, if present, or each -RHA, if present, is independently thienyl, pyrazolyl, pyridyl, pyrimidinyl, and indolyl; and is optionally substituted, for example, with one or more groups -Rx.
(65) A compound according to any one of (1 ) to (61), wherein -RHA, if present, or each -RHA, if present, is independently pyridyl; and is optionally substituted, for example, with one or more groups -Rx
(66) A compound according to any one of (1) to (61), wherein -RHA, if present, or each -RHA, if present, is independently pyrid-2-yl; and is optionally substituted, for example, with one or more groups -Rx.
(67) A compound according to any one of (1) to (61), wherein -RHA, if present, or each -RHA, if present, is independently pyrid-3-yl; and is optionally substituted, for example, with one or more groups -Rx. (68) A compound according to any one of (1) to (61), wherein -RHA, if present, or each -RHA, if present, is independently pyrid-4-yl; and is optionally substituted, for example, with one or more groups -Rx.
(69) A compound according to any one of (1) to (61), wherein -RHA, if present, or each -RHA, if present, is independently thienyl; and is optionally substituted, for example, with one or more groups -Rx.
(70) A compound according to any one of (1) to (61), wherein -RHA, if present, or each -RHA, if present, is independently thien-3-yl; and is optionally substituted, for example, with one or more groups -Rx.
(71) A compound according to any one of (1) to (61), wherein -RHA, if present, or each -RHA, if present, is independently pyrazolyl; and is optionally substituted, for example, with one or more groups -Rx
(72) A compound according to any one of (1) to (61), wherein -RHA, if present, or each -RHA, if present, is independently 1H-pyrazolyl; and is optionally substituted, for example, with one or more groups -Rx. (73) A compound according to any one of (1) to (61), wherein -R , if present, or each -R^, if present, is independently 1 H-pyrazol-4-yl; and is optionally substituted, for example, with one or more groups -Rx.
(74) A compound according to any one of (1) to (61), wherein -RHA, if present, or each -R^, if present, is independently 1H-pyrazol-1-yl; and is optionally substituted, for example, with one or more groups -Rx. (75) A compound according to any one of (1) to (61), wherein -RHA, if present, or each -RHA, if present, is independently pyrimidinyl; and is optionally substituted, for example, with one or more groups -Rx.
(76) A compound according to any one of (1) to (61), wherein -RHA, if present, or each -RHA, if present, is independently pyrimidin-5-yl; and is optionally substituted, for example, with one or more groups -Rx.
(77) A compound according to any one of (1) to (61), wherein -RHA, if present, or each -RHA, if present, is independently indolyl; and is optionally substituted, for example, with one or more groups -Rx.
(78) A compound according to any one of (1) to (61), wherein -RHA, if present, or each -RHA, if present, is independently indol-5-yl; and is optionally substituted, for example, with one or more groups -Rx.
(79) A compound according to any one of (1) to (61), wherein -RHA, if present, or each -RHA, if present, is independently benzo[d]isoxazo)-3-one-5-yl; and is optionally substituted, for example, with one or more groups -Rx. (80) A compound according to any one of (1) to (61), wherein -RHA, if present, or each -RHA, if present, is independently benzo[d]isoxazol-3-one-5-yl.
(81 ) A compound according to any one of (1 ) to (61 ), wherein -R^, if present, or each -RHA, if present, is independently 3,4-dihydro-2H-isoquinolin-1-one-7-yl; and is optionally substituted, for example, with one or more groups -R
(82) A compound according to any one of (1) to (61), wherein -RHA, if present, or each -RHA, if present, is independently 3,4-dihydro-2H-isoquinolin-1-one-7-yl. The Group -Rcc
(83) A compound according to any one of (1) to (82), wherein -Rcc, if present, or each -Rcc, if present, is non-aromatic C3.7cycloalkyl; and is optionally substituted, for example, with one or more groups -R .
(84) A compound according to any one of (1) to (82), wherein -Rcc, if present, or each -Rcc, if present, is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; and is optionally substituted, for example, with one or more groups -Rx
(85) A compound according to any one of (1) to (82), wherein -Rcc, if present, or each -Rcc, if present, is cyclopentyl or cyclohexyl; and is optionally substituted, for example, with one or more groups -Rx. (86) A compound according to any one of (1) to (82) wherein -Rcc, if present, or each -Rcc, if present, is non-aromatic C3.7cycloalkenyl; and is optionally substituted, for example, with one or more groups -Rx.
(87) A compound according to any one of (1) to (82), wherein -Rcc, if present, or each -Rcc, if present, is cyclopropenyl, cyclobutenyl, cyclopentenyl, or cyclohexenyl; and is optionally substituted, for example, with one or more groups -Rx.
(88) A compound according to any one of (1) to (82), wherein -Rcc, if present, or each -Rcc, if present, is cyclopentenyl or cyclohexenyl; and is optionally substituted, for example, with one or more groups -Rx
(89) A compound according to any one of (1) to (82), wherein -Rcc, if present, or each -Rcc, if present, is cyclohexenyl; and is optionally substituted, for example, with one or more groups -R .
The Group -RHC
(90) A compound according to any one of (1) to (89), wherein -RHC, if present, or each -RHC, if present, is non-aromatic C3-7heterocyclyl; and is optionally substituted, for example, with one or more groups -Rx; wherein the non-aromatic C3.7heterocyclyl has from 3 to 7 ring atoms, wherein exactly 1 or exactly 2 of said ring atoms is N, and the remaining ring atoms are C.
(91) A compound according to any one of (1) to (89), wherein -RHC, if present, or each -RHC, if present, is non-aromatic Cs-eheterocyclyl; and is optionally substituted, for example, with one or more groups -Rx; wherein the non-aromatic C3.rheterocyclyl has from 5 to 6 ring atoms, wherein exactly 1 or exactly 2 of said ring atoms is N, and the remaining ring atoms are C. (92) A compound according to any one of (1) to (89), wherein -R , if present, or each -RHC, if present, is tetrahydropyranyl, azetidinyl, pyrrolidinyl, imidazolidinyl, piperidinyl, piperizinyl, morpholinyl, azepinyl, or diazepinyl; and is optionally substituted, for example, with one or more groups -Rx.
(93) A compound according to any one of (1) to (89), wherein -RHC, if present, or each -RHC, if present, is azetidino, pyrrolidino, imidazolidino, piperidino, piperizino, morpholino, azepino, or diazepino; and is optionally substituted, for example, with one or more groups -R
(94) A compound according to any one of (1) to (89), wherein -RHC, if present, or each -RHC, if present, is piperidino, piperizino, or morpholino; and is optionally substituted, for example, with one or more groups -Rx.
(95) A compound according to any one of (1) to (89), wherein -RHC, if present, or each -RHC, if present, is piperidino; and is optionally substituted, for example, with one or more groups -Rx. (96) A compound according to any one of (1) to (89), wherein -RHC, if present, or each -RHC, if present, is piperizino; and is optionally substituted, for example, with one or more groups -Rx.
(97) A compound according to any one of (1) to (89), wherein -RHC, if present, or each -RHC, if present, is morpholino; and is optionally substituted, for example, with one or more groups -Rx.
Optional Substituents on -RCA. -RHA -Rcc. and -RHC (98) A compound according to any one of (1) to (84), wherein each of -RCA, -RHA, -Rcc, and -RHC, if present, is optionally substituted with one or more substituents, -Rx, wherein each -R is independently selected from -RX1 and -R*2, wherein: each -RX1 is independently:
-Rz
-F, -CI, -Br, -I,
-CF3, -OCF3,
-OH, -RZL-OH, -0-RZL-OH, -NH-RZL-OH, -NRz-RZL-OH,
-0R -RZL-0R -0-RZL-OR2, -NH-RZL-ORz, -NR -RZL-ORz,
-SH, -SRZ,
-CN,
-N02, -C(=0)OH, -C(=0)ORz,
-C(=0)Rz,
-NH2, -NHRZ, -NRz 2, -RNZ,
-RZL-NH2l -R -NHR2, -R2L-NR2 2, -RZL-RNZ,
-0-R2L-NH2, -0-RZL-NHRz, -0-RZL-NRz 2, -0-RZL-RNZ,
-NH-RZL-NH2, -NH-RZL-NHR2, -NH-R2L-NR2 2, -NH-R2L-RNZ,
-NRZ-R2L-NH2, -NRZ-RZL-NHRZ, -NRZ-RZL-NRZ 2, -NRZ-RZL-RNZ,
-C(=0)NH2, -C(=0)NHRz, -C(=0)NRz 2, -C(=0)RNZ,
-RZL-C(=0)NH2, -RZL-C(=0)NHRz, -RZL-C(=0)NRz 2, -RZL-C(=0)RNZ,
-0-RZL-C(=0)NH2, -0-RZL-C(=0)NHRz, -0-RZL-C(=0)NRz 2, -0-RZL-C(=0)RNZ,
-NH-C(=0)R2, -NRz-C(=0)Rz,
-NH-C(=0)OH, -NRz-C(=0)OH, -NH-C(=0)ORz, -NRz-C(=0)ORz,
-OC(=0)NH2, -OC(=0)NHRz, -OC(=0)NRz 2, -OC(=0)RNZ,
-NHC(=0)NH2, -NHC(=0)NHRz -NHC(=0)NRz 2, -NHC(=0)RNZ,
-NRzC(=0)NH2, -NRzC(=0)NHRz, -NRzC(=0)NRz 2, -NRzC(=0)RNZ,
-S(=0)2Rz,
-S(=0)2NH2> -S(=0)2NHR2, -S(=0)2NRz 2, -S(=0)2RNZ,
-NH-S(=0)2Rz, -NRz-S(=0)2Rz, or
=0;
and additionally, two or more adjacent substituents -RX1, if present, may together form -OCH20-, -OCH2CH20-, or -OCH2CH2CH20-; wherein:
each -RZL- is independently saturated aliphatic C-^alkylene;
each -RNZ is independently azetidino, pyrrolidino, piperidino, piperazino, morpholino, azepino, or diazepino, and is optionally substituted with one or more substitutents selected from saturated aliphatic C^alkyl;
each -Rz is independently saturated aliphatic C^alkyl, phenyl, benzyl, or
C5.5heteroaryl, wherein phenyl, benzyl, and C5.6heteroaryl are each optionally substituted with one or more substituents selected from -F, -CI, -Br, -I, -R22, -CF3, -OH, -OR22, -OCF3, -NH2, -NHR22, -NR2^, -C(=0)OH, -C(=0)NH2, -C(=0)NHR2z, and -C(=0)NR2z 2, wherein each -R22 is independently saturated aliphatic C1-4alkyl; and each -R 2 is independently:
-NH-C(=J)-NH2, -NRK -C(=J)-NH2,
Figure imgf000033_0001
-NH-C(=0)H, -NRK1-C(=0)H,
-NH-C(=0)RKS, -NRK1-C(=0)RK5, -C(=0)-NH2, -C(=0)-NHRK2, -C(=0)-NRK2 2, -C(=0)-NRK3RK4,
-OH, or -ORKS; wherein each =J is independently =0 or =S; and wherein each -R is independently saturated aliphatic C1-4alkyl; and wherein each -R 2 is independently:
saturated aliphatic d.4alkyl, aliphatic C^alkenyl, or saturated C3.6cycloalkyl;
each optionally substituted with one or more groups independently selected from: -F, -CI, -Br, -I, -OH, -ORA1, -OCF3, -SRA1, -S(=0)RA1, -S(=0)2RA1, -NH2, -NHRA1, -NRA 2, pyrrolidinyl, piperidinyl, morpholinyl, piperizinyl, -C(=0)OH, -C(=0)ORA1, -OC(=0)RA\ -C(=0)NH2, -C(=0)NHRA1, -C(=0)NRA 2, pyrrolidino-C(=0)-, piperidino-C(=0)-, morpholino-C(=0)-, piperizino-C(=0)-, and -RAZ;
wherein:
each pyrrolidinyl, piperidinyl, morpholinyl, piperizinyl, pyrrolidino, piperidino, morpholino, and piperizino is optionally substituted with one or more groups
independently selected from -F, -RA1, -OH, -ORA , -NH2, -NHRA1, -NRA1 2, pyrrolidino, piperidino, morpholino, piperizino, -C(=0)OH, -C(=0)ORA1, -C(=0)RA1, -C(=0)NH2,
-C(=0)NHRA1, -C(=0)NRA1 2, pyrrolidino-C(=0)-, piperidino-C(=0)-, morpholino-C(=0)-, piperizino-C(=0)-, and -0CH2CH20-;
each -RA1 is independently saturated aliphatic C1-4alkyl; and
each -RA2 is independently phenyl, C5.6heteroaryl, or non-aromatic
C5.7heterocyclyl; and wherein each -NRK3RK4 is independently:
pyrrolidino, piperidino, morpholino, or piperizino;
each optionally substituted with one or more groups independently selected from -F, -RA3, -OH, -ORA3, -NH2, -NHRA3, -NRA3 2, pyrrolidino, piperidino, morpholino, piperizino, -C(=0)OH, -C(=0)ORA3, -C(=0)RA3, -C(=0)NH2, -C(=0)NHRA3, -C(=0)NRA3 2,
pyrrolidino-C(=0)-, piperidino-C(=0)-, morpholino-C(=0)-, piperizino-C(=0)-, and
-OCH2CH20-;
wherein each -RA3 is independently saturated aliphatic C^alkyl, optionally substituted with one or more groups independently selected from -OH, -ORA", -NH2, -NHRA4, -NRM 2, pyrrolidino, piperidino, morpholino, and piperizino;
wherein each -R is independently saturated aliphatic C^alkyl; and wherein each -RKS is independently saturated aliphatic Ci.4alkyl, and is optionally substituted with one or more groups independently selected from: -F, -CI, -Br, -I, -CF3, -OH, -ORA5, -OCF3, -NH2, -NHRA5, and -NRA5 2, wherein each -RAS is independently saturated aliphatic C^alkyl; and wherein each -R is independently saturated aliphatic C1-4alkyl, and is optionally substituted with one or more groups independently selected from: -F, -CI, -Br, -I, -CF3l -OH, -ORA6, -OCF3, -NH2, -NHRA6, and -NRAS 2, wherein each -RA6 is independently saturated aliphatic C^alkyl.
Optional Substituents -Rx
(99) A compound according to (98), wherein:
if exactly one group -Rx is present, then -Rx is -RX2; and
if a plurality of groups -Rx are present, then at least one -R is -RX2.
(100) A compound according to (98), wherein:
if exactly one group -Rx is present, then -Rx is -RX2; and
if a plurality of groups -Rx are present, then exactly one -Rx is -RX2.
(101) A compound according to (98), wherein:
exactly one group -R is present, and -Rx is -R*2.
(102) A compound according to (98), wherein:
exactly two groups -Rx are present, one -Rx is -RX2, and the other -Rx is -RX1.
Optional Substituents -RX
( 03) A compound according to any one of (98) to (102), wherein each -RX1, if present, is independently:
-Rz,
-F, -CI, -Br, -I,
-CF3, -OCF3,
-OH, -RZL-OH, -0-RZL-OH, -NH-RZL-OH, -NRz-RZL-OH,
-OR2, -R2L-OR2, -0-R2L-OR2 -NH-R2L-ORz, -NRz-RZL-OR2,
-CN,
-N02,
-C(=0)OH, -C(=0)ORz,
-C(=0)R2,
-NH2, -NHR2, -NR2 2, -RNZ,
-RZL-NH2, -RZL-NHR2, -RZL-NRZ 2, -RZL-RNZ,
-0-RZL-NH2, -0-RZL-NHR2, -0-RZL-NRz 2, -0-RZL-RNZ,
-NH-RZL-NH2, -NH-R2L-NHRZ, -NH-RZL-NR2 2, -NH-RZL-RNZ,
-NRZ-RZL-NH2, -NRZ-R2L-NHRZ, -NRZ-RZL-NRZ 2, -NRZ-RZL-RN2,
-C(=0)NH2, -C(=0)NHRz, -C(=0)NRz 2, -C(=0)RNZ,
-NH-C(=0)Rz, -NRz-C(=0)R2,
-S(=0)2Rz,
-S(=0)2NH2, -S(=0)2NHRz, -S(=0)2NRz 2, -S(=0)2RNZ, -NH-S(=0)2Rz, or -NR2-S(=0)2Rz
(104) A compound according to any one of (98) to (102), wherein each -RX1, if present, is independently:
-R2,
-F, -CI, -Br, -I,
-CF3> -OCF3,
-OH, -R2L-OH, -0-R2L-OH,
-ORz, -R2L-ORz, -0-RZL-ORz,
-CN,
-N02,
-C(=0)OH, -C(=0)OR2,
-C(=0)Rz,
-NH2, -NHR2, -NRz 2l -RNZ,
-R2L-NH2, -R2L-NHRZ, -R2L-NR2 2, -RZL-RN2,
-NH-RZL-NH2, -NH-R2L-NHR2, -NH-RZL-NRZ 2, -NH-RZL-RNZ,
-C(=0)NH2, -C(=0)NHR2, -C(=0)NRz 2, -C(=0)RNZ,
-NH-C(=0)R2, or -NRz-C(=0)Rz. (105) A compound according to any one of (98) to (102), wherein each -RX , if present, is independently:
-Rz,
-F, -CI, -Br, -I,
-CF3, -OCF3,
-OH, -R2L-OH,
-ORz, -RZL-ORz,
-CN,
-N02,
-C(=0)OH, -C(=0)OR2,
-C(=0)Rz,
-NH2, -NHRZ, -NRZ 2, -RNZ,
-RZL-NH2, -R2L-NHRZ, -RZL-NR2 2, -RZL-RN2,
-C(=0)NH2l -C(=0)NHR2, -C(=0)NRz 2, or
-NH-C(=0)R2
( 06) A compound according to any one of (98) to (102), wherein each -RX1, if present, is independently:
-Rz,
-F, -CI, -Br,
-OH,
-OR2,
-C(=0)OH, -C(=0)ORz,
-NH2, -NHRZ, -NRZ 2, -RNZ, -C(=0)NH2, -C(=0)NHR2, -C(=0)NR2 2, or
-NH-C(=0)R2.
(107) A compound according to any one of (98) to (102), wherein each -RX , if present, is independently -Rz, -F, -CI, -Br, -OH, or -ORz.
(108) A compound according to any one of (98) to (107), wherein each -RZL-, if present, is independently saturated aliphatic C2-4alkylene. (109) A compound according to any one of (98) to (107), wherein each -RZL-, if present, is independently -CH2CH2-.
(110) (A compound according to any one of (98) to (109), wherein each -RNZ, if present, is independently pyrrolidino, piperidino, piperazino, or morpholino, and is optionally substituted with one or more substitutents selected from saturated aliphatic Ci.4alkyl.
(1 1) A compound according to any one of (98) to (110), wherein each -Rz, if present, is independently saturated aliphatic d.6alkyl, phenyl, or benzyl. (112) A compound according to any one of (98) to (110), wherein each -Rz, if present, is independently saturated aliphatic Ci.6alkyl.
(113) A compound according to any one of (98) to (110), wherein each -Rz, if present, is independently saturated aliphatic C^alkyl.
Optional Substituents -RX2
(114) A compound according to any one of (98) to (113), wherein each -R*2, if present, is independently:
Figure imgf000037_0001
(115) A compound according to any one of (98) to (113), wherein each -RX2, if present, is independently:
-NH-C(=0)-NH2, -NRK1-C(=0)-NH2,
-NH-C(=0)-NHRK2, -NRK1-C(=0)-NHRK2 )
Figure imgf000037_0002
-NH-C(=0)-NRK3RM, or -NRK1-C(=0)-NRK3RK4 (1 16) A compound according to any one of (98) to ( 13), wherein each -RX2, if present, is independently:
-NH-C(=0)H, -NRK1-C(=0)H,
-NH-C(=0)RK5, -NRK1-C(=0)RK5,
-C(=0)-NH2, -C(=0)-NHRK2, -C(=0)-NRK2 2, or -C(=0)-NRK3R
(1 17) A compound according to any one of (98) to (1 13), wherein each -RX2, if present, is independently:
-NH-C(=0)H, -NRK1-C(=0)H,
-NH-C(=0)RK5, or -NRK -C(=0)RK5.
( 18) A compound according to any one of (98) to (1 13), wherein each -R*2, if present, is independently:
-C(=0)-NH2, -C(=0)-NHRK2, -C(=0)-NRK 2, or -C(=0)-NRK3RM
( 19) A compound according to any one of (98) to ( 13), wherein each -RX2, if present, is independently:
-OH or -ORK6. The Group -RK1
(120) A compound according to any one of (98) to (1 19), wherein each -RK , if present, is independently -Me, -Et, -nPr, -iPr, -nBu, -iBu, -sBu, or -tBu. (121) A compound according to any one of (98) to (1 19), wherein each -R \ if present, is independently -Me or -Et.
(122) A compound according to any one of (98) to (119), wherein each -RK1, if present, is independently -Me.
The Group -RK2
(123) A compound according to any one of (98) to (122), wherein each -R 2, if present, is independently: saturated aliphatic d.4alkyl, optionally substituted with one or more groups independently selected from: -F, -CI, -Br, -I, -OH, -ORA1, -OCF3, -SRA1, -S(=0)RA1, -S(=0)2RA , -NH2, -NHRA1, -NRA1 2, pyrrolidinyl, piperidinyl, morpholinyl, piperizinyl, -C(=0)OH, -C(=0)ORA1, -OC(=0)RA1, -C(=0)NH2, -C(=0)NHRA , -C(=0)NRA1 2, pyrrolidino-C(=0)-, piperidino-C(=0)-, morpholino-C(=0)-, piperizino-C(=0)-, and -RA2. (124) A compound according to any one of (98) to (122), wherein each -R , if present, is independently saturated aliphatic Ci^alkyl. (125) A compound according to any one of (98) to (122), wherein each -R 2, if present, is independently aliphatic C2.4alkenyl.
(126) A compound according to any one of (98) to (122), wherein each -R*2, if present, is independently saturated C3.6cycloalkyl.
The Group -RA1
(127) A compound according to any one of (98) to (126), wherein each -RA1, if present, is independently -Me, -Et, -nPr, -iPr, -nBu, -iBu, -sBu, or -tBu.
(128) A compound according to any one of (98) to (126), wherein each -RA1, if present, is independently -Me or -Et. (129) A compound according to any one of (98) to (126), wherein each -RA , if present, is independently -Me.
The Group -RA2 (130) A compound according to any one of (98) to (129), wherein each -R*2, if present, is independently phenyl or C5-6heteroaryl.
(131) A compound according to any one of (98) to (129), wherein each -Rw, if present, is independently phenyl.
The Group -NR 3RK4
(132) A compound according to any one of (98) to (131), wherein each -NRK3R 4, if present, is independently pyrrolidine piperidino, morpholino, or piperizino.
The Group -RA3
(133) A compound according to any one of (98) to ( 32), wherein each -RA3, if present, is independently saturated aliphatic C^alkyl.
(134) A compound according to any one of (98) to (132), wherein each -RA3, if present, is independently -Me, -Et, -nPr, -iPr, -nBu, -iBu, -sBu, or -tBu.
(135) A compound according to any one of (98) to (132), wherein each -RA3, if present, is independently -Me or -Et.
(136) A compound according to any one of (98) to ( 32), wherein each -RA3, if present, is independently -Me. The Group -RM
(137) A compound according to any one of (98) to (136), wherein each -RA4, if present, is independently -Me, -Et, -nPr, -iPr, -nBu, -iBu, -sBu, or -tBu.
( 38) A compound according to any one of (98) to ( 36), wherein each -RA4, if present, is independently -Me or -Et. (139) A compound according to any one of (98) to (136), wherein each -RA4, if present, is independently -Me.
The Group -RKS (140) A compound according to any one of (98) to (139), wherein each -RKS, if present, is independently saturated aliphatic d.4alkyl, and is optionally substituted with one or more groups independently selected from: -OH, -ORA5, -NH2, -NHRAS, and -NRA5 2.
(141 ) A compound according to any one of (98) to (139), wherein each -RK5, if present, is independently saturated aliphatic C^alkyl.
The Group -RAS
(142) A compound according to any one of (98) to (141), wherein each -RA5, if present, is independently -Me, -Et, -nPr, -iPr, -nBu, -iBu, -sBu, or -tBu.
(143) A compound according to any one of (98) to (141), wherein each -RA5, if present, is independently -Me or -Et. (144) A compound according to any one of (98) to (141), wherein each -RA5, if present, is independently -Me.
The Group -RK6 (145) A compound according to any one of (98) to (144), wherein each -RK6, if present, is independently saturated aliphatic Ci.4alkyl, and is optionally substituted with one or more groups independently selected from: -OH, -ORA6, -NH2, -NHRA6, and -NRA6 2.
(146) A compound according to any one of (98) to (144), wherein each -RK6, if present, is independently saturated aliphatic C1-4alkyl. The Group -FT6
(147) A compound according to any one of (98) to (146), wherein each -RA6, if present, is independently -Me, -Et, -nPr, -iPr, -nBu, -iBu, -sBu, or -tBu.
(148) A compound according to any one of (98) to (146), wherein each -RA6, if present, is independently -Me or -Et.
(149) A compound according to any one of (98) to ( 46), wherein each -RAe, if present, is independently -Me.
Examples of Some Specific Embodiments
(150) A compound according to (1), selected from compounds of the following formulae and pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
(151) A compound according to (1), selected from compounds of the following formulae and pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
(152) A compound according to (1), selected from compounds of the following formulae and pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000053_0002
(153) A compound according to (1), selected from compounds of the following formulae and pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000054_0001
Figure imgf000055_0001
Figure imgf000056_0001
(154) A compound according to (1), selected from compounds of the following formulae and pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000056_0002
80
-56-
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
(155) A compound according to (1), selected from compounds of the following formulae and pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000061_0003
(156) A compound according to (1), selected from compounds of the following formulae and pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000061_0002
Figure imgf000062_0001
(157) A compound according to (1 ), selected from compounds of the following formulae and pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000062_0002
Figure imgf000063_0001
Figure imgf000064_0001
(158) A compound according to (1), selected from compounds of the following formulae and pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000065_0002
(159) A compound according to (1 ), selected from compounds of the following formulae and pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000065_0001
Figure imgf000066_0001
(160) A compound according to (1), selected from compounds of the following formulae and pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000067_0001
Combinations
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the chemical groups represented by the variables (e.g., -X=, -Y-, -RY, -R26, -R27, -R28, -R29,
DQS pSSS rjX DP2 DP3 5 6
-Γν
-RX2, -R2, -R2L-, -RN2, -R22, -RK1, -RK2, -NRK3RK4, -R 5, -RK6, =J, -RA1, -RA2, -RA3, -RM, -RA5, -RA6, etc.) are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace compounds that are stable compounds (i.e., compounds that can be isolated, characterised, and tested for biological activity).
In addition, all sub-combinations of the chemical groups listed in the embodiments describing such variables are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination of chemical groups was individually and explicitly disclosed herein. Substantially Purified Forms
One aspect of the present invention pertains to FTA compounds, as described herein, in substantially purified form and/or in a form substantially free from contaminants. In one embodiment, the substantially purified form is at least 50% by weight, e.g., at least 60% by weight, e.g., at least 70% by weight, e.g., at least 80% by weight, e.g., at least 90%) by weight, e.g., at least 95% by weight, e.g., at least 97% by weight, e.g., at least 98% by weight, e.g., at least 99% by weight. Unless specified, the substantially purified form refers to the compound in any
stereoisomeric or enantiomeric form. For example, in one embodiment, the substantially purified form refers to a mixture of stereoisomers, i.e., purified with respect to other compounds. In one embodiment, the substantially purified form refers to one
stereoisomer, e.g., optically pure stereoisomer. In one embodiment, the substantially purified form refers to a mixture of enantiomers. In one embodiment, the substantially purified form refers to a equimolar mixture of enantiomers (i.e., a racemic mixture, a racemate). In one embodiment, the substantially purified form refers to one enantiomer, e.g., optically pure enantiomer. In one embodiment, the contaminants represent no more than 50% by weight, e.g., no more than 40% by weight, e.g., no more than 30% by weight, e.g., no more than 20% by weight, e.g., no more than 10% by weight, e.g., no more than 5% by weight, e.g., no more than 3% by weight, e.g., no more than 2% by weight, e.g., no more than 1% by weight. Unless specified, the contaminants refer to other compounds, that is, other than stereoisomers or enantiomers. In one embodiment, the contaminants refer to other compounds and other stereoisomers. In one embodiment, the contaminants refer to other compounds and the other enantiomer.
In one embodiment, the substantially purified form is at least 60% optically pure (i.e., 60% of the compound, on a molar basis, is the desired stereoisomer or enantiomer, and 40% is the undesired stereoisomer or enantiomer), e.g., at least 70% optically pure, e.g., at least 80% optically pure, e.g., at least 90% optically pure, e.g., at least 95% optically pure, e.g., at least 97% optically pure, e.g., at least 98% optically pure, e.g., at least 99% optically pure.
Isomers
Certain compounds may exist in one or more particular geometric, optical, enantiomeric, diastereoisomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and l-forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; a- and β-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms"). A reference to a class of structures may well include structurally isomeric forms falling within that class (e.g., d-7alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl). However, reference to a specifc group or substitution pattern is not intended to include other structural (or constitutional isomers) which differ with respect to the connections between atoms rather than by positions in space. For example, a reference to a methoxy group, -OCH3, is not to be construed as a reference to its structural isomer, a
hydroxymethyl group, -CH2OH. Similarly, a reference to ortho-chlorophenyl is not to be construed as a reference to its structural isomer, meta-chlorophenyl. The above exclusion does not pertain to tautomeric forms, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/enol (illustrated below), imine/enamine, amide/imino alcohol, amidine/amidine, nitroso/oxime, thioketone/enethiol, N-nitroso/hydroxyazo, and nitro/aci-nitro.
Figure imgf000069_0001
keto enol enolate Note that specifically included in the term "isomer" are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including 1H, 2H (D), and 3H (T); C may be in any isotopic form, including 12C, 13C, and 4C; O may be in any isotopic form, including 160 and 180; and the like.
Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including mixtures (e.g., racemic mixtures) thereof. Methods for the preparation (e.g., asymmetric synthesis) and separation (e.g., fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.
Salts It may be convenient or desirable to prepare, purify, and/or handle a corresponding salt of the compound, for example, a pharmaceutically-acceptable salt. Examples of pharmaceutically acceptable salts are discussed in Berge ei a/., 1977, "Pharmaceutically Acceptable Salts," J. Pharm. Sci.. Vol. 66, pp. 1-19. For example, if the compound is anionic, or has a functional group which may be anionic (e.g., -COOH may be -COO"), then a salt may be formed with a suitable cation.
Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na+ and K+, alkaline earth cations such as Ca2+ and Mg2+, and other cations such as Af3. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH ) and substituted ammonium ions (e.g., NH3R+, NH2R2 +, NHR3\ NR„+). Examples of some suitable substituted ammonium ions are those derived from:
ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4+
If the compound is cationic, or has a functional group which upon protonation may become cationic (e.g., -NH2 may become -NH3 +), then a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric, nitrous, phosphoric, and phosphorous.
Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, trifluoroacetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric.
Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose. Unless otherwise specified, a reference to a particular compound also includes salt forms thereof.
Solvates and Hydrates It may be convenient or desirable to prepare, purify, and/or handle a corresponding solvate of the compound. The term "solvate" is used herein in the conventional sense to refer to a complex of solute (e.g., compound, salt of compound) and solvent. If the solvent is water, the solvate may be conveniently referred to as a hydrate, for example, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.
Unless otherwise specified, a reference to a particular compound also includes solvate and hydrate forms thereof.
Chemically Protected Forms
It may be convenient or desirable to prepare, purify, and/or handle the compound in a chemically protected form. The term "chemically protected form" is used herein in the conventional chemical sense and pertains to a compound in which one or more reactive functional groups are protected from undesirable chemical reactions under specified conditions (e.g., pH, temperature, radiation, solvent, and the like). In practice, well known chemical methods are employed to reversibly render unreactive a functional group, which otherwise would be reactive, under specified conditions. In a chemically protected form, one or more reactive functional groups are in the form of a protected or protecting group (also known as a masked or masking group or a blocked or blocking group). By protecting a reactive functional group, reactions involving other unprotected reactive functional groups can be performed, without affecting the protected group; the protecting group may be removed, usually in a subsequent step, without substantially affecting the remainder of the molecule. See, for example, Protective Groups in Organic Synthesis (T. Green and P. Wuts; 4th Edition; John Wiley and Sons, 2006).
A wide variety of such "protecting," "blocking," or "masking" methods are widely used and well known in organic synthesis. For example, a compound which has two nonequivalent reactive functional groups, both of which would be reactive under specified conditions, may be derivatized to render one of the functional groups "protected," and therefore unreactive, under the specified conditions; so protected, the compound may be used as a reactant which has effectively only one reactive functional group. After the desired reaction (involving the other functional group) is complete, the protected group may be "deprotected" to return it to its original functionality. For example, a hydroxy group may be protected as an ether (-OR) or an ester
(-OC(=0)R), for example, as: a t-butyl ether; a benzyl, benzhydryl (diphenylmethyl), or trityl (triphenylmethyl) ether; a trimethylsilyl or t-butyldimethylsilyl ether; or an acetyl ester (-OC(=0)CH3, -OAc).
For example, an aldehyde or ketone group may be protected as an acetal (R-CH(OR)2) or ketal (R2C(OR)2), respectively, in which the carbonyl group (>C=0) is converted to a diether (>C(OR)2), by reaction with, for example, a primary alcohol. The aldehyde or ketone group is readily regenerated, for example, by hydrolysis using water in the presence of acid.
For example, an amine group may be protected, for example, as an amide (-NRCO-R) or a urethane (-NRCO-OR), for example, as: a methyl amide (-NHCO-CH3); a benzyloxy amide (-NHCO-OCH2C6H5, -NH-Cbz); as a t-butoxy amide (-NHCO-OC(CH3)3, -NH-Boc); a 2-biphenyl-2-propoxy amide (-NHCO-OC(CH3)2C6H4C6H5, -NH-Bpoc), as a
9-fluorenylmethoxy amide (-NH-Fmoc), as a 6-nitroveratryloxy amide (-NH-Nvoc), as a 2-trimethylsilylethyloxy amide (-NH-Teoc), as a 2,2,2-trichloroethyloxy amide (-NH-Troc), as an allyloxy amide (-NH-Alloc), as a 2-(phenylsulfonyl)ethyloxy amide (-NH-Psec); or, in suitable cases (e.g., cyclic amines), as a nitroxide radical (>Ν-0·).
For example, a carboxylic acid group may be protected as an ester for example, as: an Ci.7alkyl ester (e.g., a methyl ester; a t-butyl ester); a C1-7haloalkyl ester (e.g., a
Ci.7trihaloalkyl ester); a triC1-7alkylsilyl-C1-7alkyl ester; or a C5-20aryl-Ci.7alkyl ester (e.g., a benzyl ester; a nitrobenzyl ester); or as an amide, for example, as a methyl amide.
For example, a thiol group may be protected as a thioether (-SR), for example, as: a benzyl thioether; an acetamidomethyl ether (-S-CH2NHC(=0)CH3). Prodrugs
It may be convenient or desirable to prepare, purify, and/or handle the compound in the form of a prodrug. The term "prodrug," as used herein, pertains to a compound which yields the desired active compound in vivo. Typically, the prodrug is inactive, or less active than the desired active compound, but may provide advantageous handling, administration, or metabolic properties.
For example, some prodrugs are esters of the active compound (e.g., a physiologically acceptable metabolically labile ester). During metabolism, the ester group (-C(=0)OR) is cleaved to yield the active drug. Such esters may be formed by esterification, for example, of any of the carboxylic acid groups (-C(=0)OH) in the parent compound, with, where appropriate, prior protection of any other reactive groups present in the parent compound, followed by deprotection if required. Also, some prodrugs are activated enzymatically to yield the active compound, or a compound which, upon further chemical reaction, yields the active compound (for example, as in antibody directed enzyme prodrug therapy (ADEPT), gene directed enzyme prodrug therapy (GDEPT), lipid directed enzyme prodrug therapy (LIDEPT), etc.). For example, the prodrug may be a sugar derivative or other glycoside conjugate, or may be an amino acid ester derivative.
Chemical Synthesis
Several methods for the chemical synthesis of FTA compounds of the present invention are described herein. These and/or other well known methods may be modified and/or adapted in known ways in order to facilitate the synthesis of additional compounds within the scope of the present invention.
Compositions
One aspect of the present invention pertains to a composition (e.g., a pharmaceutical composition) comprising a FTA compound, as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.
Another aspect of the present invention pertains to a method of preparing a composition (e.g., a pharmaceutical composition) comprising admixing a FTA compound, as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient.
Uses
The compounds described herein are useful, for example, in the treatment of diseases and conditions that are ameliorated by the inhibition of LIM kinase (LIMK) activity (e.g., LIMK1 activity and/or LIMK2 activity), such as, for example, proliferative conditions, cancer, etc.
Use in Methods of Inhibiting LIM Kinase (LIMK) One aspect of the present invention pertains to a method of inhibiting LIM kinase (LIMK) activity (e.g., LIMK1 activity and/or LIMK2 activity), in vitro or in vivo, comprising contacting LIMK (e.g., LIMK1 and/or LIMK2) with an effective amount of a
FTA compound, as described herein. One aspect of the present invention pertains to a method of inhibiting LIM kinase (LIMK) activity (e.g., LIMK1 activity and/or LIMK2 activity) in a cell, in vitro or in vivo, comprising contacting the cell with an effective amount of a FTA compound, as described herein. Suitable assays for determining LI K activity inhibition are described herein and/or are known in the art.
Use in Methods of Inhibiting Cell Proliferation, Etc.
The FTA compounds described herein, e.g., (a) regulate (e.g., inhibit) cell proliferation; (b) inhibit cell cycle progression; (c) promote apoptosis; or (d) a combination of one or more of these. One aspect of the present invention pertains to a method of regulating (e.g., inhibiting) cell proliferation (e.g., proliferation of a cell), inhibiting cell cycle progression, promoting apoptosis, or a combination of one or more these, in vitro or in vivo, comprising contacting a cell with an effective amount of a FTA compound, as described herein. In one embodiment, the method is a method of regulating (e.g., inhibiting) cell proliferation (e.g., proliferation of a cell), in vitro or in vivo, comprising contacting a cell with an effective amount of a FTA compound, as described herein.
In one embodiment, the method is performed in vitro.
In one embodiment, the method is performed in vivo.
In one embodiment, the FTA compound is provided in the form of a pharmaceutically acceptable composition. Any type of cell may be treated, including but not limited to, lung, gastrointestinal
(including, e.g., bowel, colon), breast (mammary), ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas, brain, and skin.
One of ordinary skill in the art is readily able to determine whether or not a candidate compound regulates (e.g., inhibits) cell proliferation, etc. For example, assays which may conveniently be used to assess the activity offered by a particular compound are described herein.
For example, a sample of cells (e.g., from a tumour) may be grown in vitro and a compound brought into contact with said cells, and the effect of the compound on those cells observed. As an example of "effect," the morphological status of the cells (e.g., alive or dead, etc.) may be determined. Where the compound is found to exert an influence on the cells, this may be used as a prognostic or diagnostic marker of the efficacy of the compound in methods of treating a patient carrying cells of the same cellular type. Use in Methods of Therapy
Another aspect of the present invention pertains to a FTA compound, as described herein, for use in a method of treatment of the human or animal body by therapy.
Use in the Manufacture of Medicaments
Another aspect of the present invention pertains to use of a FTA compound, as described herein, in the manufacture of a medicament for use in treatment.
In one embodiment, the medicament comprises the FTA compound. Methods of Treatment Another aspect of the present invention pertains to a method of treatment comprising administering to a patient in need of treatment a therapeutically effective amount of a FTA compound, as described herein, preferably in the form of a pharmaceutical composition. Conditions Treated - Conditions Mediated by LIM Kinase (LIMK)
In one embodiment (e.g., of use in methods of therapy, of use in the manufacture of medicaments, of methods of treatment), the treatment is treatment of a disease or condition that is mediated by LIM kinase (LIMK) (e.g., LIMK1 and/or LIMK2).
Conditions Treated - Conditions Ameliorated by the Inhibition of LIMK Activity
In one embodiment (e.g., of use in methods of therapy, of use in the manufacture of medicaments, of methods of treatment), the treatment is treatment of. a disease or condition that is ameliorated by the inhibition of LIM kinase (LIMK) activity (e.g., LIMK1 activity and/or LIMK2 activity).
Conditions Treated - Proliferative Conditions In one embodiment (e.g., of use in methods of therapy, of use in the manufacture of medicaments, of methods of treatment), the treatment is treatment of: a proliferative condition.
The term "proliferative condition," as used herein, pertains to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth. ln one embodiment, the treatment is treatment of: a proliferative condition characterised by benign, pre-malignant, or malignant cellular proliferation, including but not limited to, neoplasms, hyperplasias, and tumours (e.g., histocytoma, glioma, astrocyoma, osteoma), cancers (see below), psoriasis, bone diseases, fibroproliferative disorders (e.g., of connective tissues), pulmonary fibrosis, atherosclerosis, smooth muscle cell proliferation in the blood vessels, such as stenosis or restenosis following angioplasty.
Conditions Treated - Cancer In one embodiment (e.g., of use in methods of therapy, of use in the manufacture of medicaments, of methods of treatment), the treatment is treatment of cancer.
In one embodiment, the treatment is treatment of cancer characterised by, or further characterised by, cancer cells which overexpress LIM kinase (LIMK) (e.g., LIMK1 and/or LIMK2).
In one embodiment, the treatment is treatment of lung cancer, small cell lung cancer, non-small cell lung cancer, gastrointestinal cancer, stomach cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, thyroid cancer, breast cancer, ovarian cancer, endometrial cancer, prostate cancer, testicular cancer, liver cancer, kidney cancer, renal cell carcinoma, bladder cancer, pancreatic cancer, brain cancer, glioma, sarcoma, osteosarcoma, bone cancer, nasopharyngeal cancer (e.g., head cancer, neck cancer), skin cancer, squamous cancer, Kaposi's sarcoma, melanoma, malignant melanoma, lymphoma, or leukemia.
In one embodiment, the treatment is treatment of:
a carcinoma, for example a carcinoma of the bladder, breast, colon (e.g., colorectal carcinomas such as colon adenocarcinoma and colon adenoma), kidney, epidermal, liver, lung (e.g., adenocarcinoma, small cell lung cancer and non-small cell lung carcinomas), oesophagus, gall bladder, ovary, pancreas (e.g., exocrine pancreatic carcinoma), stomach, cervix, thyroid, prostate, skin (e.g., squamous cell carcinoma); a hematopoietic tumour of lymphoid lineage, for example leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non- Hodgkin's lymphoma, hairy cell lymphoma, or Burkett's lymphoma;
a hematopoietic tumor of myeloid lineage, for example acute and chronic myelogenous leukemias, myelodysplastic syndrome, or promyelocytic leukemia;
a tumour of mesenchymal origin, for example fibrosarcoma or habdomyosarcoma; a tumor of the central or peripheral nervous system, for example astrocytoma, neuroblastoma, glioma or schwannoma;
melanoma; seminoma; teratocarcinoma; osteosarcoma; xenoderoma
pigmentoum; keratoctanthoma; thyroid follicular cancer; or Kaposi's sarcoma.
In one embodiment, the treatment is treatment of solid tumour cancer. ln one embodiment, the treatment is treatment of breast cancer, prostate cancer, melanoma, or glioma. In one embodiment, the treatment is treatment of cancer metastasis.
In one embodiment, the cancer is characterised by, or further characterised by, cancer stem cells. The anti-cancer effect may arise through one or more mechanisms, including but not limited to, the regulation of cell proliferation, the inhibition of cell cycle progression, the inhibition of angiogenesis (the formation of new blood vessels), the inhibition of metastasis (the spread of a tumour from its origin), the inhibition of cell migration (the spread of cance cells to other parts of the body), the inhibition of invasion (the spread of tumour cells into neighbouring normal structures), or the promotion of apoptosis
(programmed cell death). The compounds of the present invention may be used in the treatment of the cancers described herein, independent of the mechanisms discussed herein. Conditions Treated - Additional Conditions
In one embodiment, the treatment is treatment of vasodilation.
In one embodiment, the treatment is treatment of hypertension, angina, cerebral vasospasm, or ischemia following subarachnoid hemorrhage.
In one embodiment, the treatment is treatment of a neurodegenerative disorder.
In one embodiment, the treatment is treatment of atherosclerosis.
In one embodiment, the treatment is treatment of fibrosis.
In one embodiment, the treatment is treatment of an inflammatory disease. In one embodiment, the treatment is treatment of Crohn's disease or chronic obstructive pulmonary disease (COPD).
In one embodiment, the treatment is treatment of glaucoma (also known as ocular hypertension). Treatment
The term "treatment," as used herein in the context of treating a condition, pertains generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, alleviatiation of symptoms of the condition, amelioration of the condition, and cure of the condition. Treatment as a prophylactic measure (i.e., prophylaxis) is also included. For example, use with patients who have not yet developed the condition, but who are at risk of developing the condition, is encompassed by the term "treatment."
For example, treatment includes the prophylaxis of cancer, reducing the incidence of cancer, alleviating the symptoms of cancer, etc.
The term "therapeutically-effective amount," as used herein, pertains to that amount of a compound, or a material, composition or dosage form comprising a compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio, when administered in accordance with a desired treatment regimen.
Combination Therapies
The term "treatment" includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously. For example, the compounds described herein may also be used in combination therapies, e.g., in conjunction with other agents, for example, cytotoxic agents, anticancer agents, etc. Examples of treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g., drugs, antibodies (e.g., as in immunotherapy), prodrugs (e.g., as in photodynamic therapy, GDEPT, ADEPT, etc.); surgery; radiation therapy; photodynamic therapy; gene therapy; and controlled diets.
For example, it may be beneficial to combine treatment with a compound as described herein with one or more other (e.g., 1 , 2, 3, 4) agents or therapies that regulates cell growth or survival or differentiation via a different mechanism, thus treating several characteristic features of cancer development.
One aspect of the present invention pertains to a compound as described herein, in combination with one or more additional therapeutic agents, as described below.
The particular combination would be at the discretion of the physician who would select dosages using his common general knowledge and dosing regimens known to a skilled practitioner. The agents (i.e., the compound described herein, plus one or more other agents) may be administered simultaneously or sequentially, and may be administered in individually varying dose schedules and via different routes. For example, when administered sequentially, the agents can be administered at closely spaced intervals (e.g., over a period of 5-10 minutes) or at longer intervals (e.g., 1, 2, 3, 4 or more hours apart, or even longer periods apart where required), the precise dosage regimen being commensurate with the properties of the therapeutic agent(s). The agents (i.e., the compound described here, plus one or more other agents) may be formulated together in a single dosage form, or alternatively, the individual agents may be formulated separately and presented together in the form of a kit, optionally with instructions for their use. Other Uses
The FTA compounds described herein may also be used as cell culture additives to inhibit (LIMK) activity (e.g., LIMK1 activity and/or LIMK2 activity), e.g., to inhibit cell proliferation, etc.
The FTA compounds described herein may also be used as part of an in vitro assay, for example, in order to determine whether a candidate host is likely to benefit from treatment with the compound in question. The FTA compounds described herein may also be used as a standard, for example, in an assay, in order to identify other compounds, other LIMK activity inhibitors, other anti-proliferative agents, other anti-cancer agents, etc.
Kits
One aspect of the invention pertains to a kit comprising (a) a FTA compound as described herein, or a composition comprising a FTA compound as described herein, e.g., preferably provided in a suitable container and/or with suitable packaging; and (b) instructions for use, e.g., written instructions on how to administer the compound or composition.
The written instructions may also include a list of indications for which the active ingredient is a suitable treatment. Routes of Administration
The FTA compound or pharmaceutical composition comprising the FTA compound may be administered to a subject by any convenient route of administration, whether systemically/peripherally or topically (i.e., at the site of desired action).
Routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eyedrops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal,
intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot or reservoir, for example, subcutaneously or intramuscularly.
The Subject/Patient The subject/patient may be a chordate, a vertebrate, a mammal, a placental mammal, a marsupial (e.g., kangaroo, wombat), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse), murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., a bird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., a horse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., a cow), a primate, simian (e.g., a monkey or ape), a monkey (e.g., marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutang, gibbon), or a human.
Furthermore, the subject/patient may be any of its forms of development, for example, a foetus.
In one preferred embodiment, the subject/patient is a human. Formulations While it is possible for the FTA compound to be administered alone, it is preferable to present it as a pharmaceutical formulation (e.g., composition, preparation, medicament) comprising at least one FTA compound, as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents. The formulation may further comprise other active agents, for example, other therapeutic or prophylactic agents. Thus, the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition comprising admixing at least one FTA compound, as described herein, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, e.g., carriers, diluents, excipients, etc. If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the compound.
The term "pharmaceutically acceptable," as used herein, pertains to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit risk ratio. Each carrier, diluent, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation.
Suitable carriers, diluents, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990; and Handbook of Pharmaceutical Excipients, 5th edition, 2005.
The formulations may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association the compound with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the compound with carriers (e.g., liquid carriers, finely divided solid carrier, etc.), and then shaping the product, if necessary.
The formulation may be prepared to provide for rapid or slow release; immediate, delayed, timed, or sustained release; or a combination thereof.
Formulations may suitably be in the form of liquids, solutions (e.g., aqueous, nonaqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, mouthwashes, drops, tablets (including, e.g., coated tablets), granules, powders, losenges, pastilles, capsules (including, e.g., hard and soft gelatin capsules), cachets, pills, ampoules, boluses, suppositories, pessaries, tinctures, gels, pastes, ointments, creams, lotions, oils, foams, sprays, mists, or aerosols.
Formulations may suitably be provided as a patch, adhesive plaster, bandage, dressing, or the like which is impregnated with one or more compounds and optionally one or more other pharmaceutically acceptable ingredients, including, for example, penetration, permeation, and absorption enhancers. Formulations may also suitably be provided in the form of a depot or reservoir. The compound may be dissolved in, suspended in, or admixed with one or more other pharmaceutically acceptable ingredients. The compound may be presented in a liposome or other microparticulate which is designed to target the compound, for example, to blood components or one or more organs.
Formulations suitable for oral administration (e.g., by ingestion) include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil-in-water, water-in-oil), elixirs, syrups, electuaries, tablets, granules, powders, capsules, cachets, pills, ampoules, boluses.
Formulations suitable for buccal administration include mouthwashes, losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs. Losenges typically comprise the compound in a flavored basis, usually sucrose and acacia or tragacanth. Pastilles typically comprise the compound in an inert matrix, such as gelatin and glycerin, or sucrose and acacia. Mouthwashes typically comprise the compound in a suitable liquid carrier.
Formulations suitable for sublingual administration include tablets, losenges, pastilles, capsules, and pills.
Formulations suitable for oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g., oil- in-water, water-in-oil), mouthwashes, losenges, pastilles, as well as patches, adhesive plasters, depots, and reservoirs.
Formulations suitable for non-oral transmucosal administration include liquids, solutions (e.g., aqueous, non-aqueous), suspensions (e.g., aqueous, non-aqueous), emulsions (e.g. , oil-in-water, water-in-oil), suppositories, pessaries, gels, pastes, ointments, creams, lotions, oils, as well as patches, adhesive plasters, depots, and reservoirs.
Formulations suitable for transdermal administration include gels, pastes, ointments, creams, lotions, and oils, as well as patches, adhesive plasters, bandages, dressings, depots, and reservoirs.
Tablets may be made by conventional means, e.g., compression or moulding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the compound in a free-flowing form such as a powder or granules, optionally mixed with one or more binders (e.g., povidone, gelatin, acacia, sorbitol, tragacanth, hydroxypropylmethyl cellulose); fillers or diluents (e.g., lactose, microcrystalline cellulose, calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc, silica); disintegrants (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose); surface-active or dispersing or wetting agents (e.g., sodium lauryl sulfate); preservatives (e.g., ethy] p-hydroxybenzoate, propyl p-hydroxybenzoate, sorbic acid); flavours, flavour enhancing agents, and sweeteners. Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the compound therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile. Tablets may optionally be provided with a coating, for example, to affect release, for example an enteric coating, to provide release in parts of the gut other than the stomach.
Ointments are typically prepared from the compound and a paraffinic or a water-miscible ointment base.
Creams are typically prepared from the compound and an oil-in-water cream base. If desired, the aqueous phase of the cream base may include, for example, at least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1 ,3-diol, mannitol, sorbitol, glycerol and polyethylene glycol and mixtures thereof. The topical formulations may desirably include a compound which enhances absorption or penetration of the compound through the skin or other affected areas. Examples of such dermal penetration enhancers include dimethylsulfoxide and related analogues.
Emulsions are typically prepared from the compound and an oily phase, which may optionally comprise merely an emulsifier (otherwise known as an emulgent), or it may comprises a mixture of at least one emulsifier with a fat or an oil or with both a fat and an oil. Preferably, a hydrophilic emulsifier is included together with a lipophilic emulsifier which acts as a stabiliser. It is also preferred to include both an oil and a fat. Together, the emulsifier(s) with or without stabilisers) make up the so-called emulsifying wax, and the wax together with the oil and/or fat make up the so-called emulsifying ointment base which forms the oily dispersed phase of the cream formulations.
Suitable emulgents and emulsion stabilisers include Tween 60, Span 80, cetostearyl alcohol, myristyl alcohol, glyceryl monostearate and sodium lauryl sulfate. The choice of suitable oils or fats for the formulation is based on achieving the desired cosmetic properties, since the solubility of the compound in most oils likely to be used in pharmaceutical emulsion formulations may be very low. Thus the cream should preferably be a non-greasy, non-staining and washable product with suitable consistency to avoid leakage from tubes or other containers. Straight or branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl stearate, propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl oleate, isopropyl palmitate, butyl stearate, 2-ethylhexyl palmitate or a blend of branched chain esters known as Crodamol CAP may be used, the last three being preferred esters. These may be used alone or in combination depending on the properties required. Alternatively, high melting point lipids such as white soft paraffin and/or liquid paraffin or other mineral oils can be used.
Formulations suitable for intranasal administration, where the carrier is a liquid, include, for example, nasal spray, nasal drops, or by aerosol administration by nebuliser, include aqueous or oily solutions of the compound.
Formulations suitable for intranasal administration, where the carrier is a solid, include, for example, those presented as a coarse powder having a particle size, for example, in the range of about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose.
Formulations suitable for pulmonary administration (e.g., by inhalation or insufflation therapy) include those presented as an aerosol spray from a pressurised pack, with the use of a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichoro-tetrafluoroethane, carbon dioxide, or other suitable gases.
Formulations suitable for ocular administration include eye drops wherein the compound is dissolved or suspended in a suitable carrier, especially an aqueous solvent for the compound.
Formulations suitable for rectal administration may be presented as a suppository with a suitable base comprising, for example, natural or hardened oils, waxes, fats, semi-liquid or liquid polyols, for example, cocoa butter or a salicylate; or as a solution or suspension for treatment by enema.
Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the compound, such carriers as are known in the art to be appropriate.
Formulations suitable for parenteral administration (e.g., by injection), include aqueous or non-aqueous, isotonic, pyrogen-free, sterile liquids (e.g., solutions, suspensions), in which the compound is dissolved, suspended, or otherwise provided (e.g., in a liposome or other microparticulate). Such liquids may additional contain other pharmaceutically acceptable ingredients, such as anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, suspending agents, thickening agents, and solutes which render the formulation isotonic with the blood (or other relevant bodily fluid) of the intended recipient. Examples of excipients include, for example, water, alcohols, polyols, glycerol, vegetable oils, and the like. Examples of suitable isotonic carriers for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection.
Typically, the concentration of the compound in the liquid is from about 1 ng/ml to about 10 g/ml, for example from about 10 ng/ml to about 1 g/ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
Dosage
It will be appreciated by one of skill in the art that appropriate dosages of the
FTA compounds, and compositions comprising the FTA compounds, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular FTA compound, the route of administration, the time of administration, the rate of excretion of the FTA compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, the severity of the condition, and the species, sex, age, weight, condition, general health, and prior medical history of the patient. The amount of FTA compound and route of administration will ultimately be at the discretion of the physician, veterinarian, or clinician, although generally the dosage will be selected to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects.
Administration can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician, veterinarian, or clinician.
In general, a suitable dose of the FTA compound is in the range of about 10 pg to about 250 mg (more typically about 100 pg to about 25 mg) per kilogram body weight of the subject per day. Where the compound is a salt, an ester, an amide, a prodrug, or the like, the amount administered is calculated on the basis of the parent compound and so the actual weight to be used is increased proportionately. EXAMPLES
The following examples are provided solely to illustrate the present invention and are not intended to limit the scope of the invention, as described herein.
Chemical Synthesis General All non-aqueous reactions were performed in oven-dried glassware under an atmosphere of dry nitrogen, unless otherwise specified. Tetrahydrofuran was freshly distilled from sodium/benzophenone under N2. Dichloromethane was freshly distilled from CaH2 under N2. All other solvents were reagent grade. Petroleum ether describes a mixture of hexanes in the bp range 40-60°C. Analytical thin-layer chromatography was performed on Merck silica gel 60F254 aluminium-backed plates which were visualised by
fluorescence quenching under UV light. Flash chromatography was performed with silica gel 60 (particle size 0.040-0.063 mm). All NMR spectra were recorded on a Bruker Avance DRX 300 with the solvents indicated (1H NMR at 300 MHz). Chemical shifts are reported in ppm on the δ scale, referenced to the appropriate solvent peak. Mass spectrometry was performed on a Finnigan LCQ Advantage MAX and a Waters ZQ3100.
Method 1 : 4-Aminobenzothienof3,2-dlpyrimdine
Synthesis 1A
Ethyl 3-amin -carboxylate
Figure imgf000086_0001
2-Fluorobenzonitrile (8.25 mmol), diisopropylethylamine (8.25 mmol) and ethyl
2-mercaptoacetate (8.25 mmol) in anhydrous Ν,Ν-dimethylformamide (5 mL) was allowed to stir for 30 min at room temperature. Potassium carbonate (8.25 mmol) was added and the solution was heated to 80°C for 20 h. Ice-water was added and the resulting precipitate was filtered off, washing with water to obtain the carboxylate as a white solid (92%).
LCMS rt 7.43, M+H 222; 1H-NMR (CDCI3) δ 7.43 (1H, d, J 8.1 Hz), 7.65 (1H, d, J 8.1 Hz), 7.49-7.44 (1H, m), 7.39-7.34 (1H, m), 4.36 (2H, q, J 7.1 Hz), 1.40 (3H, t, J 7.1 Hz). Synthesis 1B
3H-[1]benzothieno[3,2-d]pyrimid-4-one
Figure imgf000087_0001
A stirred mixture of ethyl 3-aminobenzo[b]thiophene-2-carboxylate (3.62 mmol) in formamide (5 mL) was heated to 150°C. Formamidine acetate (3.62 mmol) was added and the mixture heated at 150°C for 45 min. The addition of formamidine acetate (3.62 mmol) was repeated every 45 min for 6 h. Ice-water was added and the precipitate that was formed was filtered off, washed with water, and dried in a vacuum oven to obtain the pyrimidine as a tanned solid (95%).
LCMS rt 5.20; M+H 203; 1H-NMR (DMSO) δ D8.33 (1H, s), 8.26-8.21 (1H, m), 8.16-8.12 (1H, m), 7.69-7.56 (1H, m).
Synthesis 1C
4-Chlorobenzothieno[3,2-d]pyrimidine
Figure imgf000087_0002
A mixture of 3H-benzothieno[3,2-d]pyrimid-4-one (2.48 mmol) and
N,N-dimethylformamide (0.025 mmol) in phosphorous oxychloride (6 mL) was allowed to stir at 90°C for 20 h. The phosphorous oxychloride was removed in vacuo. Ice-water was added and the solution neutralized by portionwise addition of solid sodium hydrogen carbonate. The resulting precipitate was filtered off, washed with water and dried in a vacuum oven to give the chloro pyrimidine as a tanned solid (86%).
LCMS rt 7.73, M+H 221; H-NMR (DMSO) δ 9.17 (1H, s), 8.48 (1H, d, J 7.8 Hz), 8.29 (1H, d, J 8.1 Hz), 7.84 (1H, dt, J 7.2 and 1.2 Hz), 7.71 (1H, dt, J 7.2 Hz and 0.9).
Synthesis 1D
4-Aminobenzothieno[3,2-d]pyrimidine
Figure imgf000087_0003
A suspension of 4-chlorobenzothieno[3,2-d]pyrimidine (0.45 mmol) in concentrated ammonium hydroxide (1 mL) and dioxane (3 mL) was heated to 100°C under microwave irradiation (250 W) for 2 h, Ice-water was added and the precipitate that resulted was filtered off, washed with water, and dried in a vacuum oven to obtain the amino pyrimidine as a pale-yellow solid (95%). LCMS rt 4.08, M+H 402; H-NMR (DMSO) δ 8.51 (1H,s), 8.30 (1H, s, J 7.2 Hz), 8.12 (1H, s, J 8.1 Hz), 7.68-7.51 (4H, m).
Method 2: 7-Phenyl-4-aminobenzothienof3,2-d1pyrimidine (AE-001)
Synthesis 2A
7-Bromo-4-aminobenzothieno[3,2-d]pyrimidine
Figure imgf000088_0001
The 7-bromo-4-aminobenzothieno[3,2-d]pyrimidine was synthesised from
4-bromo-2-fluorobenzonitrile in the same manner as Method 1.
LCMS rt 5.50, M+H 280; 1H-NMR (DMSO) δ 8.51 (1H, s), 8.47 (1H, d, J 1.7 Hz), 8.18 (1H, d, J 8.4 Hz), 7.70 (1H, dd, J 8.5 and 1.8 Hz), 7.60 (1H, bs).
Synthesis 2B
7-Phenyl-aminobenzothieno[3,2-d]pyrimidine (AE-001)
Figure imgf000088_0002
A mixture of 7-bromo-aminobenzothieno[3,2-d]pyrimidine (0.36 mmol), potassium carbonate (0.89 mmol), phenylboronic acid (0.4 mmol), tetrabutylammonium bromide (0.036 mmol) and PdCI2(PPh3)4 (0.036 mmol) in a solution of dioxane (4 mL) and water (1 mL) were heated at 120°C under microwave irradiation for 2 h. 10% Citric acid solution (10 mL) was added and the precipitate that resulted was filtered off, washed with water, and dried in a vacuum oven. The solid was dissolved in a dilute
methanol/tetrahydrofuran mixture with warming, and filtered. The filtrate was
concentrated to dryness in vacuo. The resulting residue is triturated with diethylether, and filtered off to give a tanned solid (70%). Alternatively, the crude residue is applied to column chromatography gradient eluting with 100% dichloromethane to 15% methanol dichloromethane. LCMS rt 7.53, M+H 278; 'H-NMR (DMSO) δ 8.52 (1H, s), 8.46-8.45 (1 H, m), 8.33 (1H, d, J 8.8 Hz), 7.88-7.81 (3H, m), 7.54-7.41 (5H, m).
The following 6-phenyl-, 8-phenyl-, and 9-pheny)- 4-aminobenzothieno[3,2-d]pyrimidines were synthesised in the same manner as Method 2, using the corresponding nitrites (listed) in place of 4-bromo-2-fluorobenzonitrile.
Figure imgf000089_0001
The following substituted 9-phenyl-4-aminobenzothieno[3,2-d]pyrimidines were synthesised in the same manner as Method 2, using the corresponding boronic acids (listed) in place of phenylboronic acid.
Figure imgf000089_0002
Figure imgf000090_0001
The following substituted 7-phenyl-4-aminobenzothieno[3,2-d]pyrimidines were synthesised in the same manner as Method 2, using the corresponding boronic acids (listed) in place of phenylboronic acid.
Figure imgf000090_0002
Figure imgf000091_0001
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
ester
Method 3: Substituted amido 7-phenyl 4-aminobenzothienof3,2-dlPyrimidine
Synthesis 3A
7-(3-N,N-morpholinylbenzamide) 4-aminobenzothieno[3,2-d) pyrimidine (AB-036)
Figure imgf000098_0002
A mixture of 7-(3-carboxyphenyl)-4-aminobenzothieno[3,2-d]pyrimidine AA-003 (0.09 mmol), diisopropylethylamine (0.42 mmol) and HBTU (0.13 mmol) in
N,N-dimethylformamide (0.3 mL), was allowed to stir for 5 min. Morpholine (0.18 mmol) was added and the solution was allowed to stir for 16 h. Ice-water was added and the precipitate that formed was filtered off, washing with water and dried in a vacuum oven to give the amide as a tanned solid (90%). LCMS rt 6.66, M+H 391 ; 1H-N R (DMSO) δ 08.58 (1H, s), 8.55 (1H, s), 8.37, (1H, s), 7.94-7.42 (7H, m), 3.62 (4H, bs), 2.97 (4H, bs).
The following substituted amido 7-phenyl-4-aminobenzothieno[3,2-d]pyrimidines were synthesised in the same manner as Method 3, using the corresponding amines (listed) in place of morpholine.
Figure imgf000099_0001
Figure imgf000100_0001
Figure imgf000101_0001
Figure imgf000102_0001
The following substituted amido 7-aryl-4-aminobenzothieno[3,2-d]pyrimidines were synthesised in the same manner as Method 3, using the corresponding amines (listed) in place of morpholine and starting from the appropriate substituted 7-(carboxyaryl)-4- aminobenzothieno[3,2-d]pyrimidine starting material.
Figure imgf000102_0002
Figure imgf000103_0001
Figure imgf000104_0001
Method 5: Substituted ureido 7-phenyl 4-aminobenzothienof3,2-dl pyrimidine
Synthesis 5A
7-(3-ethylurea)-4-aminobenzothieno[3,2-d]pyrirnidine (AA-003)
Figure imgf000104_0002
A solution of 7-(3-amino)-4-aminobenzothieno[3,2-d]pyrimidine AE-0 8 (0.07 mmol) and ethyl isocyanate (1.09 mmol) in anhydrous THF (1 mL) was stirred at 50°C for 16 h. After this time, the solvent was removed in vacuo. The resulting residue was triturated with diethyl ether, and filtered to give the urea as a tanned solid (80%).
LCMS rt 4.86, M+H 363; 1H-NMR (DMSO) δ 8.55 (1H, s), 8.53 (1H, s), 8.36 (1H, s), 8.33 (1H,s), 7.90 (1H, s), 7.81-7.77 (1H, m), 7.54 (2H, bs), 7.38-7.34 (3H, m), 6.18-6.14 (1H, bs), 3.40-3.20 (2H, m), 1.07 (3H, t J 7.2 Hz). Method 6: Substituted ureido 7-phenyl 4-aminobenzothienof3,2-dI pyrimidine
Synthesis 6A
7-(3-cydopropylurea)-4-aminobenzothienoI3,2-d]pyrimidine (AA-032)
Figure imgf000105_0001
A solution of 7-(3-aminophenyl)-4-aminobenzothieno[3,2-d]pyrimidine AE-018 (0.51 mmol), pyridine (1.54 mmol) and phenyl chloroformate (0.59 mmol) in
N,N-dimethylformamide (3 mL) containing under nitrogen was stirred at 0°C for 45 min and then warmed to 25°C for 2 h. After this time, water was added and a precipitate formed. This was filtered, washed with water and then oven dried to give the
7-(3-phenylcarbamate)-4-aminobenzothieno[3,2-d]pyrimidine as an olive solid (80%). A solution of the carbamate (0.05 mmol) in 1 ,4-dioxane (0.5 mL) containing excess cyclopropylamine (6.91 mmol) was stirred at 70°C for 4 h. After this time the solvent and excess amine was removed in vacuo. Diethyl ether was added and the precipitate that resulted was filtered, washing with diethylether, to give the urea as a light brown solid (80%).
LCMS rt 6.86, M+H 376; 1H-NMR (DMSO) δ 8.34 (1H, 8.53), 8.43 (1H, s), 8.37-8.33 (3H, m), 7.93 (1H, s), 7.82-7.78 (1 H, m), 7.54 (2H, bs), 7.41-7.35 (3H, m), 6.46 (1 H, bs), 2.57- 2.54 (1H, m), 0.66-0.64 (2H, m), 0.44-0.42 (2H, m).
The following substituted ureido 7-phenyl-4-aminobenzothieno[3,2-d]pyrimidines were synthesised in the same manner as Method 5, using the corresponding isocyanates (listed) in place of ethyl isocyanate, or in the same manner as Method 6 using the corresponding amines (listed) in place of cyclopropylamine.
Figure imgf000105_0002
Figure imgf000106_0001
Figure imgf000107_0001
Figure imgf000108_0001
The following substituted ureido 7-phenyl-4-aminobenzothieno[3,2-d]pyrimidines were synthesised in the same manner as Method 5, using the corresponding isocyanates (listed) in place of ethyl isocyanate, or in the same manner as Method 6 using the corresponding amines (listed) in place of cyclopropylamine, and replacing 7-(3- aminophenyl)-4-aminobenzothieno[3,2-d]pyrimidine (AE-018) with an appropriately substituted analogue.
Figure imgf000108_0002
Figure imgf000109_0001
The following substituted ureido 7-phenyl-4-aminobenzothieno[3,2-d]pyrimidines were synthesised in the same manner as Method 5, using the corresponding isocyanates (listed) in place of ethyl isocyanate, or in the same manner as Method 6 using the corresponding amines (listed) in place of cyclopropylamine, and replacing
7-(3-aminophenyl)-4-aminobenzothieno[3,2-d]pyrimidine (AE-018) with
7-(4-aminophenyl)-4-aminobenzothieno[3,2-d]pyrimidine (AE-014).
Figure imgf000110_0001
Method 7: 4-Amino-5H-pyrimidor5,4-b]indole Synthesis 7A
Preparation of N-(2-cyanophenyl)acetamide
Figure imgf000110_0002
A mixture of anthranilamide (42.3 mmol), acetic anhydride (42.3 mmol) and dimethylamino pyridine (2.1 mmol) in dichloromethane (50 mL) was allowed to stir for 20 h at room temperature. Petroleum ether was added to the reaction mixture. A precipitate formed and was filtered off, washing with petroleum ether to obtain the acetamide as a white solid (88%). LCMS rt 3.76, M+H 376. 1H-NMR (CDCI3) δ 8.35 (1H, d, J 8.7 Hz), 7.75 (1H, bs), 7.60- 7.55 (2H, m), 7.19-7.13 (1H, m), 2.26 (3H, s).
Synthesis 7B
1 -acetyl-3-amino-1 H-indole-2-carbonitrile
Figure imgf000111_0001
A mixture of N-(2-cyanophenyl)acetamide (28 mmol), chloroacetonitrile (28 mmol) and potassium tert-butoxide (28 mmol) in N,N-dimethylformamide (20 mL) was allowed to stir for 20 h at room temperature. Ice-water was added to the reaction mixture. The mixture was extracted with ethyl acetate (2 x 30 mL). The organic layer was dried (MgS04) and the organic layer was concentrated in vacuo to obtain an oil. The oil was subjected to silica chromatography gradient eluting with 100% petroleum ether to 60% ethyl acetate /petroleum ether to obtain a pale orange solid (54%). LCMS rt 5.62, M+H 200. 1H-NMR (DMSO) δ 8.09 (1H, d, J 8.7 Hz), 7.93 (1H, d, J 8.0 Hz), 7.51 (1H, t, J 8.4 Hz), 7.31 (1H, t, J 7.9 Hz), 6.69 (2H, bs), 2.71 (3H, s).
Synthesis 7C
3-amino rbonitrile
Figure imgf000111_0002
A mixture of 1-acetyl-3-amino-1H-indole-2-carbonitrile (13.6 mmol), and potassium carbonate (27.2 mmol) in a solution of water (20 mL) and ethanol (20 mL) was allowed to reflux for 4 h. Ice-water was added to the reaction mixture. The mixture was extracted with ethyl acetate (2 x 40 mL). The organic layer was dried (MgS0 ) and the organic layer was concentrated in vacuo to obtain an oil. The oil was subjected to silica chromatography gradient eluting with 00% dichloromethane to 5% methanol / dichloromethane to obtain a white solid (24%).
LCMS rt 5.62, M+H 158. 1H-NMR (DMSO) δ D10.63 (1h, bs), 7.71 (1H, d, J 8.1 Hz), 7.25- 7.14, (2H, m), 6.96-6.90 (1H, m), 5.65 (2H, bs). Synthesis 7D
4-Amino-5H-pyrimido[5,4-b]indole
Figure imgf000112_0001
3-Amino-1H-indole-2-carbonitrile was used in a similar process to that in Synthesis 8.
LCMS Π 2.42, M+H 185. 'H-NMR (D SO) δ 010.94 (1H, bs), 8.28 (1H, s), 8.05 (1H, d, J 7.9 Hz), 7.61 (1H, d, J 8.3 Hz), 7.49 (1H, t, J 7.0 Hz), 7.20 ( H, t, J 7.1 Hz), 6.89 (2H, bs).
Method 8: 4-Amino5-methyl-5H-pyrimido[5,4-b1indole
Synthesis 8A
2-((2-cyanophenyl)(methyl)amino)acetic acid
Figure imgf000112_0002
2-Fluoro-benzonitrile (4.13 mmol), sarcosine (4.54 mmol), potassium carbonate (10.0 mmol), and copper acetate (0.41 mmol) in dimethylsulfoxide (4 mL) was allowed to stir at 140°C for 20 h. Ice-water is added, followed by ethyl acetate (10 mL) and the layers were separated. The aqueous layer is then acidified to pH 2 with concentrated hydrochloric acid. The aqueous layer is then extracted with ethyl acetate (2 x 10 mL). The organic layer is dried (MgS04) and concentrated in vacuo to afford the aniline as an orange oil (89%).
LCMS rt 5.38, M+H 191. 1H-NMR (DMSO) δ 12.70 (1H, bs), 7.44 (1H, dd, J 6.0 and 1.5 Hz), 7.50-7.44 (1H, m), 6.96 (1H, d, J 8.7 Hz), 6.89-6.84 (1H, m), 4.19 (2H, s), 3.06 (3H, s).
Synthesis 8B
Methyl 3-amino-1-methyl-1 H-indole-2-carboxylate
Figure imgf000112_0003
2-((2-Cyanophenyl)(methyl)amino)acetic acid (3.42 mmol) was heated to reflux in thionyl chloride (10 mL) for 30 min. The solution was cooled and evaporated to dryness in vacuo. Anhydrous methanol (10 mL) was added to the resulting residue and allowed the solution was allowed to sit for 5 min, The methanol was then removed in vacuo and the residue dissolved in ethyl acetate (20 mL). The organic solution was then washed with 10% sodium hydrogen carbonate solution (20 mL), dried (MgS04) and concentrated under vacuum to afford an oily residue. The residue was dissolved in N,N- dimethylformamide (5 mL) and potassium carbonate (3.42 mmol) was added. This mixture was then heated at 60°C for 24 h. Ice-water was added and the mixture extracted with ethyl acetate (2 x 10 mL). The organic layer was dried (MgS04) and concentrated in vacuo. The resulting residue was subjected to column chromatography eluting with 00% petroleum ether to 40% ethyl acetate/petroleum ether to afford the indole as an oil (53%).
1H-NMR (DMSO) δ D7.81-7.77 (1H, m), 7.31-7.28 (2H, m), 6.95-6.90 (1H, m), 5.88 (2H, bs), 3.81 (3H, s), 3.76 (3H, s).
Synthesis 8C
5-methyl- indol-4-ol
Figure imgf000113_0001
Methyl 3-amino-1-methyl-1H-indole-2-carboxylate was used in a similar process to that in Synthesis 1B.
LCMS rt 5.07, M+H 200. H-NMR (DMSO) δ 012.35 (1H, bs), 8.02-7.99 (1H, m), 7.96 (1H, s), 7.67-7.64 (1H, m), 7.56-7.50 (1H, m), 7.29-7.24 <1H, m), 4.15 (3H, s).
Synthesis 8D
4-Chloro-5-methyl-5H-pyrimido[5,4-b]indole
Figure imgf000113_0002
5-Methyl-5H-pyrimido[5,4-b]indol-4-ol was used in a similar process to that in
Synthesis 1C.
LCMS rt 6.50, M+H 218. 1H-NMR (DMSO) δ D8.81 (1H, s), 8.28-8.25 (1H, m), 7.85-7.77 (2H, m), 7.43-7.40 (1H, m), 4.18 (3H, s).
Synthesis 8E
4-Amino5-methyl-5H-pyrimido[5,4-b]indole
Figure imgf000113_0003
A suspension of 4-chloro-5-methyl-5H-pyrimido[5,4-b]indole (0.45 mmol) in concentrated ammonium hydroxide (1 mL) and dimethylsulfoxide (3 mL) was heated to 150°C in a sealed pressure vessel for 20 h. Ice-water was added and the precipitate that resulted was filtered off, washed with water, and dried in a vacuum oven to obtain the amino pyrimidine as a pale-yellow solid (90%). LCMS rt 5.52, M+H 199.
Method 9: 7-Phenyl-4-amino-5-methyl-5H-pyrimidof5,4-b)indole
Synthesis 9A
7-Bromo~ -amino-5-methyl-5H-pyrimido[5,4-b]indole
Figure imgf000114_0001
7-Bromo-4-amino-5-methyl-5H-pyrimido[5,4-b]indole was synthesised from
4-bromo-2-fluorobenzonitrile in the same manner as Method 8. LCMS rt 4.58, M+H 277. 1H-NMR (DMSO) δ 08.27 (1H, s), 7.98-7.97 (2H, m), 7.34 (1H, dd, J 8.10 and 1.8 Hz), 6.96 <2H, bs), 4.05 (3H, s).
Synthesis 9B
7-Phenyl-4-amino-5-methyl-5H-pyrimido[5,4-b]indole (CC-004)
Figure imgf000114_0002
The title compound was synthesised from 7-bromo-4-amino-5-methyl-5H-pyrimido[5,4- bjindole in the same manner as Method 2 (AE-001).
LCMS rt 6.21, M+H 275. 1H-NMR (DMSO) δ D8.28 (1H, s), 8.12 ( H, d, J 8.1 Hz), 7.95 (1H, s), 7.84-7.81 (2H, m), 7.55-7.39 (4H, m), 6.87 (2H, bs).
Substituted 7-phenyl-4-amino-5-methyl-5H-pyrimido[5,4-b]indoles were synthesised from the corresponding boronic acids in the same manner as Method 9 (CC-005). Table 6
Substituted 7-phenyl-4-amino-5-methyl-5H-pyrimido[5,4-b]indoles
Code Structure Reagent used LCMS
3-Aminocarbonyl rt 5.97,
CC-001
phenyiboronic acid M+H 318
3-Formylaminophenyl
rt 6.05,
CC-002 boronic acid, pinacol
M+H 318 ester
H
4-Hydroxyphenyl rt 6.05,
CC-003
boronic acid M+H 291
Method 10: Pyridof3\2\4:51thienof3,2-dlpyrimidine
Synthesis 10A
Pyndo[3', ':4:5]thieno(3,2-d]pyrimidine
Figure imgf000115_0001
The title compound was synthesised from 2-chloro-3-cyanopyridine in the same manner as in Synthesis 1D. LCMS rt 2.98, M+H 203. 1H-NMR (DMSO) δ 8.81 (1H, dd, J 4.7 and 1.7 Hz), 8.61 (1H, dd, J 7.9 and 1.7 Hz), 8.54 (1H, s), 7.64-7.60 (3H, m).
Method 11: 7-Phenyl-pyridof3',2':4:51thienof3,2-dlpyrimidine (BB-001)
Synthesis 1A
2-Chloro-6-phenylnicotinic acid
Figure imgf000115_0002
A mixture of 2,6-nicotinic acid (4.4 mmol), phenyiboronic acid (4.4 mmol), potassium carbonate (15.5 mmol) and PdCI2(PPh3)2 (5 mol %) in a solution of dimethoxyethane (5 mL), ethanol (5 mL) and water (5 mL) was allowed to reflux for 4 h. The mixture was partitioned between ethyl acetate and water. The layers were then separated. The aqueous layer was then acidified with 2 N hydrochloric acid solution to pH 5 and extracted with ethyl acetate (2 x 20 mL). The organic layer was dried ( gS04) and concentrated in vacuo to obtain the acid as a solid (70%).
1H-NMR (CDCI3) δ 8.13 (1H, d, J 8.0 Hz), 8.09-8.05 (2H, m), 7.99 (1 H, d, J 8.0 Hz), 7.53- 7.47 (3H, m).
Figure imgf000116_0001
A mixture of 2-chloro-6-phenylnicotinic acid in thionyl chloride was allowed to reflux for 1 h. The reaction mixture was concentrated to dryness in vacuo. The residue was dissolved in dioxane (5 mL) and NH4OH solution was added. Ice-water was added to the reaction mixture. The precipitate that formed was filtered off, washing with water and dried in a vacuum oven to obtain the amide as a white solid (90%).
1H-NMR (CDCI3) δ 8.07-7.94 (5H, m), 7.73 (1H, bs), 7.54-7.48 (3H, m).
Synthesis 1 C
2-Chloro-6-phenylnicotinonitri]e
Figure imgf000116_0002
A mixture of 2-chloro-6-phenylnicotinamide in acetic anhydride was allowed to reflux for 2 h. The reaction mixture was concentrated to dryness in vacuo. Ice-water was added to the reaction mixture. The mixture was partitioned between ethyl acetate and 10% sodium hydrogencarbonate solution. The layers were then separated. The organic layer was dried ( gS04) and the organic layer was concentrated in vacuo to obtain an oil. The oil was subjected to silica chromatography gradient eluting with 10% ethyl acetate I petroleum ether to 50% ethyl acetate / petroleum ether to obtain the nitrile as a white solid (49%). H-NMR (CDCIa) δ D8.05-8.00 (2H, m), 7.99 (1H, d, J 8.4 Hz), 7.76 (1H, d, J 8.1 Hz), 7.51-7.49 (3H, m). Synthesis D
6-Phenyl-2-thiox 'ihydropyridine-3-carbonitrile
Figure imgf000117_0001
A mixture of 2-chloro-6-phenylnicotinonitrile (0.2 mmol) and sodium hydrosulfide
(0.2 mmol) in ethanol (3 mL) was allowed to reflux for 2 h. 10% Citric acid solution was added to the reaction mixture. The precipitate that formed was filtered off, washing with water and dried in a vacuum oven to obtain a bight yellow solid. The solid was suspended between ethyl acetate and 1 N sodium hydroxide solution. The layers were then separated. The aqueous layer was then acidified with 2 N hydrochloric acid solution to pH 5 and the precipitate that formed was filtered off, washing with water and dried in a vacuum oven to obtain the pyrid-2-thione as a bight yellow solid (60%).
'H-NMR (DMSO) δ 08.06 (1 H, d, J 7.8 Hz), 7.76-7.73 (2H, m), 7.55-7.52 (2H, m), 7.06
(1H, d, J 7.8 Hz).
Synthesis 1 1 E
3-Amino-6-phenylthieno[2,3-b]pyridine-2-carbonitrile
Figure imgf000117_0002
A mixture of 6-p tenyi-2-th/oxo-1 ,2-d/hydropyridine-3-carbonitrile (0.7 mmol),
diisopropylethylamine (0.7 mmol) and chloroacetonitrile (0.7 mmol) in
N,N-dimethylformamide (3 mL) was allowed to stir for 1 h at room temperature. Ice-water was added to the reaction mixture. The precipitate that formed was filtered off, washing with water and dried in a vacuum oven to obtain a white solid (the solid was a mixture of acyclic and cyclic product). The solid was dissolved in N,N-dimethylformamide (3 mL) and potassium carbonate (0.7 mmol) was added. The mixture was allowed to stir at 50°C for 8 h. Ice-water was added to the reaction mixture. The precipitate that formed was filtered off, washing with water and dried in a vacuum oven to obtain the thieno pyridine as a white solid (68 %).
1H-NMR (DMSO) δ 08.55 (1H, d, J 8.7 Hz), 8.17-8.14 (2H, m), 8.1 1 (1 H, d, J 8.4 Hz), 7.53-7.50 (3H, m), 7.33 (2H, bs). Synthesfs F
7-Pheny(-pyrido idine (BB-001)
Figure imgf000118_0001
The title compound was synthesised from 3-amino-6-phenylthieno[2,3-b]pyridine-2- carbonitrile in the same manner as Synthesis 1B.
LCMS rt 5.87, M+H 279. 1H-NMR (DMSO) δ 08.66 ( H, d, J 8.1 Hz), 8.52 (1H, s), 8.19- 8.15 (3H, m), 7.63 (2H, bs), 7.54-7.52 (2H, m).
Method 12: 7-Morpholino-4-amino-9-methyl-pyridof3',2'.4.5lthienof3,2-dlpyrimidine
(BB-008)
Synthesis 12A
Morpholine 3-cyano-4-m ahydropyridine-2-thiofate
Figure imgf000118_0002
Morpholine (36.3 mmol) is added to a solution of 2-cyanothioacetamide (36.3 mmol) in ethanol (20 mL) and the solution stirred at 25°C for 5 min. Ethyl acetoacetate (36.3 mmol) is added and the solution was then stirred at 25°C for 24 h. Acetone was added to the solution and the precipitate that formed was filtered off washing with acetone to afford the pyridine as a white solid (61%).
1H-NMR (DMSO) δ 5.36 (1H, s), 3.75-3.72 (4H, m), 3.09-3.06 (4H, m), 1.97 (3H, s).
Synthesis 12B
2-(Cyanomethylthio)-4-methyl-6-oxo-1,6-dihydropyridine-3-carbonitrile
Figure imgf000118_0003
To a solution of morpholine 3-cyano-4-methyl-6-oxo-1 ,2,3,6-tetrahydropyridine-2-thiolate (23.8 mmol) in Ν,Ν-dimethylformamide (15 mL), was added chloroacetonitrile (23.8 mmol). The solution was stirred at 25°C for 1 h and ice-water was added. The precipitate that formed was filtered off, washing with water to give the nitrile as a light brown solid (88%). LC S rt 7.25, +H 206. 1H-NMR (DMSO) δ 6.51 ( H,s), 4.28 (2H, s), 2.36 (3H, s).
Synthesis 12C
5-cyano-6-(cyanomethylthio)-4-methylpyridin-2-yl methanesulfonate
Figure imgf000119_0001
To a solution of 2-(cyanomethylthio)-4-methyl-6-oxo-1,6-dihydropyridine-3-carbonitrile (14.6 mmol) and methanesulfonyl chloride ( 6.0 mmol) in tetrahydrofuran (15 mL), was added diisopropylethylamine (16.0 mmol). The solution was then stirred at 25°C for 24 h. Water was added and the solution was extracted with ethyl acetate (2 x 40 mL). The organic solvent was dried (MgS04) and concentrated in vacuo. The residue was triturated with dichloromethane and the precipitate filtered off washing with
dichloromethane. The filtrate was concentrated in vacuo to afford the mesylate as a solid (35%).
1H-NMR (DMSO) δ 7.28 (1H, s), 4.36 (2H, s), 3.69 (3H, s), 2.52 (3H, s).
Synthesis 12D
2-(Cyanomethylthio)-4-methyl-6-morpholinonicotinonitrile
Figure imgf000119_0002
A mixture of 5-cyano-6-(cyanomethylthio)-4-methylpyridin-2-yl methanesulfonate
(0.35 mmol), diisopropylethylamine (1.06 mmol), and morpholine (1.06 mmol) in dioxane (4 mL) was stirred at 90°C for 16 h. Ice-water was added and the precipitate that resulted was filtered off, washing water to give the aminopyridine as a solid (70%).
1H-NMR (CDCI3) δ 6.24 (1H,s ), 3.86 (2H, s), 3.83-3.80 (4H, m), 3.71-3.68 (4H, m), 2.39 (3H, s).
Synthesis 12E
3-Amino-4-methyl-6-morpholinothieno[2,3-b]pyridine-2-carbonitrile
Figure imgf000119_0003
A mixture of 2-(cyanomethylthio)-4-methyl-6-morpholinonicotinonitrile (0.25 mmol), potassium carbonate (0.50 mmol), in N,N-dimethylformamide (2 mL) was heated at 90°C for 2 h. Ice-water was added and the precipitate that resulted was filtered off, washing water and dried in a vacuum oven to give the thienopyridine as a cream coloured solid (92%).
LCMS rt 6.38, M+H 275. 1H-NMR (DMSO) δ 06.39 (1H, s), 3.70 (4H , m), 3.55-3.34 (4H, m), 2.58 (3H, s). Synthesis 2F
7-Morpholino-4-amino-9-methyl-pyrido[3',2':4:5]thieno[3,2-d]pyrimidine (BB-014)
Figure imgf000120_0001
7- orpholino-4-amino-9-methyl-pyrido[3',2''.4:5]thieno[3,2-d]pyrimidine was synthesised from 3-amino-4-methyl-6-morpholinothieno[2,3-b]pyridine-2-carbonitrile in the same manner as Synthesis 1 B .
LCMS rt 6.39, M+H 302. 1H-NMR (DMSO) δ 08.42 (1H, s), 7.19 (2H, bs), 6.84 (1H, s), 3.71-3.69 (4H, m), 3.63-3.61 (4H, m), 2.82 (3H, s). Substituted 4-amino-9-methyl-pyrido[3',2':4:5]thieno[3,2-d]pyrimidines were synthesised from the corresponding amines in the same manner as Method 12 (AA-022).
Table 7
7-amino 4-amino-9-methyl-pyrido[3, 12':4:5]thieno[3,2-d]pyrimidines
Code Structure Reagent used LCMS rt- 5.39,
BB-003 N-Methyl piperazine
M+H 315
N,N-Dimethyl-2- rt- 0.90,
BB-004 (piperazin-l-yl)ethyl
M+H 372 amine
Figure imgf000121_0001
Biological Methods
Assay 1 - LIMK1 (Kinase Domain) Kinase Glo
1. Introduction
The serine kinases LIMK-1 and LIMK-2 are kinases which have LIM domains or protein- motifs that are frequently found in cytosolic proteins which interact with the actin cytoskeleton involved in cell division and motility. The purpose of the assay was to screen for inhibitors of LIMK-1 whereby kinase activity was measured by a luminescence ATP detection system in a single-point 384 well screening assay measuring ATP consumption by the kinase-substrate phosphorylation reaction. The reaction measured was characterized by two steps as outlined below: APT/Mg^
LIMK-1 + cofilin cofilin-PP + ATP/AMP
Luciferase/ g2+ Oxyluciferin + AMP +
ATP + D-Luciferin
+ CO* + Light
In the first step, LIMK-1 catalyses the phosphorylation of its substrate cofilin thereby consuming ATP. The second step involves the detection of the remaining ATP using the Luminescence ATP detection system, Kinase-Glo® (Promega). In the detection reaction, the firefly luciferase enzyme produces light by its reaction with its substrate D-Luciferin and ATP. The light which is produced as a result of the reaction is proportional to the amount of ATP that is present in the initial kinase reaction. 2. Materials and Methods
2.1. Reagents and Materials
LimK-1 and cofilin were supplied by Ora Bernard, St Vincent's Medical Institute. ATP (99% purity) supplied by Sigma Aldrich whilst the Kinase-Glo detection kit was obtained from Promega Corp (Cat #V6714). Common assay buffer reagents such as HEPES, KCI, MgCI2, MnCI2, NaF, NaVO, and BSA were purchased from Sigma Aldrich. Library compounds were pre-prepared in Metrical 50 μΐ, V-bottom polypropylene plates. The assay was carried out in PerkinElmer Lifesciences 384 well Proxiplates™ (white, shallow well of 28μΙ total volume, inverted chimney design) with Matrical deep well plates
(Millennium Science) and Axygen open reservoir plates (Proscience) used for automated reagent additions. The MiniTrak IX (PerkinElmer Lifesciences) was used to automate the dilution of compounds, the MultiDrop reagent dispenser (Thermo Corporation) was used to perform all other additions and measurements were detected using the Envision 2103 (PerkinElmer Lifesciences).
2.2. Method
2.2.1. Kinase buffer 1X
20 mM HEPES, pH 7.4
150 mM NaCI
10 mM MgCI2
0.25 mM EGTA
0.01% Triton X-100
0.01 % (w/v) ovalbumine
1 mM DTT All solutions were prepared using MilliQ or a comparable quality of water. The kinase buffer was stored as stock at room temperature except for ovalbumine and DTT added on the day of screening from stock solutions. 2.2.2. Assay Protocol
• 4.9 pL of LimK-1 in kinase buffer (5 ng/reaction).
• + 0.1 pL of compound dilution.
• incubate at RT for 20 min.
· + 5 pL of cofilin ( .9 pg/reaction = 10 pM final concentration) with ATP (10 pM final concentration) in kinase buffer.
• incubate at room temperature for 30 min.
• + 10 pL Kinase-Glo solution.
• incubate at room temperature for 10 min in dark.
· read luminescence.
Automation was used utilized to run the assay. A MultiDrop reagent dispenser added 4.9 pL of LIMK-1 in kinase buffer to all wells except negative control wells. Those were filled with the same volume of assay buffer.
On a separate plate a dilution series was prepared: 10 pL of compounds in DMSO (concentration 10 mM) were dispensed into the wells of column 1 and 12. A MiniTrak program was used to dispense 5 pL DMSO into a new column and mix with 5 pL from the previous one. The remaining columns 23 and 24 were filled with DMSO only.
Using the 384-pintool 100 nL of the dilutions were transferred to the assay plate.
Compounds were incubated with the enzyme for at least 20 min to ensure homogenous distribution in the well before 5 pL of cofilin/ATP in kinase buffer was added to all wells (MultiDrop dispenser). Prepared in that way, final concentrations of the compounds ranged between 100 pM and 9.7 nM. The plates were lidded and incubated for 30 min at room temperature.
The plates were de-lidded and 10 pL Kinase-Glo reagent per well were added with a MultiDrop. The plates were lidded again and incubated for further 10 min in the dark to allow for stabilization of the luminescence signal. Afterwards, the plates were read in the EnVision 2103 reader using the program "Ultrasensitive Luminescence". Each well was read for 0.1 sec. Because the Kinase-Glo reagents were sensitive to light, all handlings were done under modest light conditions. 2.2.3. Data Analysis
Data were analysed in Microsoft Excel or IDBS BioBook templates and the quality of the assay results were monitored by determination of the Z'-factor for each assay plate (Zhang et al, J Biomol Screening, 4:67-73, 1999). The results were calculated as percent inhibition of the enzyme by the compound according to the following equation:
Volnhibition
Figure imgf000124_0001
where:
x = signal in samples after compound treatment.
ff = mean signal of the controls (no compound).
μ* = mean signal of the blanks (no LI K-1).
3. Appendix
3.1. Parameters for the PerkinElmer Envision 2103 read program "US Lum" General:
Program Name: LUM384 for LIMK-1.
Label 1 : Ultra sensitive Luminescence.
Plate filter: 384 wells, 16 * 24.
Counting time: 0.1 sec.
Correcf/ons:
Background correction: Yes.
Counting Control:
Measure each sample: 1 time.
Measure each plate: 1 time.
Delay between plate: 0 min.
Plate orientation: Normal. 3.2. Kinase-Glo substrate solution (10x100 ml kit)
Most of the reagent was prepared fresh for each screen run. Any left over solution from previous occasions was stored at -20°C and only thawed once. The substrate solution was prepared according to the supplier's manual, e.g., bringing the solutions to room temperature and reconstituting the lyophilized substrate solution. This was carried out in the dark due to the reagent's sensitivity to light. The reconstituted substrate solution contained a mutant of firefly luciferase and its substrate D-Luciferin which were necessary for the detection of ATP levels after the kinase reaction. 3.3. Automation Protocol
The MiniTrak program "LimK1 pintool addition only" was utilized to run the assay. Assay 2 - Lim-kinase 1 (LimK-1) Transcreener FP
1. Introduction
This protocol describes a method to quantify the effect of inhibitors of LIMK1. The 384 well format assay is based on the Transcreener FP technology to measure ADP formed during the reaction.
2. Materials and Methods 2. 1. Materials
Figure imgf000125_0001
2.2. Solutions: All solutions were prepared using MilliQ or a comparable quality of water.
LIMK1 assay buffer 1X:
20 mM HEPES, pH 7.4
150 mM NaCI 10 mM MgCI2
0.25 mM EGTA
0.01% Triton X-100 Stored at room temperature.
Added on the day of the assay from 1000X stock solutions:
0.01% (w/v) ovalbumine.
1 mM DTT.
ATP stock solution 10 mM:
ATP 10 mM.
HEPES 10 mM.
EDTA 1mM.
titrated to pH 7.00.
single-use aliquots stored at -20 .
LIMK1 dilution:
LIMK1: 0.8 ng/5 pL will give a final concentration of 1.9 nM during the kinase reaction. LIMK1 assay buffer 1X.
Suosfrafe dilution:
Cofilin: 1.9 pg/5 pL will give 10 μΜ final concentration during the kinase reaction.
ATP: 40 pM in dilution to give 20 pM during the kinase reaction.
LIMK1 assay buffer 1X.
Stop & Detect mix:
The concentration of the ADP antibody is dependent on the ATP concentration present due to a limited selectivity.
ADP-antibody: 28.1 pg/mL in the mix.
Tracer: 8 nM.
Dilute in Stop & Detect buffer 1X, contained in the pack as 10X. 2.3. Assay Protocol:
Preparation of dilution series: a - volume of compound containing solution.
b - volume of diluent.
The Minitrak program for pin tool transfer is optimized to pick up the dilution out of 7.5 pL of DMSO, therefore 6 is usually 7.5 ul. Choose a to achieve any desired dilution factor of
1 :(a+6)/a. Protocol:
• into columns 1 and 12 of the dilution plate add a+o μΙ of 10 mM compound stock.
• add a+o μΙ DMSO into columns 23+24.
· the following steps are performed by the Min/trak program 11_pt_titration_P30.
• dilute by aspirating a pi from columns 1/12, mixing with b μΙ DMSO, and dispensing the entire volume into columns 2/13.
• continue till columns 11/22. Assay setup:
• add 5 μί. UMK1 dilution to all wells except Blank and Buffer only.
• transfer 0.1 μΙ compound dilution into assay plate using Minitrak program
Limk1_Pintool_Addition_Only.
· incubate 10 minutes to allow for binding of compounds to enzyme.
• add 5 μΙ substrate dilution to all wells except Buffer only.
• incubate 60 min at room temperature.
• add 10 μΙ Stop & Detect mix.
• seal plate with Microamp seal.
· incubate at room temperature 2-4 h (over night is acceptable).
• read fluorescence polarization.
Note for the MultiDrop: prior to all additions flush the MultiDrop cassette with assay buffer containing ovalbumine to minimize loss of the enzyme/ substrate in the tubings.
Data Analysis:
Data was analysed in Microsoft Excel or IDBS BioBook templates and the quality of the assay results was monitored by determination of the Z'-factor for each assay plate (Zhang ef a/, J Biomol Screening, 4:67-73, 1999). The results were calculated as percent inhibition of the enzyme by the compound according to the following equation:
%Inhibition = \W*{\ ~ ) where:
x = signal in samples after compound treatment
μ' ~ mean signal of the controls (no compound)
μ* = mean signal of the blanks (no LIMK-1) 3. Appendix
3.1. Automation details The MiniTrak programs "Titration J 1 pt_P30" and "LimK1_pintool_addition_only" were utilized to run the assay.
3.2. Accessories for the PerkinElmer Envision 2103 Filter set Cy5 FP label (#2100-8380) consisting of:
excitation filter 620/40 nm.
mirror module D658.
emission filters 688/45 nm for S- and P-polarisation. 3.3. Program and Label settings for PerkinElmer Envision 2103
Program Name: FP Transcreener.
Label: Transcreener FP.
Plate layout: 384 wells, 16 * 24.
Background correction: Yes.
Reading mode: Fluorescence polarization.
Calculation: mP value, with blank correction.
Excitation light: 50%.
G factor: 0.63.
Detector gain (1 st and 2nd): 800.
Number of flashes: 150.
Number of flashes per A/D conversion: 1.
Assay 3 - LIMK2 (Full Length) Enzyme Binding Assay
1. Reagents
1X Kinase Buffer A: 50 mM HEPES at pH 7.5, 10 mM gCI2> 1 m EGTA, 0.01 % Brij-35.
Kinase Enzyme: LIM Kinase 2, full length human N-terminal GST-fusion protein (99 kDa) with human His-tagged ROCK2 using baculovirus expression system purchased from Carna Biosciences (Product code 09-106) (Lot # 08CBS-04 6 C); Stock concentration = 279 pg/mL. Aliquots stored at -80°C.
LanthaScreen™ Eu Kinase Binding Assay Reagents: Kinase tracer 178 (Product code PV5593) and Eu-anti GST antibody (Product code PV5594) were obtained from
Invitrogen. Both reagents were stored at -20°C. Prior to IC50 determinations, a Tracer Kd experiment was run to optimise the concentration of each new batch of Tracer 78 to use to ensure that the concentration of Tracer selected was near or slightly below Kd to ensure sensitive detection of inhibitors. 2. Method
Dry spot 5 μΐ_ of compound in 3% DMSO to test wells, or 3% DMSO to blanks and controls into a white polystyrene non-binding surface 384-well low volume microplate (Matrix, product code 4365) using a BioMek Nx. A 15 nM kinase and 6 nM antibody kinase/antibody solution (3 X the desired final assay concentration) was prepared in 1 X Kinase Buffer A. The antibody tube was centrifuged at approximately 10,000 x g for 0 minutes and the desired volume aspirated from the top of the solution to eliminate spurious data points that can arise on occasion due to any particulates in the product. A working solution of tracer 178 was prepared at 3x the final desired concentration in 1 X Buffer A. 5 pL of kinase/antibody solution was added to all test compound wells and positive control wells. An antibody solution containing no UMK2 kinase was added to the final two columns to give negative control wells. 5 uL of Tracer 78 was added to all wells of the plate and then incubated at room temperature for 60 minutes. Following the incubation period the plate was read on a Tecan Ultra plate reader where two sequential measurements were measured: one at 620 nm for the antibody/donor emission, and the other at 665 nm for the acceptor/tracer emission. The emission ratio is calculated by the ratio of the two fluorescence intensities (acceptor/donor).
3. Tecan settings
Measurement 1: Excitation Filter 337 nm, Emission Filter 620 nm, Mirror Dichroic2 (e.g., Fl 96), Lag time 100 ps, Integration time 200 ps, optimal Gain, optimal Z-position.
Measurement 2: Excitation Filter 337 nm, Emission Filter 665 nm, Mirror Dichroic2 (e.g., Fl 96), Lag time 100 ps, Integration time 200 ps, optimal Gain, optimal Z-position.
Biological Data - Assay 1 - LIMK1 (Kinase Domain) Enzyme Activity Assay
The following compounds were examined using Assay 1 :
AA-003, AA-004, AA-006, AA-021 , AA-022, AA-024, AA-025, AA-026, AA-027, AA-029, AA-030, AA-031 , AA-034, AA-035, AA-038, AA-039, AA-040, AB-003, AB-008, AC-001, AC-002, AC-003, AC-004, AD-001, AD-002, AD-003, AD-004, AD-005, AD-006, AD-008, AD-009, AD-010, AD-011 , AD-012, AD-013, AD-014, AD-015, AE-00 , AE-003, AE-01 1, AE-012, AE-013, AE-014, AE-015, AE-016, AE-017, AE-018, AE-020, AE-021 , AE-022, AE-023, AE-025, AE-026, AE-027, AE-028, AE-029, AE-034, AF-001, AG-001, AG-002, AG-003, AG-004, AG-005, AH-001 , AH-002, AH-003, AH-005, AH-007, AH-008, AH-0 2, AH-013, AH-014, AH-015, 68-001, BB-002, BB-003, BB-004, BB-005, BB-006, BB-007, BB-008, BB-009, BB-010, CC-001, CC-002, CC-003, CC-004.
All of these compounds were found to have an IC50 of less than 20 μ .
The following compounds were found to have an IC50 of less than 5 μ :
AA-003, AA-004, AA-006, AA-021, AA-022, AA-024, AA-025, AA-026, AA-027, AA-029, AA-030, AA-034, AA-035, AA-038, AA-039, AA-040, AB-003, AB-008, AC-00 , AC-002, AC-003, AC-004, AD-001 , AD-002, AD-003, AD-004, AD-005, AD-006, AD-008, AD-009, AD-010, AD-011, AD-012, AD-013, AD-014, AD-015, AE-001 , AE-003, AE-011, AE-012, AE-013, AE-014, AE-015, AE-016, AE-017, AE-018, AE-021 , AE-025, AE-026, AE-027, AE-028, AE-029, AE-034, AG-003, AG-005, AH-001, AH-002, AH-003, AH-005, AH-007, AH-008, AH-012, AH-013, AH-015, BB-001 , BB-002, BB-006, BB-008, BB-009, BB-010, CC-001 , CC-002, CC-003, CC-004.
The following compounds were found to have an IC50 of less than 0.5 μ :
AA-003, AA-004, AA-006, AA-021, AA-024, AA-025, AA-026, AA-027, AA-029, AA-030, AA-034, AA-038, AA-039, AA-040, AB-003, AB-008, AC-001 , AC-002, AC-003, AC-004, AD-001 , AD-002, AD-003, AD-005, AD-006, AD-008, AD-009, AD-010, AD-011 , AD-013, AE-001 , AE-003, AE-0 1 , AE-014, AE-015, AE-017, AE-018, AE-021 , AE-025, AE-027, AE-029, AE-034, AH-002, AH-003, AH-007, AH-012, AH-015, CC-001, CC-002, CC-004. The following compounds were found to have an IC50 of less than 0.2 μ :
AA-003, AA-021 , AA-024, AA-029, AA-030, AA-034, AA-038, AA-040, AB-003, AB-008, AC-001 , AC-002, AC-004, AD-005, AD-008, AD-009, AE-003, AE-011 , AE-014, AE-021 , AE-029, AE-034, AH-002, CC-004.
Biological Data - Assay 2 - LI K1 (Kinase Domain) Enzyme Binding Assay The following compounds were examined using Assay 2: AA-001 , AA-002, AA-003, AA-005, AA-006, AA-007, AA-008, AA-009, AA-010, AA-0 1 , AA-012, AA-013, AA-014, AA-015, AA-016, AA-017, AA-0 8, AA-0 9, AA-020, AA-021 , AA-023, AA-028, AA-029, AA-032, AA-033, AA-036, AA-037, AA-038, AA-040, AB-001 , AB-002, AB-003, AB-004, AB-005, AB-006, AB-007, AB-009, AB-010, AB-011 , AB-012, AB-013, AB-014, AB-015, AB-016, AB-017, AB-0 8, AB-019, AB-020, AB-021 , AB-022, AB-023, AB-024, AB-025, AB-026, AB-027, AB-028, AB-029, AB-030, AB-031, AB-032, AB-033, AB-034, AB-035, AB-036, AB-037, AC-001, AC-002, AD-007, AD-008, AD-009, AD-016, AD-017, AE-001 , AE-002, AE-003, AE-004, AE-005, AE-006, AE-007, AE-008, AE-009, AE-010, AE-019, AE-024, AE-030, AE-031 , AE-032, AE-033, AH-003, AH-004, AH-006, AH-009, AH-010, AH-011 , AJ-001 , BB-009, CC-001 , CC-002, CC-003, CC-004.
All of these compounds were found to have an IC5o of less than 10 μ .
The following compounds were found to have an IC50 of less than 1 μΜ:
AA-001, AA-002, AA-003, AA-005, AA-006, AA-007, AA-008, AA-009, AA-010, AA-01 , AA-0 2, AA-013, AA-0 4, AA-015, AA-016, AA-017, AA-018, AA-019, AA-020, AA-021 , AA-023, AA-028, AA-029, AA-032, AA-033, AA-036, AA-037, AA-038, AA-040, AB-001, AB-002, AB-003, AB-004, AB-005, AB-006, AB-007, AB-009, AB-010, AB-011 , AB-012, AB-013, AB-014, AB-015, AB-016, AB-017, AB-018, AB-019, AB-020, AB-021 , AB-022, AB-023, AB-024, AB-025, AB-026, AB-027, AB-028, AB-029, AB-030, AB-031 , AB-032, AB-033, AB-034, AB-035, AB-037, AC-001 , AC-002, AD-007, AD-008, AD-009, AD-016, AD-017, AE-001 , AE-002, AE-003, AE-004, AE-005, AE-006, AE-019, AE-024, AE-030, AE-031, AE-032, AE-033, AH-003, AH-004, AH-006, AH-009, AH-0 1, BB-009, CC-001, CC-002, CC-003, CC-004.
The following compounds were found to have an IC50 of less than 0.2 μ :
AA-001 , AA-003, AA-005, AA-007, AA-008, AA-009, AA-010, AA-014, AA-015, AA-016, AA-018, AA-020, AA-02 , AA-023, AA-028, AA-029, AA-032, AA-033, AA-036, AA-037, AA-038, AA-040, AB-002, AB-003, AB-004, AB-005, AB-010, AB-012, AB-013, AB-014, AB-0 5, AB-016, AB-019, AB-020, AB-021 , AB-022, AB-025, AB-026, AB-027, AB-028, AB-029, AB-034, AB-035, AC-001 , AC-002, AD-007, AD-008, AD-009, AE-002, AE-030, AE-031 , AE-032, AE-033.
Biological Data - Assay 3 - LI K2 (Full Length) Enzyme Binding Assay The following compounds were examined using Assay 3: AA-040 and AB-027.
The IC50 for AA-040 was 0.202 μΜ.
The IC50 for AA-027 was 0.547 μ . Biological Data - Selected Compounds
Data for some of the compounds is shown in the following table. Assay 1 Assay 2 Assay 3
Code
ICso (μΜ) ICso (μ ) ICS0 (μ )
AA-040 0.062 0.155 0.202
AB-003 0.083 0.094 -
AC-001 0.077 0.066 -
AD-009 0.061 0.111 -
AE-003 0.135 0.258 -
AF-001 5.704 - -
AG-005 2.459 - -
AH-003 0.418 0.442 -
AJ-001 - 8.011 -
BB-009 0.793 0.859 -
CC-00 0.327 0.433 -
* * *
The foregoing has described the principles, preferred embodiments, and modes of operation of the present invention. However, the invention should not be construed as limited to the particular embodiments discussed. Instead, the above-described embodiments should be regarded as illustrative rather than restrictive, and it should be appreciated that variations may be made in those embodiments by workers skilled in the art without departing from the scope of the present invention.
REFERENCES
A number of patents and publications are cited herein in order to more fully describe and disclose the invention and the state of the art to which the invention pertains. Full citations for these references are provided below. Each of these references is incorporated herein by reference in its entirety into the present disclosure, to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.
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Claims

CLAI S
A compound selected from compounds of the following formula, and
pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000140_0001
wherein:
-X= is independently -CRZ6= or -N=;
-Y- is independently -S-, -NRY-, or -0-;
-RY is independently -H or saturated aliphatic d.6alkyl; and wherein, if -X= is -CR =, then:
-RZ6 is independently -H, -RQS, or -RQL;
-R27 is independently -H, -RQS, or -RQL;
-RZ8 is independently -H, -RQS, or -R0L; and
-R2S is independently -H , -RQS, or -RQL;
with the proviso that at least one of -Rza, -RZ7, -R28, and -RZ9 is -R' and wherein, if -X= is -N= then:
-RZ7 is independently -H, -RQS, or -RQL;
-R2S is independently -H, -RQS, or -RQL; and
-R29 is independently -H, -RQS, or -RQL;
with the proviso that at least one of -R27, -RZ8, and -RZ9 is -RQL; wherein:
each -RQL is independently -RCA, -RHA, -Rcc, or -RHC;
wherein:
-RCA is independently phenyl or naphthyl, and is optionally substituted with one or more substituents, -Rx;
-RHA is independently C^heteroaryl, and is optionally substituted with one or more substituents, -Rx;
-Rcc is independently non-aromatic C3.7cycloalkyl or non-aromatic C3.7cycloalkenyl, and is optionally substituted with one or more substituents, -Rx;
-RHC is independently non-aromatic C3.7heterocyclyl, and is optionally substituted with one or more substituents, -Rx; and wherein:
each -RQS is independently -F, -CI, -Br, -I, -Rs, -CF3, -OH, -ORs, -OCF3, -NH2, -NHRS, -NRS2, -NHC(=0)Rs, -NHC(=0)ORs, -C(=0)OH, -C(=0)ORs, -C(=0)NH2, -C(=0)NHRs, -C(=0)NRs 2, -SH, -SRS, -CN, or -N02;
wherein each -Rs is independently saturated aliphatic C^alkyl, phenyl, -CH phenyl, -CH2CH2-phenyl, or -CH=CH-phenyl, wherein each phenyl is optionally substituted with one or more groups selected from: -F, -CI, -Br, -I, -R! -CF3, -OH, -ORss, and -OCF3, wherein each -Rss is independently saturated aliphatic C^alkyl; and wherein each -Rx is independently selected from -RX and -RX2, wherein: each -R is independently: -R2,
-F, -CI, -Br, -I,
-CF3, -OCF3,
-OH, -RZL-OH, -0-RZL-OH, -NH-RZL-OH, -NRz-RZL-0H,
-ORz, -RZL-ORz, -0-RZL-ORz, -NH-R^-OR2, -NRz-RZL-0Rz,
-SH, -SRZ,
-CN,
-N02,
-C(=0)OH, -C(=0)0Rz,
-C(=0)Rz,
-NH2, -NHRZ, -NRZ 2, -RNZ,
-RZL-NH2, -RZL-NHRZ, -RZL-NRZ 2, -RZL-RNZ,
-0-RZL-NH2, -0-RZL-NHRz, -0-RZL-NRz 2, -0-RZL-RNZ,
-NH-RZL-NH2, -NH-R2L-NHRZ, -NH-R2L-NRZ 2, -NH-RZL-RNZ,
-NRZ-RZL-NH2, -NRZ-RZL-NHRZ, -NRZ-RZL-NRZ 2, -NRZ-RZL-RNZ,
-C(=0)NH2, -C(=0)NHRz, -C(=0)NRz 2, -C(=0)RNZ,
-RZL-C(=0)NH2, -RZL-C(=0)NHRz, -RZL-C(=0)NRz 2, -RZL-C(=0)RNZ,
-0-RZL-C(=0)NH2, -0-RZL-C(=0)NHRz, -0-RZL-C(=0)NRz 2, -0-RZL-C(=0)RNZ, -NH-C(=0)Rz, -NRz-C(=0)Rz,
-NH-C(=0)OH, -NRz-C(=0)0H, -ISIH-C(=0)0Rz, -NRz-C(=0)0Rz,
-OC(=0)NH2, -0C(=0)NHRz, -OC(=0)NRz 2, -0C(=0)RNZ,
-NHC(=0)NH2, -NHC(=0)NHRz, -NHC(=0)NRz 2, -NHC(=0)RNZ,
-NRzC(=0)NH2, -NRzC(=0)NHRz, -NRzC(=0)NRz 2, -NRzC(=0)RNZ,
-S(=0)2Rz,
-S(=0)2NH2, -S(=0)2NHRz, -S(=0)2NRz 2, -S(=0)2RNZ,
-NH-S(=0)2Rz, -NRz-S(=0)2Rz, or
=0;
and additionally, two or more adjacent substituents -RX1, if present, may together form -0CH20-, -0CH2CH20-, or -0CH2CH2CH20-; wherein:
each -R2L- is independently saturated aliphatic C^alkylene;
each -RN2 is independently azetidino, pyrrolidino, piperidino, piperazino, morpholino, azepino, or diazepino, and is optionally substituted with one or more substitutents selected from saturated aliphatic d.4alkyl;
each -R2 is independently saturated aliphatic C1-6alkyl, phenyl, benzyl, or C5.6heteroaryl, wherein phenyl, benzyl, and C5.6heteroaryl are each optionally substituted with one or more substituents selected from -F, -CI, -Br, -I, -R22, -CF3, -OH
Figure imgf000142_0001
and -C(=0)NRZ22, wherein each -R22 is independently saturated aliphatic C^alkyl; and
-R is independently:
-NH-C(=J)-NH2> -NRK -C(=J)-NH2,
-NH-C(=J)-NHRK2, -NRK -C(=J)-NHRK2,
-NH-C(=J)-NRK2 2, -NR^-Ci^-NR^,
-NH-C(=J)-NR 3RK4, -NRK1-C(=J)-NRK3R'
-NH-C(=0)H, -NR 1-C(=0)H,
-NH-C(=0)RK6, -NRK1-C(=0)RK5,
-C(=0)-NH2, -C(=0)-NHRK2, -C(=0)-NR 2 2> -C(=0)-NRK3R 4,
-OH, or -0R S; wherein each =J is independently =0 or =S; and wherein each -RK1 is independently saturated aliphatic Ci.4alkyl; and wherein each -R 2 is independently:
saturated aliphatic C^alkyl, aliphatic C^alkenyl, or saturated
C3.6cycloalkyl;
each optionally substituted with one or more groups independently selected from: -F, -CI, -Br, -I, -OH, -ORA1, -0CF3, -SRA1, -S(=0)RA , -S(=0)2RA1, -NH2, -NHRA1, -NRA12, pyrrolidinyl, piperidinyl, morpholinyl, piperizinyl, -C(=0)OH, -C(=0)ORA1, -OC(=0)RA1, -C(=0)NH2, -C(=0)NHRA1, -C(=0)NRA1 2,
pyrrolidino-C(=0)-, piperidino-C(=0)-, morpholino-C(=0)-, piperizino-C(=0)-, and -R^;
wherein:
each pyrrolidinyl, piperidinyl, morpholinyl, piperizinyl, pyrrolidino, piperidino, morpholino, and piperizino is optionally substituted with one or more groups independently selected from -F, -FT, -OH, -OR , -NH2, -NHR , -NRA 2, pyrrolidino, piperidino, morpholino, piperizino, -C(=0)OH, -C(=0)ORA1, -C(=0)R' -C(=0)NH2, -C(=0)NHRA1, -C(=0)NRA1 2, pyrrolidino-C(=0)-, piperidino-C(=0)-, morpholino-C(=0)-, piperizino-C(=0)-, and -OCH2CH20-;
each -RA is independently saturated aliphatic Chalky!; and
each -RA2 is independently phenyl, Cs.sheteroaryl, or non-aromatic Cs.7heterocyclyl; and wherein each -NR R is independently:
pyrrolidino, piperidino, morpholino, or piperizino;
each optionally substituted with one or more groups independently selected from -F, -RA3, -OH, -ORA3, -NH2, -NHRA3, -NRA3 2, pyrrolidino, piperidino, morpholino, piperizino, -C(=0)OH, -C(=0)ORA3, -C(=0)RA3, -C(=0)NH2, -C(=0)NHRA3, -C(=0)NRA3 2, pyrrolidino-C(=0)-, piperidino-C(=0)-,
morpholino-C(=0)-, piperizino-C(=0)-, and -OCH2CH20-;
wherein each -RA3 is independently saturated aliphatic Chalky!, optionally substituted with one or more groups independently selected from -OH, -ORA4, -NH2, -NHRM, -NRA42, pyrrolidino, piperidino, morpholino, and piperizino;
wherein each ~RM is independently saturated aliphatic d^alkyl; and wherein each -RK5 is independently saturated aliphatic Chalky!, and is optionally substituted with one or more groups independently selected from: -F, -CI, -Br, -I, -CF3, -OH, -ORA5, -OCF3, -NH2( -NHRA5, and -NRA5 2, wherein each -R' is independently saturated aliphatic C1-4alkyl; and wherein each -RK6 is independently saturated aliphatic C^alkyl, and is optionally substituted with one or more groups independently selected from: -F, -CI, -Br, -I, -CF3, -OH, -ORAe, -OCF3, -NH2, -NHRA6, and -NRA6 2l wherein each -R' is independently saturated aliphatic
Figure imgf000143_0001
with the proviso that the compound is not a compound selected from: compounds PP-001 to PP-034, and pharmaceutically acceptable salts, hydrates, and solvates thereof.
2. A compound according to claim 1 , wherein -Y- is -S-.
3. A compound according to claim 1, wherein -Y- is -0-. 4. A compound according to claim 1, wherein -Y- is -NRY-.
5. A compound according to any one of claims 1 to 4, wherein ~X= is -CRZ6=.
6. A compound according to any one of claims 1 to 4, wherein -X= is -N=.
7. A compound according to claim 1 , wherein the compound is selected from compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000144_0001
A compound according to claim 7, wherein:
-RZ6 is independently -H or -RQS;
-RZ7 is independently -RQL;
-RZ8 is independently -H or -RQS; and
-RZ9 is independently -H or -RQS.
A compound according to claim 7, wherein:
-RZ6 is independently -H;
-RZ7 is independently -RQL;
-RZ8 is independently -H; and
-RZ9 is independently -H or -RQS.
A compound according to claim 7, wherein.
-RZ6 is independently -H;
-RZ7 is independently -RQL;
-RZ8 is independently -H; and
-RZ9 is independently -H. A compound according to claim 1, wherein the compound is selected from compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof.
Figure imgf000145_0001
with the proviso that the compound is not a compound selected from: compounds PP-001 to PP-034, and pharmaceutically acceptable salts, hydrates, and solvates thereof.
A compound according to claim 11, wherein:
Figure imgf000145_0002
-RZ7 is independently -RQL;
-R29 is independently -H or -RQS; and
-R29 is independently -H or -RQS.
A compound according to claim 11, wherein:
Figure imgf000145_0003
-R27 is independently -RQL;
-R28 is independently -H; and
-R29 is independently -H or -RQS
14. A compound according to claim 11 , wherein:
-R27 is independently -RQL;
-R28 is independently ~H; and
-R29 is independently -H.
A compound according to claim 1 , wherein the compound is selected from
Figure imgf000145_0004
compounds of the following formula, and pharmaceutically acceptable salts, hydrates, and solvates thereof:
Figure imgf000145_0005
A compound according to claim 15, wherein:
Figure imgf000145_0006
-R26 is independently -H or -RQS;
-R27 is independently -RQL;
-R28 is independently -H or -RQS; and
-R29 is independently -H or -Ros. A compound according to claim 15, wherein:
-R26 is independently -H;
-RZ7 is independently -RQL;
-R28 is independently -H; and
-RZ9 is independently -H or -RQS. 8. A compound according to claim 15, wherein:
-R26 is independently -H;
-R27 is independently -RQL;
-R28 is independently -H; and
-R29 is independently -H.
19. A compound according to any one of claims 1 to 18, wherein each -RQS, if present, is independently -F, -CI, -Br, -I, -Rs, -CF3, -OH, -ORs, -OCF3, -NH2) -NHRS, -NRS 2, -NHC(=0)Rs, -NHC(=0)ORs, -C(=0)OH, -C(=0)ORs, -C(=0)NH2, -C(=0)NHRs, or -C(=0)NRs 2.
20. A compound according to any one of claims 1 to 18, wherein each -RQS, if present, is independently -F, -CI, -Br, -I, -Rs, -CF3, -OH, -ORs, or -OCF3.
21. A compound according to any one of claims 1 to 18, wherein each -RQS, if present, is independently -Rs.
22. A compound according to any one of claims 1 to 21 , wherein each -Rs, if present, is independently saturated aliphatic C^alkyl or phenyl, wherein said phenyl is optionally substituted with one or more groups selected from: -F, -CI, -Br, -I, -Rsss, -CF3, -OH, -ORsss, or -OCF3, wherein each -Rsss is independently saturated aliphatic C1-4alkyl. 23. A compound according to any one of claims 1 to 21 , wherein each -Rs, if present, is independently saturated aliphatic CMalkyl or phenyl.
24. A compound according to any one of claims 1 to 21 , wherein each -Rs, if present, is independently -Me, -Et, -nPr, -iPr, -nBu, -iBu, or -tBu.
25. A compound according to any one of claims 1 to 21 , wherein each -Rs, if present, is independently -Me.
26. A compound according to any one of claims 1 to 25, wherein -RQL, or each -RQL, is independently -RCA or -RHA.
27. A compound according to any one of claims 1 to 25, wherein -RQL, or each -RQL, is independently -RCA.
28. A compound according to any one of claims 1 to 25, wherein -R , or each -R , is independently -RHA. 29. A compound according to any one of claims 1 to 28, wherein -RCA, if present, or each -RCA, if present, is independently phenyl; and is optionally substituted with one or more substituents, -Rx.
30. A compound according to any one of claims 1 to 28, wherein -RCA, if present, or each -RCA, if present, is independently;
Figure imgf000147_0001
wherein each of -R , -R , and -R is independently -H or a ring substituent, wherein each ring substituent is independently -R*. 31. A compound according to any one of claims 1 to 28, wherein -RCA, if present, or each -RCA, if present, is independently:
Figure imgf000147_0002
wherein each of -R and -R is independently -H or a ring substituent, wherein each ring substituent is independently -Rx.
32. A compound according to any one of claims 1 to 28, wherein -RCA, if present, or each -RCA, if present, is independently:
Figure imgf000147_0003
wherein -R 3 is independently a ring substituent, wherein the ring substituent is independently -Rx
33. A compound according to any one of claims 1 to 28, wherein -RCA, if present, or each -RCA, if present, is independently.
Figure imgf000147_0004
wherein -RP4 is independently a ring substituent, wherein the ring substituent is independently -Rx.
34. A compound according to any one of claims 1 to 28, wherein -R , if present, or each -RCA, if present, is independently:
Figure imgf000148_0001
wherein each of -R and -R is independently a ring substituent, wherein each ring substituent is independently -Rx.
A compound according to any one of claims 1 to 28, wherein -RCA, if present, or each -RCA, if present, is independently:
Figure imgf000148_0002
wherein each of -R and -R is independently -H or a ring substituent, wherein each ring substituent is independently -R .
36. A compound according to any one of claims 1 to 28, wherein -R , if present, or each -RCA, if present, is independently:
Figure imgf000148_0003
wherein each of -R and -R is independently a ring substituent, wherein each ring substituent is independently -Rx.
A compound according to any one of claims 1 to 28, wherein -RCA, if present, or each -RCA, if present, is indepe
Figure imgf000148_0004
wherein each of -R and -R is independently a ring substituent, wherein each ring substituent is independently -Rx. A compound according to any one of claims 1 to 28, wherein -R , if present, or each -RCA, if present, is independently:
Figure imgf000149_0001
wherein each of -Rp and -R is independently a ring substituent, wherein each ring substituent is independently -Rx.
39. A compound according to any one of claims 1 to 28, wherein -RCA, if present, or each -RCA, if present, is indepe
Figure imgf000149_0002
wherein -R is independently a ring substituent, wherein the ring substituent is independently -Rx.
40. A compound according to any one of claims 1 to 28, wherein -RCA, if present, or each -RCA, if present, is independently:
Figure imgf000149_0003
wherein each of -RP3, -RP4, and -R is independently a ring substituent, wherein each ring substituent is independently -R .
41. A compound according to any one of claims 1 to 28, wherein - ", if present, or each -RCA, if present, is indepe
Figure imgf000149_0004
42. A compound according to any one of claims 1 to 41 , wherein -R , if present, or each -RHA, if present, is independently furanyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrimidinyl, pyridazinyl, and indolyl; and is optionally substituted with one or more substituents, -Rx. A compound according to any one of claims 1 to 42, wherein:
if exactly one group -Rx is present, then -Rx is -RX2; and
if a plurality of groups -Rx are present, then at least one -Rx is -R:
A compound according to any one of claims 1 to 42, wherein:
if exactly one group -R is present, then -R is -Rx2; and
if a plurality of groups -R are present, then exactly one -Rx is -R:
A compound according to any one of claims 1 to 42, wherein:
exactly one group -Rx is present, and -R is -R*2.
46. A compound according to any one of claims 1 to 42, wherein:
exactly two groups -Rx are present, one -Rx is -RX2, and the other -Rx is -RX1. 47. A compound according to any one of claims 1 to 46, wherein each -RX , if present, is independently:
-Rz,
-F, -CI, -Br, -I,
Figure imgf000150_0001
-OH, -RZL-OH, -0-RZL-OH,
-OR2, -RZL-ORz, -0-RZL-ORz,
-CN,
-N02,
-C(=0)OH, -C(=0)ORz,
-C(=0)Rz,
-NH2, -NHRZ, -NRZ2, -RNZ,
-RZL-NH2, -RZL-NHRZ, -RZL-NRZ 2, -RZL-RNZ
-NH-RZL-NH2, -NH-RZL-NHRZ, -NH-RZL-NRZ 2, -NH-RZL-RNZ,
-C(=0)NH2, -C(=0)NHRz, -C(=0)NRz 2, -C(=0)RNZ,
-NH-C(=0)Rz, or -NRz-C(=0)R2.
48. A compound according to any one of claims 1 to 46, wherein each -RX1, if present, is independently:
-Rz,
-F, -CI, -Br, -I,
Figure imgf000150_0002
-OH, -RZL-OH,
-OR2, -RZL-ORz,
-CN,
-N02,
-C(=0)OH, -C(=0)ORz,
-C(=0)Rz,
-NH2, -NHR2, -NRZ 2, -RNZ, -RZL-NH2l -RZL-NHRZ, -RZL-NRz 2l -RZL-RNZ,
-C(=0)NH2) -C(=0)NHRz, -C(=0)NRz 2, or
-NH-C(=0)Rz.
A compound according to any one of claims 1 to 46, wherein each -RX1, if present, is independently: -Rz, -F, -CI, -Br, -OH, -ORz, -C(=0)OH, -C(=0)ORz, -NH2, -NHR2, -NRZ2, -RNZ, -C(=0)NH2, -C(=0)NHRz, -C(=0)NRz 2, or -NH-C(=0)Rz.
A compound according to an one of claims 1 to 46, wherein each -RXi, if present, is independently -Rz, -F, -CI, -Br, -OH, or -ORz.
A compound according to any one of claims 1 to 50, wherein:
each -RZL-, if present, is independently -CH2CH2-;
each -RNZ, if present, is independently pyrrolidino, piperidino, piperazino, or morpholino, and is optionally substituted with one or more substitutents selected from saturated aliphatic Chalky!; and
each -Rz, if present, is independently saturated aliphatic C^alkyl, phenyl, or benzyl.
A compound according to any one of claims 1 to 51, wherein each -RX2, if present, is independently:
-NH-C(=J)-NH2, -NRK1-C(=J)-NH2,
Figure imgf000151_0001
A compound according to any one of claims 1 to 51 , wherein each -RX2, if present, is independently:
-NH-C(=0)-NH2, -NRK1-C(=0)-NH2,
Figure imgf000151_0002
-NH-C(=0)-NRK3RK4, or -NRK1-C(=0)-NRK3RK4.
A compound according to any one of claims 1 to 51 , wherein each -RX2, if present, is independently:
-NH-C(=0)H, -NRK -C(=0)H,
-NH-C(=0)RK5, -NRK1-C(=0)RK5,
-C(=0)-NH2l -C(=0)-NHRK2, -C(=0)-NR 2 2, or -C(=0)-NRK3RH 55. A compound according to any one of claims 1 to 51 , wherein each -RX2, if present, is independently: -NH-C(=0)H, -NRK1-C(=0)H, -NH-C(=0)R 5, or -NRK1-C(=0)RK5.
56. A compound according to any one of claims 1 to 51 , wherein each -R , if present, is independently: -C(=0)-NH2, -C(=0)-NHRK2, -C(=0)-NR 2 2, or -C(=0)-NRK3R
57. A compound according to any one of claims 1 to 51 , wherein each -RX2, if present, is independently: -OH or -ORK6
58. A compound according to any one of claims 1 to 57, wherein each -RK1, if present, is independently -Me, -Et, -nPr, -iPr, -nBu, -I'BU, -sBu, or -tBu. 59. A compound according to any one of claims 1 to 57, wherein each -RK1, if present, is independently -Me.
60. A compound according to any one of claims 1 to 59, wherein each -RK2, if present, is independently: saturated aliphatic
Figure imgf000152_0001
optionally substituted with one or more groups independently selected from: -F, -CI, -Br, -I, -OH, -ORA1, -OCF3,
-SRA1, -S(=0)RA1, -S(=0)2RA , -NH2, -NHRA1, -NRA1 2, pyrrolidinyl, piperidinyl, morpholinyl, piperizinyl, -C(=0)OH, -C(=0)ORA1, -OC(=0)RA1, -C(=0)NH2,
Figure imgf000152_0002
morpholino-C(=0)-, piperizino-C(=0)-t and -RA2.
61. A compound according to any one of claims 1 to 59, wherein each -RK2, if present, is independently saturated aliphatic C1- alkyl.
62. A compound according to any one of claims 1 to 59, wherein each -RK2, if present, is independently aliphatic C^alkenyl.
63. A compound according to any one of claims 1 to 59, wherein each -R 2, if present, is independently saturated C3.6cycloalkyl. 64. A compound according to any one of claims 1 to 63, wherein each -RA1, if present, is independently -Me, -Et, -nPr, -iPr, -nBu, -iBu, -sBu, or -tBu.
65. A compound according to any one of claims 1 to 63, wherein each -RA , if present, is independently -Me.
66. A compound according to any one of claims 1 to 65, wherein each -RA2, if present, is independently phenyl or C5.6heteroaryl.
67. A compound according to any one of claims 1 to 65, wherein each -RA2, if present, is independently phenyl.
68. A compound according to any one of claims 1 to 67, wherein each -NRK3RM, if present, is independently pyrrolidine piperidino, morpholino, or piperizino.
69. A compound according to any one of claims 1 to 68, wherein each -RA3, if present, is independently saturated aliphatic C alkyl. 70. A compound according to any one of claims 1 to 68, wherein each -RA3, if present, is independently -Me, -Et, -nPr, -iPr, -nBu, -iBu, -sBu, or -tBu.
71. A compound according to any one of claims 1 to 68, wherein each -RA3, if present, is independently -Me.
72. A compound according to any one of claims 1 to 71 , wherein each -RA", if present, is independently -Me, -Et, -nPr, -iPr, -nBu, -iBu, -sBu, or -tBu.
73. A compound according to any one of claims 1 to 71 , wherein each -RA4, if present, is independently -Me.
74. A compound according to any one of claims 1 to 73, wherein each -RK5, if present, is independently saturated aliphatic CMalkyl, and is optionally substituted with one or more groups independently selected from: -OH, -ORAS, -NH2, -NHRAS, and -NRA5 2.
75. A compound according to any one of claims 1 to 73, wherein each -RK5, if present, is independently saturated aliphatic C alkyl. 76. A compound according to any one of claims 1 to 75, wherein each -RA5, if present, is independently -Me, -Et, -nPr, -iPr, -nBu, -iBu, -sBu, or -tBu.
77. A compound according to any one of claims 1 to 75, wherein each -RAS, if present, is independently -Me.
78. A compound according to any one of claims 1 to 77, wherein each -RK6, if present, is independently saturated aliphatic C alkyl, and is optionally substituted with one or more groups independently selected from: -OH, -ORA6, -NH2, -NHRA6, and
-NR A 66
2·
A compound according to any one of claims 1 to 77, wherein each -R , if present, is independently saturated aliphatic C alk l.
80. A compound according to any one of claims 1 to 79, wherein each -RA6, if present, is independently -Me, -Et, -nPr, -iPr, -nBu, -iBu, -sBu, or -tBu.
81. A compound according to any one of claims 1 to 79, wherein each -RA6, if present, is independently -Me. A compound according to claim 1, selected from the following compounds and pharmaceutically acceptable salts, hydrates, and solvates thereof:
AA-00 , AA-002, AA-003, AA-004, AA-005, AA-006, AA-007, AA-008, AA-009, AA-010, AA-011, AA-012, AA-013, AA-014, AA-015, AA-016, AA-017, AA-018, AA-019, AA-020, AA-021 , AA-022, AA-023, AA-024, AA-025, AA-026, AA-027, AA-028, AA-029, AA-030, AA-031, AA-032, AA-033, AA-034, AA-035, AA-036, AA-037, AA-038, AA-039,
AB-001 , AB-002, AB-003, AB-004, AB-005, AB-006, AB-007, AB-008, AB-009, AB-010, AB-011 , AB-012, AB-013, AB-014, AB-015, AB-016, AB-017, AB-018, AB-019, AB-020, AB-021 , AB-022, AB-023, AB-024, AB-025, AB-026, AB-027, AB-028, AB-029, AB-030, AB-031, AB-032, AB-033, AB-034, AB-035, AB-036, AB-037,
AC-001 , AC-002, AC-003, AC-004,
AD-001 , AD-002, AD-003, AD-004, AD-005, AD-006, AD-007, AD-008, AD-009, AD-010, AD-011 , AD-012, AD-013, AD-014, AD-015, AD-016, AD-017,
AE-001 , AE-002, AE-003, AE-004, AE-005, AE-006, AE-007, AE-008, AE-009, AE-010, AE-011 , AE-012, AE-013, AE-014, AE-015, AE-016, AE-017, AE-018, AE-019, AE-020, AE-021, AE-022, AE-023, AE-024, AE-025, AE-026, AE-027, AE-028, AE-029, AE-030, AE-031 , AE-032, AE-033, AE-034,
AF-001 ,
AG-001 , AG-002, AG-003, AG-004, AG-005,
AH-001, AH-002, AH-003, AH-004, AH-005, AH-006, AH-007, AH-008, AH-009, AH-010, AH-011 , AH-012, AH-0 3, AH-014, AH-0 5, and
AJ-001.
A compound according to claim 1 , selected from the following compounds and pharmaceutically acceptable salts, hydrates, and solvates thereof:
BB-00 , BB-002, BB-003, BB-004, BB-005, BB-006, BB-007, BB-008, BB-009, and BB-010.
A compound according to claim 1 , selected from the following compounds and pharmaceutically acceptable salts, hydrates, and solvates thereof:
CC-001 , CC-002, CC-003, and CC-004.
A composition comprising a compound according to any one of claims 1 to 84, and a pharmaceutically acceptable carrier, diluent, or excipient.
86. A method of preparing a composition comprising admixing a compound according to any one of claims 1 to 84 and a pharmaceutically acceptable carrier, diluent, or excipient. A compound as defined in any one of claims 1 to 84, but without the recited proviso regarding compounds PP-001 to PP-034, for use in a method of treatment of the human or animal body by therapy.
A compound as defined in any one of claims 1 to 84, but without the recited proviso regarding compounds PP-001 to PP-034, for use in a method of treatment of:
a disease or condition that is mediated by LIM kinase (LI K);
a disease or condition that is ameliorated by the inhibition of LIM kinase (LIMK) activity;
a proliferative condition;
cancer;
cancer characterised by, or further characterised by, cancer cells which overexpress LIM kinase (LIMK);
solid tumour cancer;
breast cancer; prostate cancer; melanoma; glioma;
vasodilation; hypertension; angina; cerebral vasospasm; ischemia following subarachnoid hemorrhage;
a neurodegenerative disorder;
atherosclerosis;
fibrosis;
inflammatory disease; Crohn's disease; chronic obstructive pulmonary disease (COPD); or
glaucoma.
Use of a compound as defined in any one of claims 1 to 84, but without the recited proviso regarding compounds PP-001 to PP-034, in the manufacture of a medicament for the treatment of:
a disease or condition that is mediated by LIM kinase (LIMK);
a disease or condition that is ameliorated by the inhibition of LIM kinase (LIMK) activity;
a proliferative condition;
cancer;
cancer characterised by, or further characterised by, cancer cells which overexpress LIM kinase (LIMK);
solid tumour cancer;
breast cancer; prostate cancer; melanoma; glioma;
vasodilation; hypertension; angina; cerebral vasospasm; ischemia following subarachnoid hemorrhage;
a neurodegenerative disorder;
atherosclerosis;
fibrosis; inflammatory disease; Crohn's disease; chronic obstructive pulmonary disease (COPD); or
glaucoma.
A method of inhibiting LIM kinase (LIMK), in vitro or in vivo, comprising contacting LIMK with an effective amount of a compound as defined in any one of claims 1 to 84, but without the recited proviso regarding compounds PP-001 to PP-034.
A method of inhibiting LIM kinase (LIMK) activity in a cell, in vitro or in vivo, comprising contacting the cell with an effective amount of a compound as defined in any one of claims 1 to 84, but without the recited proviso regarding compounds PP-001 to PP-034.
A method of regulating cell proliferation, inhibiting cell cycle progression, promoting apoptosis, or a combination of one or more these, in vitro or in vivo, comprising contacting a cell with an effective amount of a compound as defined any one of claims 1 to 84, but without the recited proviso regarding compounds PP-001 to PP-034.
PCT/GB2012/000280 2011-03-28 2012-03-27 Pyrido [3',2' :4,5] thieno [3, 2-d] pyrimidin- 4 - ylamine derivatives and their therapeutical use Ceased WO2012131297A1 (en)

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