WO2009146013A1 - Myosin light chain phosphatase inhibitors - Google Patents

Myosin light chain phosphatase inhibitors Download PDF

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Publication number
WO2009146013A1
WO2009146013A1 PCT/US2009/038783 US2009038783W WO2009146013A1 WO 2009146013 A1 WO2009146013 A1 WO 2009146013A1 US 2009038783 W US2009038783 W US 2009038783W WO 2009146013 A1 WO2009146013 A1 WO 2009146013A1
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phenyl
salt form
alkyl
light chain
compound according
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PCT/US2009/038783
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French (fr)
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Milton L. Brown
Scott Grindrod
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Georgetown University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/38—Nitrogen atoms
    • C07D277/44—Acylated amino or imino radicals
    • C07D277/48—Acylated amino or imino radicals by radicals derived from carbonic acid, or sulfur or nitrogen analogues thereof, e.g. carbonylguanidines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D277/38—Nitrogen atoms
    • C07D277/42—Amino or imino radicals substituted by hydrocarbon or substituted hydrocarbon radicals

Definitions

  • This disclosure relates to myosin light chain phosphatase inhibitors including in particular fluorescent myosin light chain phosphatase inhibitors, compositions including the same, and methods for preparing and using the same.
  • Cancers are among the most common causes of death in developed countries. Despite continuing advances, the existing treatments exhibit undesirable side effects and limited efficacy. Identifying new effective cancer drugs is a continuing focus of medical research.
  • Cell motility is an important property in cancer progression, metastasis and proliferation, potentially representing a significant target for new cancer therapies.
  • the aggressiveness of cancer is directly related to the ability of cancer cells to migrate and invade surrounding tissues. Migration is achieved through a dynamic cycle of extension and contraction of the cell body controlled by the cytoskeleton. Lontay, et al., Cell. Sig., 2005, 17, 1265-1275; Somolyo, et al., Physiol. Rev., 2003, 83, 1325-1358; Xia, et al., Exper. Cell Res., 2005, 304, 506-517.
  • myosin light chain phosphorylation of which is controlled by a delicate balance between kinase and phosphatase activities.
  • myosin light chain phosphorylation is regulated not only through the activation of myosin light chain kinase, but also through the inhibition of myosin light chain phosphatase activity.
  • Phosphorylation of MLC by myosin light chain kinase (MLCK) enhances motor activity of myosin as well as stability of myosin filaments, so that the dephosphorylation by myosin light chain phosphatase (MLCP) produces a relaxation and reorganization of actomyosin filaments.
  • Myosin light chain phosphatase is a serine/threonine phosphatase and consists of a 130 kDa regulatory subunit that binds myosin (MYPTl), a 37 kDa catalytic subunit protein phosphatase 1C (PPlC) and a 21 kDa subunit (M-21) of undetermined function.
  • MYPTl myosin
  • PlC 37 kDa catalytic subunit protein phosphatase 1C
  • M-21 21 kDa subunit
  • PPlC is the catalytic subunit of myosin light chain phosphatase, is a member of the PPP superfamily of phosphatases.
  • Microcystin, okadaic acid and cantharidin are all extremely toxic. Microcystin covalently binds to protein phosphatases and causes hepatotoxicity. Okadaic acid causes diarrhetic shellfish poisoning. Cantharidin is a very promiscuous phosphatase inhibitor and has a LD50 of approximately 0.5 mg/kg and can be lethal at doses as low as 10 mg. The natural product inhibitors of PP1C/PP2A are also tumor promoting.
  • This disclosure is directed to compounds that function as myosin light chain phosphatase inhibitors. Such compounds are useful in the treatment of a variety of diseases or disorders.
  • the compositions and methods can be used in treating diseases or disorders associated with aberrant myosin light chain phosphatase activity or levels, including, for example, cancer.
  • the disclosure is also directed to the finding that certain of the myosin light chain phosphatase inhibitors described herein are fluorescent and can be used as theragnostic agents, i.e. an agent having combined therapeutic and diagnostic properties, to detect and/or treat a variety of disorders associated with aberrant (e.g., increased) myosin light chain phosphatase activity or levels.
  • a fluorescent myosin light chain phosphatase inhibitor is used as a diagnostic agent (e.g.
  • myosin light chain phosphatase to diagnose diseases or conditions in which aberrant levels or activity of myosin light chain phosphatase, or of myosin light chain phosphorylation levels are associated
  • a combined diagnostic/therapeutic agent e.g., to indicate the presence of one or more cells exhibiting aberrant myosin light chain phosphatase activity and/or levels and to treat such cells
  • a tracking/therapeutic agent e.g., to determine the location of the fluorescent inhibitor in order to help direct and/or potentiate an additional therapy such as surgery or radiation.
  • B is selected from the group consisting of a bond, -0-, -NR 3 -, and -N(-A- Ar 1 )-;
  • composition comprising a pharmaceutically acceptable carrier and a compound according to formula I, or a pharmaceutically acceptable salt form thereof.
  • a method of inhibiting myosin light chain phosphatase comprising contacting an effective amount of a compound according to formula I, or a salt form thereof, with myosin light chain phosphatase.
  • a method of inhibiting protein phosphatase 1C comprising contacting an effective amount of a compound according to formula I, or a salt form thereof, with protein phosphatase 1C.
  • methods of increasing the level of phosphorylation of myosin light chain or Ras-1 in a cell comprising contacting an effective amount of a compound according to formula I, or a salt form thereof, with the cell.
  • a method is also provided of increasing the level of phosphorylation of Ras-1 in a cell, wherein the method comprises contacting an effective amount of a compound according to formula I, or a salt form thereof, with the cell.
  • a method of treating a disease or condition associated with myosin light chain phosphatase activity comprising causing an effective amount of a compound according to formula I, or a salt form thereof, to be present in an individual in need of such treatment.
  • a method of inducing cell-cycle arrest and/or apoptosis of a cell comprising contacting the cell with a myosin light chain phosphatase inhibitor, for example a compound according to formula I, or a salt form thereof.
  • a method for treating cancer comprising administering an effective amount of a myosin light chain phosphatase inhibitor, for example a compound according to formula I, or a salt form thereof, to an individual in need of such treatment.
  • a myosin light chain phosphatase inhibitor for example a compound according to formula I, or a salt form thereof
  • a method of killing a tumor cell comprising: contacting the tumor cell with an effective amount of a myosin light chain phosphatase inhibitor, for example a compound according to formula I, or a salt form thereof; and irradiating the tumor cell with an effective amount of ionizing radiation and/or contacting the tumor cell with an effective amount of at least one further chemotherapeutic agent.
  • a myosin light chain phosphatase inhibitor for example a compound according to formula I, or a salt form thereof.
  • a myosin light chain phosphatase inhibitor for example a compound according to formula I, or a salt form thereof
  • a method is provided of detecting the presence of an elevated amount of myosin light chain phosphatase in a subject cell comprising: providing a fluorescent myosin light chain phosphatase inhibitor; contacting the myosin light chain phosphatase inhibitor with the subject cell and with a control cell; observing fluorescence of the cells after the contacting; wherein an elevated level of fluorescence of the subject cells relative to the level of fluorescence of the control cells is indicative of an elevated amount of myosin light chain phosphatase in the subject cell as compared to the control cell.
  • a method is provided of detecting diseased cells, wherein the diseased cells comprise elevated amounts of myosin light chain phosphatase comprising: providing a fluorescent myosin light chain phosphatase inhibitor; contacting the fluorescent myosin light chain phosphatase inhibitor with tissue; observing for fluorescence of the cells of the tissue after the contacting; wherein an elevated level of fluorescence of the some of the cells relative to others in the tissue or relative to control non-diseased cells that have been contacted with the fluorescent myosin light chain phosphatase inhibitor is indicative that the fluorescent cells may be diseased cells comprising elevated amounts of myosin light chain phosphatase.
  • a method of detecting diseased cells in a tissue of an individual comprising: providing a fluorescent myosin light chain phosphatase inhibitor; contacting the fluorescent myosin light chain phosphatase inhibitor with the tissue; observing for fluorescence of at least some cells of the tissue after the contacting; wherein an elevated level of fluorescence of the some of the cells relative to others in the tissue or relative to control non-diseased cells that have been contacted with the fluorescent myosin light chain phosphatase inhibitor is indicative that the fluorescent cells may be diseased cells comprising elevated amounts of myosin light chain phosphatase.
  • a method is provided of radiotherapy of tumors, wherein the tumors comprise cells comprise an elevated amount of myosin light chain phosphatase relative to non-tumor cells, comprising: providing a fluorescent myosin light chain phosphatase inhibitor; causing the fluorescent myosin light chain phosphatase inhibitor to be present in the tumor cells in an effective amount to inhibit myosin light chain phosphatase and for fluorescence to be observable; observing the fluorescence; and directing an effective amount of ionizing radiation to the fluorescent tumor cells.
  • Also described is a method of surgery to remove tumor tissue from an individual comprising: providing a fluorescent myosin light chain phosphatase inhibitor; causing the fluorescent myosin light chain phosphatase inhibitor to be present in at least some cells of the tumor tissue in an effective amount for fluorescence of at least some of the tumor tissue to be observable; observing the fluorescence; and surgically removing at least some of the fluorescent tumor tissue, whereby at least a portion of the tumor that comprises fluorescent tumor cells is removed.
  • Figure 1 shows the results of molecular modeling studies with 4-(2-guanidinothiazol- 4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate (Example 28, 17e) docked into the crystal structure of PPlC the guanidine group forming hydrogen bonds with a backbone carbonyl of Arg 221 in the hydrophobic groove and the dansyl portion of the molecule aligned in the ⁇ l2-13 loop region of the catalytic pocket.
  • Figure 3 shows a Western blot analysis of relative levels of MYPT, PPl, MLC and ⁇ - actin in androgen receptor positive prostate cancer cell lines (LNCaP, C4-2 and C4-2B) and androgen receptor negative prostate cancer cell lines (CWR, DU145, PC-3 and PC-3M).
  • Figure 4 shows the effect of the compound of 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (Example 28, 17e) on the chemotaxis of PC-3 cells.
  • the absorbance represents the amount of stained cells that migrated through the 8 ⁇ M membrane using insulin-like growth factor- 1 as a chemoattractant.
  • Figure 5 shows the effect of the compound of 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (Example 28, 17e) on the cell-cycle distribution of PC-3 cells.
  • the distribution is shown after PC-3 cells were treated with the compound (or control - untreated) at 6, 24, and 48 hours.
  • the sub-Gl populations of PC-3 cells treated with compound for 48h as compared to control (untreated) is shown.
  • Figure 6 shows levels of phosphorylated substrates in PC-3 cells treated with 1 ⁇ M A- (2-guanidinothiazol-4-yl)phenyl 5 -(dimethylamino)naphthalene-l -sulfonate (17e) or 10O nM nocodazole .
  • 4-(2-guanidinothiazol-4-yl)phenyl 5 -(dimethylamino)naphthalene- 1 -sulfonate (17e) is seen to be a selective myosin light chain phosphatase inhibitor since only phospho- MLC changes compared to control upon treatment with the compound.
  • Phospho-BAD is a substrate of another PPlC containing holoenzyme, but not myosin light chain phosphatase.
  • P70S6 is a substrate of PP2A holoenzymes.
  • Figure 7 shows the BrdU uptake of PC-3 cells treated with 1 ⁇ M 5- (dimethylamino)naphthalene-l -sulfonate (17e) for 24 hours as compared to control (no compound treatment). Data points represent pooling of three separate wells.
  • Figure 8 shows fluorescence imaging of PC-3 and LnCap cells treated with 1 ⁇ M A- (2-guanidinothiazol-4-yl)phenyl 5 -(dimethylamino)naphthalene-l -sulfonate (17e) for 60 min.
  • Figures (a) and (b) show (a) PC-3, and (b) LNCap cells stained with 4',6-diamidino-2- phenylindole (DAPI) (the nuclei of the cells show blue fluorescence), and phalloidin (the microfilaments in the cytoplasm of the cells show red fluorescenece.
  • DAPI 4',6-diamidino-2- phenylindole
  • Figures (c) and (d) show (c) PC-3, and (d) LNCap cells treated with 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (shown fluorescing (yellow fluorescence)) and stained with phalloidin (red fluorescence) and showing microfilament destabilization.
  • Figures (e) and (f) show PC-3 cells stained with (e) 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (yellow fluorescence - seen in the cytoplasm only) and DAPI (blue staining of the nucleus) and (f) 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (17e) (yellow fluorescence - seen in the cytoplasm only, with the nuclei seen to be dark).
  • Figure 9 shows the results of imaging studies of PtK2 cells stained for microtubules (red fluorescence in the cytoplasm in figures (a), (c), (e), (h), Q) and (I)) and microfilaments (green fluorescence in the cytoplasm in figures (b), (d), (f), (i), (k), and (m)).
  • the nucleus is also stained (DAPI - blue).
  • Figures (a) and (b) show control cells stained for (a) microtubules and (b) microfilaments.
  • Figures (c) and (d) show cells treated with 0.2 ⁇ M colchinine stained for (c) microtubules and (d) microfilaments, showing disruption of microtubules.
  • Figures (e) and (f) show cells treated with 0.2 ⁇ M jasplakinolide stained for (e) microtubules and (f) microfilaments, showing disruption of microfilaments.
  • Figures (h)- (m) show cells treated with 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (17e) at 1 ⁇ M ((h),(i)), 5 ⁇ M (Q), (k)), and 10 ⁇ M ((l),(m)), and stained for microtutules ((h),(j),(l)) and microfilaments ((i),(k),(m)), showing that the compound demonstrates disruption of both microfilaments and microtubules.
  • Cells were treated for 16 h with each drug.
  • Figure 10 shows Giemsa- stained CA46 Burkitt's lymphoma cells: (a) untreated controls; and cells treated with (b) 0.2 ⁇ M colchinine; or (c) and (d) 4-(2-guanidinothiazol-4- yl)phenyl 5 -(dimethylamino)naphthalene-l -sulfonate (17e) at (c) 5 ⁇ M and (d) 0.8 ⁇ M.
  • Figure 11 shows two sets of images of human prostate cancer tissue slices stained with 100 ⁇ M 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate (17e).
  • Figure HA shows a bright field (DIC) image of the tissue
  • Figure HB shows the tissue stained with compound 17e (green fluorescence)
  • Figure HC shows a merger of images 1 IA and 1 IB.
  • Figure 12 shows control images of human prostate cancer tissue slices stained with 100 ⁇ M l-dimethylamino-5-sulfamoylnaphthaline ("dansylamide").
  • Figure 12A is a bright field (DIC) image of the tissue
  • Figure 12B shows tissue stained with l-dimethylamino-5- sulfamoylnaphthaline (very faint fluorescence)
  • Figure 12C shows a merger of images A and B.
  • Figure 13 shows the localization of 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (17e) and MYPTl in human prostate cancer tissue.
  • Figures 13A to 13C show expression of MYPTl in prostate cancer cells.
  • Figure 13A shows human prostate cancer tissue slices with MYPTl distribution imaged by immunofluorescence with a MYPTl antibody (red fluorescence)
  • Figure 13B shows a bright field (DIC) image of the same cells
  • Figure 13C shows a merger of images 13A and 13B.
  • Figures 13E to 13H show a comparison of the distribution of compound 17e and MYPTl protein.
  • Figure 13E shows human prostate cancer tissue slices imaged by immunofluorescence using a MYPTl antibody
  • Figure 13F shows a bright field (DIC) image
  • Figure 13G shows the tissue stained with 100 ⁇ M of 17e (green fluorescence)
  • Figure 13H is a merger of images 13E, 13F and 13G.
  • Figures 14 and 15 both show co-localization of 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (17e) and MYPTl in prostate cancer cells.
  • the cancer shown in Figure 15 is less aggressive (more differentiated cells).
  • Figures 14A and 15A show immunofluorescence using a MYPTl antibody (red fluorescence).
  • Figures 14B and 15B show a bright field (DIC) image
  • Figures 14C and 15C shows staining with compound 17e (green fluorescence)
  • Figures 14D and 15D show a merger of images 14A, 14B, and 14C (14D) and of images 15A, 15B, and 15C (15D) respectively.
  • Figures 16, 17 and 18 show distribution of 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (17e) and MYPTl immunofluorescence in prostate cancer tissue and cells. Shown are merged images of cells stained with compound 17e (green fluorescence), MYPTl immunofluorescence (red fluorescence), and 4',6-diamidino-2- phenylindole (DAPI) with the brightest regions (purple fluorescence) indicating co- localization of MYPTl and compound 17e.
  • Figure 16 shows low grade prostate cancer cells showing co-localization of MYPTl and compound 17e.
  • Figure 17 shows moderately invasive prostate cancer cells showing co-localization of MYPTl and compound 17e.
  • Figure 18 shows highly invasive prostate cancer cells showing co-localization of MYPTl and compound 17e.
  • Inhibition of myosin light chain phosphatase causes an increase of phosphorylation of MLC in smooth muscle, without kinase activation. Somolyo, et al., Physiol. Rev., 2003, 83, 1325-1358; Xia, et al., Exper. Cell Res., 2005, 304, 506-517. An increased level of phosphorylated myosin light chain, the substrate of myosin light chain phosphatase, can alone initiate apoptotic cell death. Mills, et al., J. Cell. Biol. 1998, 140, 627-636.
  • novel compounds that are active as selective myosin light chain phosphatase inhibitors.
  • Certain of the novel compounds are fluorescent inhibitors of myosin light chain phosphatases.
  • novel methods of using the provided compounds including methods of treatment and screening methods.
  • novel methods related to inhibition of myosin light chain phosphatase, using fluorescent myosin light chain phosphatase inhibitors are also provided.
  • an amount of compound or radiation applied in a method refers to the amount of a compound that achieves the desired pharmacological effect or other effect, for example an amount that inhibits the abnormal growth or proliferation, or induces apoptosis of cancer cells, resulting in a useful effect.
  • treating and “treatment” mean causing a therapeutically beneficial effect, such as ameliorating existing symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, postponing or preventing the further development of a disorder and/or reducing the severity of symptoms that will or are expected to develop.
  • mammals as used herein, "individual” (as in the subject of the treatment) means both mammals and non-mammals. Mammals include, for example, humans; non-human primates, e.g. apes and monkeys; cattle; horses; sheep; dogs; cats; dogs; rats; mice; pigs; and goats. Non-mammals include, for example, fish and birds.
  • (C x -C y )alkyl (wherein x and y are integers) by itself or as part of another substituent means, unless otherwise stated, an alkyl group containing between x and y carbon atoms.
  • An alkyl group formally corresponds to an alkane or cycloalkane with one C-H bond replaced by the point of attachment of the alkyl group to the remainder of the compound.
  • An alkyl group may be straight-chained or branched.
  • Alkyl groups having 3 or more carbon atoms may be cyclic. Cyclic alkyl groups having 7 or more carbon atoms may contain more than one ring and be polycyclic.
  • Examples of straight-chained alkyl groups include methyl, ethyl, n-propyl, n-butyl, and n-octyl.
  • Examples of branched alkyl groups include /-propyl, t- butyl, and 2,2-dimethylethyl.
  • Examples of cyclic alkyl groups include cyclopentyl, cyclohexyl, cyclohexylmethyl, and 4-methylcyclohexyl.
  • Examples of polycyclic alkyl groups include bicyclo[2.2.1]heptanyl, norbornyl, and adamantyl.
  • Preferred (C x -C y )alkyl groups are (Ci-C6)alkyl.
  • (Ci-C3)alkyl More preferred are (Ci-C3)alkyl. Most preferred are methyl and ethyl.
  • the term "(C x -C y )alkylene" (wherein x and y are integers) refers to an alkylene group containing between x and y carbon atoms. An alkylene group formally corresponds to an alkane with two C-H bond replaced by points of attachment of the alkylene group to the remainder of the compound.
  • Preferred (C x -C y )alkylene are (C 1 - Ce)alkylene. More preferred are (Ci-C3)alkylene.
  • (C x -C y ) alkenyl denotes a radical containing x to y carbons, wherein at least one carbon-carbon double bond is present (therefore x must be at least 2). Some embodiments are 2 to 4 carbons, some embodiments are 2 to 3 carbons, and some embodiments have 2 carbons. Both E and Z isomers are embraced by the term “alkenyl.” Furthermore, the term “alkenyl” includes di- and tri-alkenyls.
  • bonds may be all E or Z or a mixtures of E and Z
  • alkenyl examples include vinyl, allyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexanyl, 2,4-hexadienyl and the like.
  • (C x -C y ) alkynyl (wherein x and y are integers) denotes a radical containing 2 to 6 carbons and at least one carbon-carbon triple bond, some embodiments are 2 to 4 carbons, some embodiments are 2 to 3 carbons, and some embodiments have 2 carbons.
  • alkynyl examples include ethynyl, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2- butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3- hexynyl, 4-hexynyl, 5-hexynyl and the like.
  • alkynyl includes di- and tri-ynes.
  • (C x -C y ) alkoxy (wherein x and y are integers) employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined above, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs and isomers.
  • halo or halogen by themselves or as part of another substituent mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine, chlorine, or bromine, more preferably, fluorine or chlorine.
  • aromatic refers to a carbocycle or heterocycle having one or more polyunsaturated rings having aromatic character (i.e. having (4n + 2) delocalized ⁇ (pi) electrons where n is an integer).
  • aryl employed alone or in combination with other terms, means, unless otherwise stated, a carbocyclic aromatic system containing one or more rings (typically one, two or three rings), wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene. Examples include phenyl; anthracyl; and naphthyl. Preferred are phenyl and naphthyl, most preferred is phenyl.
  • heterocycle or “heterocyclyl” or “heterocyclic” by itself or as part of another substituent means, unless otherwise stated, an unsubstituted or substituted, stable, mono- or multi-cyclic heterocyclic ring system which consists of carbon atoms and at least one heteroatom selected from the group consisting of N, O, and S, and, wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen atom may be optionally quaternized.
  • the heterocyclic system may be attached, unless otherwise stated, at any heteroatom or carbon atom which affords a stable structure.
  • heteroaryl or “heteroaromatic” refers to a heterocycle having aromatic character.
  • a polycyclic heteroaryl may include one or more rings which are partially saturated. Examples include tetrahydroquinoline and 2,3-dihydrobenzofuryl.
  • the attachment point on ring Ar 1 or Ar 2 is understood to be on an atom which is part of an aromatic monocyclic ring or a ring component of a polycyclic aromatic which is itself an aromatic ring.
  • non-aromatic heterocycles include monocyclic groups such as: aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, imidazoline, pyrazolidine, dioxolane, sulfolane, 2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydropyridine, 1 ,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran, 1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane, 4,7-dihydro-l,3-dioxepin and hexam
  • heteroaryl groups include: pyridyl, pyrazinyl, pyrimidinyl, particularly 2- and 4-pyrimidinyl, pyridazinyl, thienyl, furyl, pyrrolyl, particularly 2-pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, particularly 3- and 5-pyrazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl.
  • polycyclic heterocycles include: indolyl, particularly 3-, A-, 5-, 6- and 7-indolyl, indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl, particularly 1- and 5-isoquinolyl, 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl, particularly 2- and 5-quinoxalinyl, quinazolinyl, phthalazinyl, 1,5-naphthyridinyl, 1,8-naphthyridinyl, 1 ,4-benzodioxanyl, coumarin, dihydrocoumarin, benzofuryl, particularly 3-, A-, 5-, 6- and 7-benzofuryl, 2,3-dihydrobenzofuryl, 1 ,2-benzisoxazolyl, benzothienyl, particularly 3-, A-, 5-, 6-, and 7-benzothienyl, benzothi
  • heterocyclyl and heteroaryl moieties are intended to be representative and not limiting.
  • substituted means that an atom or group of atoms formally replaces hydrogen as a "substituent" attached to another group.
  • substituted refers to any level of substitution, namely mono-, di-, tri-, terra-, or penta-substitution, where such substitution is permitted.
  • the substituents are independently selected, and substitution may be at any chemically accessible position.
  • valency of a chemical group refers to the number of bonds by which it is attached to other groups of the molecule.
  • B is selected from the group consisting of a bond, -0-, -NR 3 -, and -N(-A- Ar 1 )-;
  • A is -SO 2 -.
  • B is -O-.
  • B is -NR -, for example NH.
  • D is -H.
  • each of R a , R b , R c , and R d is hydrogen.
  • Ar 1 is unsubstituted or substituted phenyl. In some sub-embodiments thereof, Ar 1 is substituted phenyl substituted in at least the 4-position. In some sub-embodiments thereof, Ar 1 is monosubstituted phenyl substituted in the 4-position.
  • Ar 1 is unsubstituted or substituted biphenyl-4-yl. In some sub-embodiments thereof, Ar 1 is unsubstituted or substituted biphenyl-4-yl which is unsubstituted in at least the phenylene ring thereof (i.e. the ring which has the attachment point of the of the biphenyl group to the remainder of the molecule).
  • Ar 1 is unsubstituted or substituted naphthyl.
  • Sub-embodiments thereof include those wherein the naphthyl is unsubstituted.
  • Other sub-embodiments are those wherein the naphthyl is substituted.
  • Sub- embodiments thereof also include those wherein the naphthyl is a 1 -naphthyl, which may be substituted or unsubstituted, sub-embodiments being those wherein the 1 -naphthyl is substituted.
  • the 1 -naphthyl is monosubstituted.
  • Ar 1 is 1 -naphthyl
  • Embodiments of the compounds according to Formula I include those wherein Ar 1 is 5-dimethylamino- 1 -naphthyl.
  • Examples thereof include: 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene- 1 -sulfonate; and salt forms thereof.
  • Embodiments of the compounds according to Formula I include those wherein D is hydrogen and each of R a , R b , R c and R d is hydrogen. Examples thereof include:
  • any of the embodiments thereof, as well as intermediates used in making compounds according to formula I may take the form of salts.
  • salts embraces addition salts of free acids or free bases which are compounds described herein.
  • pharmaceutically-acceptable salt refers to salts which possess toxicity profiles within a range that affords utility in pharmaceutical applications. Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which may render them useful, for example, in processes of synthesis, purification or formulation of compounds described herein.
  • Suitable pharmaceutically-acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid.
  • inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids.
  • organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, ⁇ -hydroxybutyric, sal
  • Suitable pharmaceutically acceptable base addition salts include, for example, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts.
  • Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, ⁇ -dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (7V-methylglucamine) and procaine.
  • Examples of pharmaceutically unacceptable base addition salts include lithium salts and cyanate salts.
  • All of these salts may be prepared by conventional means from the corresponding compound according to formula I by reacting, for example, the appropriate acid or base with the compound according to formula I.
  • the salts are in crystalline form, which may be prepared by crystallization of the salt from a suitable solvent. Preparaition and selection of suitable salt forms is described in Handbook of Pharmaceutical Salts: Properties, Selection, and Use By P. H. Stahl and C. G. Wermuth (Wiley- VCH 2002).
  • the compounds according to formula I, and salts thereof as well as intermediates used in making compounds according to formula I, and salts thereof may take the form of solvates, including hydrates.
  • the useful properties of the compounds described herein do not depend on whether the compound or salt thereof is or is not in the form of a solvate, so unless clearly indicated otherwise reference in the specification to compounds of formula I should be understood as encompassing solvate forms of the compound, whether or not this is explicitly stated.
  • prodrug is meant, for example, any compound (whether itself active or inactive) that is converted chemically in vivo into a biologically active compound of the formula I following administration of the prodrug to a subject.
  • prodrug is a covalently bonded carrier which releases the active parent drug when administered to a subject.
  • Prodrugs can be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compounds.
  • Prodrugs include compounds wherein hydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxyl, amino, sulfhydryl, or carboxyl group respectively.
  • Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups in the compounds according to formula I.
  • the compounds provided for by formula I may encompass various stereochemical forms and tautomers.
  • the formula also encompasses diastereomers as well as optical isomers, e.g. mixtures of enantiomers including racemic mixtures, as well as individual enantiomers and diastereomers, which arise as a consequence of structural asymmetry in certain compounds of formula I. Separation of the individual isomers or selective synthesis of the individual isomers is accomplished by application of various methods which are well known to practitioners in the art.
  • Certain compounds of formula I possess an olef ⁇ nic double bond.
  • the stereochemistry of compounds possessing an olef ⁇ nic double bond is designated using the nomenclature using E and Z designations.
  • the compounds are named according to the Cahn-Ingold-Prelog system, described in the IUPAC 1974 Recommendations, Section E: Stereochemistry, in Nomenclature of Organic Chemistry, John Wiley & Sons, Inc., New York, NY, 4 th ed., 1992, pp. 127-38, the entire content of which is incorporated herein by reference.
  • Certain compounds of formula I may contain one or more chiral centers, and may exist in, and may be isolated as pure enantiomeric or diastereomeric forms or as racemic mixtures.
  • Formula I therefore encompasses any possible enantiomers, diastereomers, racemates or mixtures thereof which are biologically active in the inhibiting myosin light chain phosphatase.
  • the isomers resulting from the presence of a chiral center comprise a pair of non-superimposable isomers that are called "enantiomers.”
  • enantiomers Single enantiomers of a pure compound are optically active, i.e., they are capable of rotating the plane of plane polarized light.
  • Single enantiomers are designated according to the Cahn-Ingold-Prelog system.
  • Formula I encompasses diastereomers as well as their racemic and resolved, diastereomerically and enantiomerically pure forms and salts thereof. Diastereomeric pairs may be resolved by known separation techniques including normal and reverse phase chromatography, and crystallization.
  • isolated optical isomer means a compound which has been substantially purified from the corresponding optical isomer(s) of the same formula.
  • the isolated isomer is at least about 80%, more preferably at least 90% pure, even more preferably at least 98% pure, most preferably at least about 99% pure, by weight.
  • Isolated optical isomers may be purified from racemic mixtures by well-known chiral separation techniques. According to one such method, a racemic mixture of a compound having the structure of formula I, or a chiral intermediate thereof, is separated into 99% wt.% pure optical isomers by HPLC using a suitable chiral column, such as a member of the series of DAICEL ® CHIRALP AK ® family of columns (Daicel Chemical Industries, Ltd., Tokyo, Japan). The column is operated according to the manufacturer's instructions.
  • Formula I therefore includes any possible stable rotamers of formula I which are biologically active in inhibiting myosin light chain phosphatase.
  • Certain compounds may exist in tautomeric forms, which differ by the location of a hydrogen atom and typically are in rapid equilibrium. In such circumstances, molecular formulae drawn will typically only represent one of the possible tautomers even though equilibration of these tautomeric forms will occur in equilibrium in the compound. Examples include keto-enol tautomerism and amide-imidic acid tautomerism. Tautomerism is frequently also seen in heterocyclic compounds. All tautomeric forms of the compounds according to formula I are to be understood as being included within the scope of the formula.
  • the compounds of formula I may be administered in the form of a pharmaceutical composition, in combination with a pharmaceutically acceptable carrier.
  • the active ingredient in such formulations may comprise from 0.1 to 99.99 weight percent.
  • “Pharmaceutically acceptable carrier” means any carrier, diluent or excipient which is compatible with the other ingredients of the formulation and not deleterious to the recipient.
  • the active agent may be administered with a pharmaceutically acceptable carrier selected on the basis of the selected route of administration and standard pharmaceutical practice.
  • the active agent may be formulated into dosage forms according to standard practices in the field of pharmaceutical preparations. See Alphonso Gennaro, ed., Remington: The Science and Practice of Pharmacy, (20th Edition, Mack Publishing Co., Easton, PA, 2003).
  • Suitable dosage forms may comprise, for example, tablets, capsules, solutions, parenteral solutions, troches, suppositories, or suspensions.
  • the active agent may be mixed with a suitable carrier or diluent such as water, an oil (particularly a vegetable oil), ethanol, saline solution, aqueous dextrose (glucose) and related sugar solutions, glycerol, or a glycol such as propylene glycol or polyethylene glycol.
  • Solutions for parenteral administration preferably contain a water soluble salt of the active agent.
  • Stabilizing agents, antioxidant agents and preservatives may also be added. Suitable antioxidant agents include sulfite, ascorbic acid, citric acid and its salts, and sodium EDTA. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorbutanol.
  • the composition for parenteral administration may take the form of an aqueous or non-aqueous solution, dispersion, suspension or emulsion.
  • the active agent may be combined with one or more solid inactive ingredients for the preparation of tablets, capsules, pills, powders, granules or other suitable oral dosage forms.
  • the active agent may be combined with at least one excipient such as fillers, binders, humectants, disintegrating agents, solution retarders, absorption accelerators, wetting agents absorbents or lubricating agents.
  • the active agent may be combined with carboxymethylcellulose calcium, magnesium stearate, mannitol and starch, and then formed into tablets by conventional tableting methods.
  • the specific dose of a compound according to formula I required to obtain therapeutic benefit in the methods of treatment described herein will, of course, be determined by the particular circumstances of the individual patient including the size, weight, age and sex of the patient, the nature and stage of the disease being treated, the aggressiveness of the disease disorder, and the route of administration of the compound.
  • a daily dosage from about 0.05 to about 50 mg/kg/day may be utilized, for example, a dosage from about 0.1 to about 10 mg/kg/day. Higher or lower doses are also contemplated as it may be necessary to use dosages outside these ranges in some cases.
  • the daily dosage may be divided, such as being divided equally into two to four times per day daily dosing.
  • the compositions may be formulated in a unit dosage form, each dosage containing from about 1 to about 500mg, more typically, about 10 to about lOOmg of active agent per unit dosage.
  • unit dosage form refers to physically discrete units suitable as a unitary dosage for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
  • compositions described herein may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydropropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes and/or microspheres.
  • a controlled-release preparation is a pharmaceutical composition capable of releasing the active ingredient at the required rate to maintain constant pharmacological activity for a desirable period of time.
  • dosage forms provide a supply of a drug to the body during a predetermined period of time and thus maintain drug levels in the therapeutic range for longer periods of time than conventional non-controlled formulations.
  • U.S. Patent No. 5,674,533 discloses controlled-release pharmaceutical compositions in liquid dosage forms for the administration of moguisteine, a potent peripheral antitussive.
  • U.S. Patent No. 5,059,595 describes the controlled-release of active agents by the use of a gastro-resistant tablet for the therapy of organic mental disturbances.
  • U.S. Patent No. 5,591,767 describes a liquid reservoir transdermal patch for the controlled administration of ketorolac, a non-steroidal anti-inflammatory agent with potent analgesic properties.
  • U.S. Patent No. 5,120,548 discloses a controlled-release drug delivery device comprised of swellable polymers.
  • U.S. Patent No. 5,639,476 discloses a stable solid controlled-release formulation having a coating derived from an aqueous dispersion of a hydrophobic acrylic polymer. Biodegradable microparticles are known for use in controlled-release formulations.
  • U.S. Patent No. 5,354,566 discloses a controlled-release powder that contains the active ingredient.
  • U.S. Patent No. 5,733,566 describes the use of polymeric microparticles that release antiparasitic compositions.
  • controlled-release of the active ingredient may be stimulated by various inducers, for example pH, temperature, enzymes, water, or other physiological conditions or compounds.
  • various mechanisms of drug release exist.
  • the controlled-release component may swell and form porous openings large enough to release the active ingredient after administration to a patient.
  • the term "controlled-release component” means a compound or compounds, such as polymers, polymer matrices, gels, permeable membranes, liposomes and/or microspheres that facilitate the controlled-release of the active ingredient in the pharmaceutical composition.
  • the controlled-release component is biodegradable, induced by exposure to the aqueous environment, pH, temperature, or enzymes in the body.
  • sol-gels may be used, wherein the active ingredient is incorporated into a sol-gel matrix that is a solid at room temperature.
  • This matrix is implanted into a patient, preferably a mammal, having a body temperature high enough to induce gel formation of the sol-gel matrix, thereby releasing the active ingredient into the patient.
  • the components used to formulate the pharmaceutical compositions are of high purity and are substantially free of potentially harmful contaminants (e.g., at least National Food grade, generally at least analytical grade, and more typically at least pharmaceutical grade).
  • the composition is preferably manufactured or formulated under Good Manufacturing Practice standards as defined in the applicable regulations of the U.S. Food and Drug Administration.
  • suitable formulations may be sterile and/or substantially isotonic and/or in full compliance with all Good Manufacturing Practice regulations of the U.S. Food and Drug Administration.
  • Suitable leaving groups L include: halogen, OSO 2 AIlCyI, OSO 2 Aryl, and OSO 2 CF 3 .
  • a procedure involves treatment of a compound of formula II with a compound of formula III at about 0-120 0 C in a suitable solvent.
  • the preferred solvents are those which will promote the nucleophilic displacement and cyclization reactions and will not engage in competing displacement reactions.
  • Suitable solvents include polar solvents, including protic solvents such as alcohols, for example ethanol, and aprotic solvents such as ketones, for example acetone.
  • Suitable solvents for the reaction include amide solvents such as N,N-dimethylformamide and N-methylpyrrolidone, dimethylsulfoxide, ether solvents such as tetrahydrofuran, and halogenated hydrocarbons such as chloroform.
  • the preferred solvent is ethanol or acetone.
  • an acid or base catalyst may be beneficial.
  • the reaction is preferably performed at elevated temperature, for example the from about 20 0 C, 30 0 C, or 50 0 C to about 100 0 C or 120 0 C, for example at about the reflux temperature of the solvent.
  • Suitable halogenating agents include elemental halogens and N-halogen compounds, for example N-haloimides, for example N-halosuccinimides.
  • the preferred halogen is bromine, and the preferred halogenating agents are elemental bromine, and N- bromosuccinimide.
  • the reaction can be performed under neutral or acidic conditions. The reaction may be carried out, for example, at a temperature in the range from about -20 0 C to about 80 0 C, about -10 0 C to about 35 0 C, or about 0 0 C to about 25 0 C.
  • Suitable solvents for conducting the reaction include halogenated solvents, for example chloroform or dichloromethane, carboxylic acids, for example acetic acid. It may be advantageous for the halogenation to be performed upon a derivative of the ketone such as an enolate, enol ether, enol silane, or enol ester.
  • Compounds of formula II wherein L is OSO 2 Alkyl, OSO 2 Aryl, and OSO 2 CF 3 may be prepared from a compound of formula II wherein L is OH by reaction with a suitable sulfonyl chloride.
  • Compounds of formula II wherein L is OH may be prepared from compounds of formula IV by oxidation.
  • Compounds of formula IV may be prepared by an acylation or sulfonylation reaction between a compound of formula V, wherein X is a suitable leaving group with a phenolic or aminoaromatic compound according to formula VI as illustrated in Scheme 3.
  • the reaction is a sulfonylation reaction and the leaving group X is preferably halogen, preferably chlorine.
  • the coupling between a sulfonamide and an aromatic amine or phenol is typically performed using a basic catalyst or reagent in a suitable solvent at a suitable temperature.
  • Suitable bases include tertiary amines such as triethylamine or JV,jV-diisopropylethylamine, or pyridine. Typically, at least one equivalent of base would be used because hydrogen chloride is formed in the reaction.
  • Suitable solvents include pyridine or dichloromethane.
  • the reactions are typically carried out at a temperature between 0 0 C and the reflux temperature of the solvent, which is typically about 100 0 C.
  • the reaction is preferably carried out at about between 0 0 C and about 10 0 C.
  • the reactions would be conducted by adding the sulfonyl chloride to a solution containing the aromatic amine or phenol and triethylamine in dichloromethane at about 10 0 C.
  • A is a carbonyl group (CO)
  • Suitable bases for the reaction include: 4-( ⁇ /, ⁇ /-dimethylamino)pyridine, pyridine, triethylamine, ⁇ /,jV-diisopropylethylamine.
  • the preferred base is dimethylaminopyridine.
  • carbodiimides for example 1,3- dicyclohexylcarbodiimide or l-(3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride
  • the preferred coupling agents are carbodiimides, for example 1,3-dicyclohexylcarbodiimide.
  • Suitable solvents for the reaction include amide solvents such as ⁇ /,N-dimethylformamide, dimethylsulfoxide, ether solvents such as tetrahydrofuran, or halogenated hydrocarbons such as chloroform.
  • the preferred solvent is ⁇ /, ⁇ /-dimethylformamide.
  • the reaction is preferably performed at a temperature of 0-50 0 C, and most preferably at a temperature of 20-30 0 C.
  • the compounds according to formula III are either commercially available, known in the art or may be prepared by a variety of methods which are known to the person skilled in the art.
  • sulfonyl chlorides may be prepared by the chlorosulfonation of aromatic compounds, or by the chlorination of various aromatic derivatives (e.g. Johnson, Proc. Natl. Acad. ScL USA, 1939, 25(9): 448-452).
  • sulfonyl chlorides see, for example G. Hilgetag and A. Martini, Preparative Organic Chemistry (J. Wiley and Sons, 1972) p.670, U.S.
  • suitable methods include reaction of organometallic compounds with carbon dioxide, oxidation of formyl derivatives (which are available through reaction of organometallic compounds with formamides, or electrophilic formylation, e.g.
  • the compounds according to formula VI are either commercially available, known in the art or may be prepared by methods known to the person skilled in the art.
  • the methyl ketone group may be introduced via Friedel Crafts acylation of the aromatic ring starting from an arene compound of formula VII as shown in Scheme 4.
  • the reaction is typically formed in the by reacting with acetyl chloride in the presence of a Lewis acid catalyst, for example aluminium trichloride.
  • a Lewis acid catalyst for example aluminium trichloride.
  • the aromatic amine or phenolic group would be protected with a protecting group (PG) for such a reaction (for example an amine protected as an amide, e.g. acetamido, and a phenol protected as alkoxy).
  • PG protecting group
  • the ortho- para directing effect of the oxygen or nitrogen-containing group represented by B renders the selective formation of the compound with the desired regiochemistry possible.
  • the synthesis scheme described above represents a convergent strategy whereby the compound according to formula I is constructed by the assembly of simpler compounds (e.g. the compounds of formula III, V, and VII as starting materials) which may be regarded as "building blocks" for the synthesis.
  • simpler compounds e.g. the compounds of formula III, V, and VII as starting materials
  • building blocks for the synthesis.
  • variations of the synthesis scheme described above are feasible.
  • the order of the steps may be varied (e.g. the reaction to form the A-B bond might in some cases be performed after formation of the thiazole ring), and that other variations of the synthesis scheme described are feasible.
  • isolated compound refers to a preparation of a compound of formula I, or a mixture of compounds according to formula I, wherein the isolated compound has been separated from the reagents used, and/or byproducts formed, in the synthesis of the compound or compounds. "Isolated” does not mean that the preparation is technically pure (homogeneous), but it is sufficiently pure to compound in a form in which it can be used therapeutically.
  • an “isolated compound” refers to a preparation of a compound of formula I or a mixture of compounds according to formula I, which contains the named compound or mixture of compounds according to formula I in an amount of at least 10 percent by weight of the total weight.
  • the preparation contains the named compound or mixture of compounds in an amount of at least 50 percent by weight of the total weight; more preferably at least 80 percent by weight of the total weight; and most preferably at least 90 percent, at least 95 percent or at least 98 percent by weight of the total weight of the preparation.
  • the compounds according to formula I and intermediates may be isolated from their reaction mixtures and purified by standard techniques such as filtration, liquid-liquid extraction, solid phase extraction, distillation, recrystallization or chromatography, including flash column chromatography, or HPLC.
  • the preferred method for purification of the compounds according to formula I or salts thereof comprises crystallizing the compound or salt from a solvent to form, preferably, a crystalline form of the compounds or salts thereof. Following crystallization, the crystallization solvent is removed by a process other than evaporation, for example filtration or decanting, and the crystals are then preferably washed using pure solvent (or a mixture of pure solvents).
  • Preferred solvents for crystallization include water, alcohols, particularly alcohols containing up to four carbon atoms such as methanol, ethanol, isopropanol, and butan-1-ol, butan-2-ol, and 2-methyl-2-propanol, ethers, for example diethyl ether, diisopropyl ether, t-butyl methyl ether, 1,2-dimethoxyethane, tetrahydrofuran and 1,4-dioxane, carboxylic acids, for example formic acid and acetic acid, and hydrocarbon solvents, for example pentane, hexane, toluene, and mixtures thereof, particularly aqueous mixtures such as aqueous ethanol.
  • alcohols particularly alcohols containing up to four carbon atoms such as methanol, ethanol, isopropanol, and butan-1-ol, butan-2-ol, and 2-methyl-2-propanol
  • ethers for example
  • the compounds according to formula I or salt thereof, and pharmaceutical compositions thereof are preferably in or prepared from a crystalline form, preferably prepared according to such a process.
  • aromatic substituents in the compounds of the invention, intermediates used in the processes described above, or precursors thereto may be introduced by employing aromatic substitution reactions to introduce or replace a substituent, or by using functional group transformations to modify an existing substituent, or a combination thereof. Such reactions may be effected either prior to or immediately following the processes mentioned above.
  • the reagents and reaction conditions for such procedures are known in the art.
  • procedures which may be employed include, but are not limited to, electrophilic functionalization of an aromatic ring, for example via nitration, halogenation, or acylation; transformation of a nitro group to an amino group, for example via reduction, such as by catalytic hydrogenation; acylation, alkylation, or sulfonylation of an amino or hydroxyl group; replacement of an amino group by another functional group via conversion to an intermediate diazonium salt followed by nucleophilic or free radical substitution of the diazonium salt; or replacement of a halogen by another group, for example via nucleophilic or organometallically-catalyzed substitution reactions.
  • a protecting group is a derivative of a chemical functional group which would otherwise be incompatible with the conditions required to perform a particular reaction which, after the reaction has been carried out, can be removed to re-generate the original functional group, which is thereby considered to have been "protected".
  • Any chemical functionality that is a structural component of any of the reagents used to synthesize compounds of this invention may be optionally protected with a chemical protecting group if such a protecting group is useful in the synthesis of compounds of this invention.
  • sensitive functional groups may be introduced as synthetic precursors to the functional group desired in the intermediate or final product.
  • An example of this is an aromatic nitro (-NO 2 ) group.
  • the aromatic nitro group goes not undergo any of the nucleophilic reactions of an aromatic amino group.
  • the nitro group can serve as the equivalent of a protected amino group because it is readily reduced to the amino group under mild conditions that are selective for the nitro group over most other functional groups.
  • Compounds according to formula I are therapeutically useful. There are therefore provided uses of the compounds according to formula I in therapy and diagnostics, and therapeutic and diagnostic methods comprising administering a compound according to formula I, or a pharmaceutically acceptable salt form thereof, to an individual.
  • Compounds according to formula I are effective as myosin light chain phosphatase inhibitors. Therefore, also provided is a method of inhibiting a myosin light chain phosphatase, comprising contacting an effective amount of a compound according to formula I, or a salt form thereof, with a myosin light chain phosphatase.
  • the method of inhibiting a myosin light chain phosphatase may be performed by contacting the myosin light chain phosphatase with a compound according to formula I, or a salt form thereof, in vitro, thereby inhibiting myosin light chain phosphatase in vitro.
  • the contacting may be performed in the presence of cells, wherein, optionally, the myosin light chain phosphatase is present within the cells, or alternatively may be performed in a cell free medium.
  • Uses of such an in vitro method of inhibiting a myosin light chain phosphatase include, but are not limited to use in a screening assay (for example, wherein the compound according to formula I is used as a positive control or standard compared to compounds of unknown activity or potency in inhibiting myosin light chain phosphatase).
  • the method of inhibiting a myosin light chain phosphatase may be performed by contacting the myosin light chain phosphatase with a compound according to formula I, or a salt form thereof, in vivo, thereby inhibiting the myosin light chain phosphatase in vivo.
  • the contacting is achieved by causing the compound according to formula I, or a salt form thereof, to be present in the individual in an effective amount to achieve inhibition of the myosin light chain phosphatase. This may be achieved, for example, by administering an effective amount of the compound according to formula I, or a pharmaceutically acceptable salt form thereof, to the individual, or by administering a prodrug of the compound according to formula I, or a pharmaceutically acceptable salt form thereof.
  • myosin light chain phosphatase Uses of such an in vivo method of inhibiting a myosin light chain phosphatase include, but are not limited to use in methods of treating a disease or condition, wherein inhibiting myosin light chain phosphatase is beneficial, or treating or preventing diseases, wherein myosin light chain phosphatase activity, for example aberrant myosin light chain phosphatase activity, or a deficient level of phosphorylated myosin light chain contributes to the pathology and/or symptomology of the disease, as described in greater detail below.
  • a method of inhibiting protein phosphatase 1C comprising contacting an effective amount of a compound according to formula I, or a salt form thereof, with protein phosphatase 1C.
  • the method may be performed by contacting protein phosphatase 1C with the compound according to formula I, or a salt form thereof, in vitro or in vivo.
  • the in vitro method may be performed in the presence of cells, wherein, optionally, the protein phosphatase 1C is present within the cells, or alternatively may be performed in a cell free medium.
  • an in vitro method include, but are not limited to use in a screening assay (for example, wherein the compound according to formula I is used as a positive control or standard compared to compounds of unknown activity or potency in inhibiting protein phosphatase 1C).
  • the in vivo method may be performed by causing the compound according to formula I, or a salt form thereof, to be present in the individual in an effective amount to achieve inhibition of the protein phosphatase 1C, for example, by administering an effective amount of the compound according to formula I, or a pharmaceutically acceptable salt form thereof, to the individual, or administering a prodrug of the compound according to formula I, or a pharmaceutically acceptable salt form thereof.
  • Uses of such an in vivo method of inhibiting a protein phosphatase 1C include use in methods of treating a disease or condition, wherein inhibiting protein phosphatase 1C is beneficial, or treating or preventing diseases, wherein protein phosphatase 1C activity contributes to the pathology and/or symptomology of the disease.
  • the compounds according to formula I are useful for increasing the level of phosphorylation of myosin light chain in a cell (or inhibiting the dephosphorylation of myosin light chain). Accordingly, there is also provide a method of increasing the level of phosphorylation of myosin light chain in a cell comprising contacting an effective amount of a compound according to formula I, or a salt form thereof, with the cell. The method may be performed by contacting the cell with a compound according to formula I, or a salt form thereof, in vitro, thereby increasing the amount of myosin light chain phosphorylation in vitro.
  • Uses of such an in vitro method of increasing the amount of myosin light chain phosphorylation include, but are not limited to use in a screening assay (for example, wherein the compound according to formula I is used as a positive control or standard compared to compounds of unknown activity or potency in increasing myosin light chain phosphorylation).
  • the method of increasing the amount of myosin light chain phosphorylation may also be performed by contacting the cell with a compound according to formula I, or a salt form thereof, in vivo, thereby increasing the amount of myosin light chain phosphorylation in vivo.
  • the contacting is achieved by causing the compound according to formula I, or a salt form thereof, to be present in the individual in an effective amount to achieve an increase in the amount of myosin light chain phosphorylation.
  • This may be achieved, for example, by administering an effective amount of the compound according to formula I, or a pharmaceutically acceptable salt form thereof, to the individual, or by administering a prodrug of the compound according to formula I, or a pharmaceutically acceptable salt form thereof.
  • Uses of such an in vivo method of increasing the amount of myosin light chain phosphorylation include, but are not limited to use in methods of treating a disease or condition, wherein increasing the amount of myosin light chain phosphorylation is beneficial, or treating or preventing diseases, wherein myosin light chain dephosphorylation contributes to the pathology and/or symptomology of the disease, as described in greater detail below.
  • Also provided is a method of increasing the level of phosphorylation of Ras-1 in a cell comprising contacting an effective amount of a compound according to formula I, or a salt form thereof, with the cell.
  • the method may be performed by contacting the cell with a compound according to formula I, or a salt form thereof, in vitro, thereby increasing the amount of phosphorylation of Ras-1 in vitro.
  • Uses of such an in vitro method of increasing the amount of phosphorylation of Ras-1 include, but are not limited to use in a screening assay (for example, wherein the compound according to formula I is used as a positive control or standard compared to compounds of unknown activity or potency in increasing phosphorylation of Ras-1).
  • the method of increasing the amount of phosphorylation of Ras-1 may also be performed by contacting the cell with a compound according to formula I, or a salt form thereof, in vivo, thereby increasing the amount of phosphorylation of Ras-1 in vivo.
  • the contacting is achieved by causing the compound according to formula I, or a salt form thereof, to be present in the individual in an effective amount to achieve an increase in the amount of phosphorylation of Ras-1.
  • This may be achieved, for example, by administering an effective amount of the compound according to formula I, or a pharmaceutically acceptable salt form thereof, to the individual, or by administering a prodrug of the compound according to formula I, or a pharmaceutically acceptable salt form thereof.
  • Uses of such an in vivo method of increasing the amount of phosphorylation of Ras-1 include, but are not limited to use in methods of treating a disease or condition, wherein increasing the amount of phosphorylation of Ras-1 is beneficial, or treating or preventing diseases, wherein Ras-1 dephosphorylation contributes to the pathology and/or symptomology of the disease.
  • Also provided is a method of inhibiting actin polymerization (or promoting actin depolymerization) in a cell comprising contacting an effective amount of a compound according to formula I, or a salt form thereof, with the cell.
  • the method may be performed by contacting the cell with a compound according to formula I, or a salt form thereof, in vitro, thereby decreasing the amount of phosphorylation of actin polymerization in vitro.
  • Uses of such an in vitro method of inhibiting actin polymerization of Ras-1 include, but are not limited to use in a screening assay (for example, wherein the compound according to formula I is used as a positive control or standard compared to compounds of unknown activity or potency in inhibiting actin polymerization).
  • the method of inhibiting actin polymerization may also be performed by contacting the cell with a compound according to formula I, or a salt form thereof, in vivo, thereby inhibiting microfilament formation in vivo.
  • the contacting is achieved by causing the compound according to formula I, or a salt form thereof, to be present in the individual in an effective amount to achieve inhibition of actin polymerization.
  • This may be achieved, for example, by administering an effective amount of the compound according to formula I, or a pharmaceutically acceptable salt form thereof, to the individual, or by administering a prodrug of the compound according to formula I, or a pharmaceutically acceptable salt form thereof.
  • Uses of such an in vivo method of inhibiting actin polymerization include, but are not limited to use in methods of treating a disease or condition, wherein inhibiting actin polymerization is beneficial, or treating or preventing diseases, wherein actin polymerization contributes to the pathology and/or symptomology of the disease.
  • method of inhibiting tubulin polymerization (or promoting tubulin depolymerization) in a cell comprising contacting an effective amount of a compound according to formula I, or a salt form thereof, with the cell.
  • the method may be performed by contacting the cell with a compound according to formula I, or a salt form thereof, in vitro, thereby decreasing the amount of phosphorylation of tubulin polymerization in vitro.
  • Uses of such an in vitro method of inhibiting tubulin polymerization of Ras-1 include, but are not limited to use in a screening assay (for example, wherein the compound according to formula I is used as a positive control or standard compared to compounds of unknown activity or potency in inhibiting tubulin polymerization).
  • the method of inhibiting tubulin polymerization may also be performed by contacting the cell with a compound according to formula I, or a salt form thereof, in vivo, thereby inhibiting microtubule formation in vivo.
  • the contacting is achieved by causing the compound according to formula I, or a salt form thereof, to be present in the individual in an effective amount to achieve inhibition of tubulin polymerization.
  • This may be achieved, for example, by administering an effective amount of the compound according to formula I, or a pharmaceutically acceptable salt form thereof, to the individual, or by administering a prodrug of the compound according to formula I, or a pharmaceutically acceptable salt form thereof.
  • Uses of such an in vivo method of inhibiting tubulin polymerization include, but are not limited to use in methods of treating a disease or condition, wherein inhibiting tubulin polymerization is beneficial, or treating or preventing diseases, wherein tubulin polymerization contributes to the pathology and/or symptomology of the disease.
  • the compounds according to formula I are effective to treat or prevent diseases or conditions associated with myosin light chain phosphatase activity, for example aberrant myosin light chain phosphatase activity.
  • a method of treating or prophylaxis of a disease or condition associated with myosin light chain phosphatase activity comprising causing an effective amount of a compound according to formula I, or a salt form thereof, to be present in an individual in need of such treatment. This may be achieved, for example, by administering an effective amount of the compound according to formula I, or a pharmaceutically acceptable salt form thereof, to the individual, or by administering a prodrug of the compound according to formula I, or a pharmaceutically acceptable salt form thereof.
  • a “disease or condition associated with myosin light chain phosphatase activity” (or, equivalently, a “myosin light chain phosphatase-associated disease or condition” is a disease or condition, wherein a myosin light chain phosphatase possesses activity that contributes to the pathology and/or symptomology of the disease or condition or, wherein inhibition of a myosin light chain phosphatase produces an effect which is therapeutically beneficial.
  • the disease or condition is a cancer.
  • the compound according to formula I or salt thereof used is an embodiment of the compounds according formula I, or a salt thereof, as described above.
  • a fluorescent compound according to formula I is used.
  • Myosin light chain phosphatase inhibitors are effective to induce cell cycle arrest and/or apoptosis of a cell. There is therefore also provided a method of inducing cell-cycle arrest and/or apoptosis of a cell comprising contacting the cell with a myosin light chain phosphatase inhibitor.
  • Suitable myosin light chain phosphatase inhibitors include the compound according formula I, or a salt form thereof.
  • the method of inducing cell-cycle arrest and/or apoptosis of a cell may be performed by contacting the cell with a myosin light chain phosphatase inhibitor such as the compound according to formula I, or a salt form thereof, in vitro, thereby inducing cell-cycle arrest and/or apoptosis of a cell in vitro.
  • a myosin light chain phosphatase inhibitor such as the compound according to formula I, or a salt form thereof
  • Uses of such an in vitro method of inducing cell-cycle arrest and/or apoptosis include, but are not limited to use in a screening assay (for example, wherein a known myosin light chain phosphatase inhibitor is used as a positive control or standard compared to compounds of unknown activity or potency in inducing cell-cycle arrest and/or apoptosis).
  • the cell-cycle arrest and/or apoptosis may be induced in a cancer cell.
  • the compound according to formula I or salt thereof used may be an embodiment of the compounds according formula I, or a salt thereof, as described above, for example a fluorescent compound according to formula I.
  • the method of inducing cell-cycle arrest and/or apoptosis of a cell may be performed by contacting the myosin light chain phosphatase with a myosin light chain phosphatase inhibitor, such as a compound according to formula I, in vivo, thereby inducing cell-cycle arrest and/or apoptosis in an individual in vivo.
  • the contacting is achieved by causing the myosin light chain phosphatase inhibitor, such as the compound according to formula I, or a salt form thereof, to be present in the individual in an amount effective to achieve inhibition of cell-cycle arrest and/or apoptosis.
  • This may be achieved, for example, by administering an effective amount of the myosin light chain phosphatase inhibitor, such as the compound according to formula I, or a pharmaceutically acceptable salt form thereof, to the individual, or by administering a prodrug of the myosin light chain phosphatase inhibitor, such as a compound according to formula I, or a pharmaceutically acceptable salt form thereof.
  • an in vitro method of inducing cell-cycle arrest and/or apoptosis include, but are not limited to use in methods of treating a disease or condition wherein inducing cell-cycle arrest and/or apoptosis is beneficial.
  • the cell-cycle arrest and/or apoptosis may be induced in a cancer cell, for example in a patient suffering from cancer.
  • the method may be performed by administering an effective amount of the myosin light chain phosphatase inhibitor, such as the compound according to formula I, a prodrug of a compound according to formula I, or salt form of either, to an individual who is suffering from cancer.
  • the compound according to formula I or salt thereof used may be an embodiment of the compounds according to formula I, or a salt thereof, as described above such as a fluorescent compound according to formula I.
  • a myosin light chain phosphatase inhibitor such as compounds according to formula I, or a salt form thereof
  • a method for treating cancer comprising causing an effective amount of a myosin light chain phosphatase inhibitor, such as a compound according to formula I, or a salt form thereof, to be present in an individual.
  • the causing may be achieved by administering an effective amount of a myosin light chain phosphatase inhibitor, such as a compound according to formula I, or a salt thereof, to an individual in need of such treatment, or administering a prodrug of such a compound.
  • a myosin light chain phosphatase inhibitor such as a compound according to formula I is can be used to treat a broad range of cancers and tumor types, including, but not limited to, bladder cancer, brain cancer, breast cancer, colorectal cancer, cervical cancer, gastrointestinal cancer, genitourinary cancer, head and neck cancer, lung cancer, ovarian cancer, prostate cancer, renal cancer, skin cancer, and testicular cancer.
  • Prostate cancer treated may include androgen-independent prostate cancer.
  • cancers that may be treated by myosin light chain phosphatase inhibitors include, but are not limited to, the following: cardiac cancers, including, for example sarcoma, e.g., angiosarcoma, fibrosarcoma, rhabdomyosarcoma, and liposarcoma; myxoma; rhabdomyoma; fibroma; lipoma and teratoma; lung cancers, including, for example, bronchogenic carcinoma, e.g., squamous cell, undifferentiated small cell, undifferentiated large cell, and adenocarcinoma; alveolar and bronchiolar carcinoma; bronchial adenoma; sarcoma; lymphoma; chondromatous hamartoma; and mesothelioma; gastrointestinal cancer, including, for example, cancers of the esophagus,
  • Cancers may be solid tumors that may or may not be metastatic. Cancers may also occur, as in leukemia, as a diffuse tissue. Thus, the term "tumor cell”, as provided herein, includes a cell with any one of the above identified disorders.
  • the myosin light chain phosphatase inhibitor such as a compound according to formula I can also be administered in combination with existing methods of treating disorders such as cancers, for example by chemotherapy, irradiation, or surgery.
  • a method of treating cancer comprising administering an effective amount of a myosin light chain phosphatase inhibitor, such as a compound according to formula I, or a salt thereof, to an individual in need of such treatment, wherein an effective amount of at least one further cancer chemotherapeutic agent is administered to the individual.
  • chemotherapeutic agents include any of: abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, anastrozole, arsenic trioxide, asparaginase, azacitidine, bevacizumab, bexarotene, bleomycin, bortezombi, bortezomib, busulfan intravenous, busulfan oral, calusterone, capecitabine, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dalteparin sodium, dasatinib, daunorubicin, decitabine, denileukin, denileukin diftitox, dexrazoxane, docetaxe
  • Also provided is a method of treating cancer comprising administering an effective amount of a myosin light chain phosphatase inhibitor, such as a compound according to formula I, or a salt thereof, to an individual in need of such treatment, wherein an effective amount of ionizing radiation is administered to the individual.
  • the further cancer therapeutic agent and/or the ionizing radiation may be administered concomitantly and/or non-concomitantly, e.g. sequentially, with the myosin light chain phosphatase inhibitor, such as a compound according to formula I, or a salt form thereof.
  • the myosin light chain phosphatase inhibitor such as a compound according to formula I, can also be administered to an individual in combination with surgical methods to treat cancers, e.g., resection of tumors.
  • the compounds can be administered to the individual prior to, during, or after the surgery.
  • the compounds can be administered parenterally or injected into the tumor or surrounding area after tumor removal, e.g., to minimize metastases or to treat residual tumor cells present.
  • the compound is fluorescent
  • the compound may be used to detect the presence of the tumor and to guide surgical resection.
  • Such fluorescent compounds can further therapeutically treat the cancer through their myosin light chain phosphatase inhibitory properties.
  • a method of guided surgery to remove at least a portion of a tumor from an individual comprising providing a fluorescent myosin light chain phosphatase inhibitor; causing the fluorescent myosin light chain phosphatase inhibitor to be present in at least some tumor cells in an effective amount to inhibit a myosin light chain phosphatase and for fluorescence to be observable; observing the fluorescence; and performing surgery on the individual to remove at least a portion of the tumor that comprises fluorescent tumor cells.
  • Causing the fluorescent myosin light chain phosphatase inhibitor to be present can occur by administering a compound according to formula I, or a prodrug or salt form thereof, to an individual.
  • a method of killing a tumor cell comprising contacting the tumor cell with an effective amount of a myosin light chain phosphatase inhibitor, such as a compound according to formula I, or a salt form thereof; and irradiating the tumor cell with an effective amount of ionizing radiation.
  • a myosin light chain phosphatase inhibitor such as a compound according to formula I, or a salt form thereof
  • a method of killing a tumor cell comprising contacting the tumor cell with an effective amount of a myosin light chain phosphatase inhibitor, such as a compound according to formula I, or a salt form thereof; and contacting the tumor cell with an effective amount of at least one further chemotherapeutic agent.
  • a myosin light chain phosphatase inhibitor such as a compound according to formula I, or a salt form thereof.
  • Causing an effective amount of the compound a myosin light chain phosphatase inhibitor, such as according to formula I, or a salt form thereof, may be achieved, for example, by administering an effective amount of a myosin light chain phosphatase inhibitor, such as the compound according to formula I, a prodrug of a compound according to formula I, or a pharmaceutically acceptable salt form thereof, to the individual.
  • the tumor cell may be a prostate cancer cell, for example an androgen-independent prostate cancer cell.
  • the compounds according to formula I may be administered to individuals (mammals, including animals and humans) afflicted with a disease such as such as cancer.
  • a disease such as cancer.
  • the individual treated is a human.
  • the compounds may be administered by any route, including oral, rectal, sublingual, and parenteral administration.
  • Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, intravaginal, intravesical (e.g., to the bladder), intradermal, transdermal, topical or subcutaneous administration.
  • parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, intravaginal, intravesical (e.g., to the bladder), intradermal, transdermal, topical or subcutaneous administration.
  • the instillation of a drug in the body of the patient in a controlled formulation with systemic or local release of the drug to occur at a later time.
  • the drug may be localized in a depot for controlled release to the circulation, or for release to a local site of tumor growth.
  • the compounds may be administered in the form of a pharmaceutical composition.
  • One or more compounds useful in the practice of the methods described herein may be administered simultaneously, by the same or different routes, or at different times during treatment.
  • the compounds may be administered before, along with, or after other medications, including other compounds.
  • the treatment using methods of treatment described herein may be carried out for as long a period as necessary, either in a single, uninterrupted session, or in discrete sessions.
  • the treating physician can increase, decrease, or interrupt treatment based on patient response.
  • Treatment may be carried out, for example, for from about four to about sixteen weeks.
  • the treatment schedule may be repeated as required.
  • Fluorescent compounds according to formula I provide added diagnostic and tracking functionalities to the therapeutic functionality of compounds according to formula I.
  • methods of using fluorescent myosin light chain phosphatase inhibitors including fluorescent compounds according to formula I, and salt forms thereof.
  • One such method provided is a method of detecting the presence of an elevated amount of a myosin light chain phosphatase in a subject cell, comprising: providing a fluorescent myosin light chain phosphatase inhibitor; contacting the fluorescent myosin light chain phosphatase inhibitor with the subject cell and with a control cell; observing fluorescence of the subject and control cells after the contacting; wherein an elevated level of fluorescence of the subject cell relative to the level of fluorescence of the control cell is indicative of an elevated amount of the myosin light chain phosphatase in the subject cell as compared to the control cell.
  • Fluorescence may be observed, for example, of the cytoplasm of the cells, whereby an elevated amount of the myosin light chain phosphatase in the cytoplasm of the subject cell as compared to the control cell is detected.
  • Uses of the provided method of detecting elevated myosin light chain phosphatase include, but are not limited to, detecting the presence of disease conditions associated with an elevated level of myosin light chain phosphatase activity and/or amount.
  • a method of detecting diseased (e.g., cancerous) cells in an individual comprising: providing a fluorescent myosin light chain phosphatase inhibitor; contacting the fluorescent myosin light chain phosphatase inhibitor with a tissue of the individual; and observing for fluorescence of the cells of the tissue after the contacting; wherein an elevated level of fluorescence of at least some of the cells in the tissue relative to other cells in the tissue or relative to control non-diseased cells that have been contacted with the fluorescent myosin light chain phosphatase inhibitor is indicative that the fluorescent cells may be diseased cells comprising elevated amounts of a myosin light chain phosphatase.
  • the contacting may be performed in vitro or in vivo, for example by causing an effective amount of a fluorescent myosin light chain phosphatase inhibitor to be present in the tissue, for example, by administering an effective amount of a fluorescent myosin light chain phosphatase inhibitor or a prodrug thereof to an individual.
  • a method of radiotherapy of tumors comprising: providing a fluorescent myosin light chain phosphatase inhibitor; causing the fluorescent myosin light chain phosphatase inhibitor to be present in the tumor cells in an effective amount to inhibit myosin light chain phosphatase and for fluorescence to be observable; observing the fluorescence; and directing an effective amount of ionizing radiation to the fluorescent tumor cells.
  • An advantage of the provided method of radiotherapy of tumors using fluorescent myosin light chain phosphatase inhibitors is that the fluorescent myosin light chain phosphatase inhibitor simultaneously renders the tumor cells visible while arresting growth of the cells and possibly sensitizing the cells to the effect of the ionizing radiation.
  • the irradiation can be directed to the tumor tissue, avoiding unnecessary damage to undiseased tissue.
  • the applied radiation may be more effective since the tumor cells may be sensitized to its effect.
  • the amount of radiation applied to the tumor tissue can, if desirable, be maximized since the radiation applied can be focused upon the tumor tissue made visible though its fluorescence.
  • a method of guided surgery or resection of at least a portion of a tumor comprising providing a fluorescent myosin light chain phosphatase inhibitor, causing the fluorescent myosin light chain phosphatase inhibitor to be present in at least some cells of the tumor tissue in an effective amount for fluorescence of the tumor tissue to be observable, observing the fluorescence, and surgically removing at least some of the fluorescent tumor tissue, whereby at least a portion of the tumor that comprises fluorescent tumor cells is removed.
  • Causing the fluorescent myosin light chain phosphatase inhibitor to be present can occur by administering a compound according to formula I, or a prodrug or salt form thereof, to an individual.
  • An advantage of the methods is that the fluorescent compounds simultaneously render the tumor cells visible while potentially also therapeutically treating the cancer through their myosin light chain phosphatase inhibitory properties. Since the tumor cells are visible, the surgery can be focused on the tumor tissue, avoiding unnecessary damage to undiseased tissue, while also minimizing the opportunity for some tumor to be inadvertently left behind.
  • the fluorescent myosin light chain phosphatase inhibitor is a compound according to formula I, or a salt form thereof, or any of the embodiments thereof as described above.
  • An example is 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate, or a salt form thereof.
  • NMR spectra were recorded using a Varian-400 spectrometer for 1 H (400 MHz) and 13 C (100 MHz). Chemical shifts ( ⁇ ) are given in ppm downfield from tetramethylsilane, an internal standard, and coupling constants (J-values) are in hertz (Hz). Purifications were performed by flash chromatography.
  • 3'-Methoxybiphenyl-4-carboxylic acid 500 mg, 2.03 mmol was dissolved in thionyl chloride (25 mL) and allowed to stir overnight. The thionyl chloride was then pumped off and the acid chloride was carried on crude. The acid chloride was dissolved in dichloromethane and 4-hydroxyacetophenone (276 mg, 2.03 mmol) was added to the solution. Sodium hydride (60% in mineral oil, 81 mg, 2.03 mmol) was then added slowly to the reaction and allowed to stir for 5h. The reaction was then quenched with saturated aqueous sodium bicarbonate and the solution was extracted twice with dichloromethane.
  • Oxalyl chloride(2.92 g, 22.2 mmol) was added dropwise to a solution of 4- cyclopropyl-benzoic acid (1.20 g, 7.4 mmol) in dichloromethane. The solution was allowed to stir overnight and the solvent was removed under reduced pressure. The acid chloride was then carried on crude.4-Hydroxyacetophenone (1.01 g, 7.4 mmol) was added to a slurry of NaH (60% in mineral oil, 296 mg, 7.4 mmol) in dichloromethane and allowed to stir for 10 min. 4-Cyclopropyl-benzoyl chloride (1.33 g, 7.4 mmol) was then added and the solution was allowed to stir for 3h.
  • Example 14 May be prepared by a method analogous to that described in Example 14 starting from 4-hydroxyacetophenone and 4-n-butylbenzoyl chloride.
  • Example 14 May be prepared by a method analogous to that described in Example 14 starting from 4-aminoacetophenone and 4-n-butylbenzoyl chloride.
  • Example 22 May be prepared as described in Example 10, except that the bromoketone is reacted with thiourea instead of 2-imino-4-thiobiuret in the last step as described in step (c) of Example 14.
  • Example 22 4-(2-Aminothiazol-4-yl)phenyl 2-naphthoate (14i)
  • Example 11 May be prepared as described in Example 11 , except that the bromoketone is reacted with thiourea instead of 2-imino-4-thiobiuret in the last step as described in step (c) of Example 14.
  • Example 12 May be prepared as described in Example 12, except that the bromoketone is reacted with thiourea instead of 2-imino-4-thiobiuret in the last step as described in step (c) of Example 14.
  • p-Toluenesulfonyl chloride (I g, 5.25 mmol) was added to a solution of 4- hydroxyacetophenone (715 mg, 5.25 mmol), triethylamine (1 mL) and JV,iV-dimethyl-4- aminopyridine (10 mg, cat.) in dichloromethane. The reaction was allowed to stir for 2h and was then quenched with water and washed with IM NaOH. The organic layer was then dried over magnesium sulfate, filtered and evaporated under reduced pressure to yield 1.52 g (100%) of a white solid.
  • Dansyl chloride 750 mg, 2.78 mmol was added to a solution of A- hydroxyacetophenone (379 mg, 2.78 mmol), triethylamine (1 mL) and N,N-dimethyl-4- aminopyridine (10 mg, cat.) in dichloromethane. The reaction was allowed to stir for 2h and was then quenched with water and washed with IM NaOH. The organic layer was then dried over magnesium sulfate, filtered and evaporated under reduced pressure to yield a yellow solid which was carried on crude.
  • the compound exhibited fluorescence with an absorption wavelength of 360 nm and an emission wavelength of 560 nm.
  • p-Toluenesulfonyl chloride (I g, 5.25 mmol) was added to a solution of 4- aminoacetophenone (710 mg, 5.25 mmol), triethylamine (1 mL) and N,N-dimethyl-4- aminopyridine (10 mg, cat.) in dichloromethane. The reaction was allowed to stir for 2h and was then quenched with water and washed with IM HCl. The organic layer was then dried over magnesium sulfate, filtered and evaporated under reduced pressure to yield 1.04 g (69%) of a white solid.
  • Modelling of potential myosin light chain kinase inhibitor was performed using PPlC structural data, beginning with calyculin, a potent inhibitor for myosin light chain phosphatase.
  • the crystal structure of PPlC was obtained from the Protein Data Bank (structure 1JK7).
  • the binding pocket was defined by a 15 angstrom sphere from the residue selection (Arg 96, He 130, lie 133, Tyr 134, Trp 206, Arg 221 and Tyr 272). Molecules to be docked into the crystal structure were built in the Sybyl shell and minimized using the conjugate gradient method with 1000 iterations or a stopping point of 0.01 kJ energy differential between conformers.
  • the FlexX suite was then used to dock the ligands into the binding pocket of PPlC with an output of 30 conformers.
  • the X-ray crystal structure of the PPlC catalytic pocket is comprised of a bimetallic center and three adjoining grooves of which the most important region involved in substrate recognition is the hydrophilic groove Gupta, et al., J. Med. Chem. 1997, 40, 3199-3206; Goldberg, et al., Nature, 1995, 376, 745-753; Maynes, et al.,. J. Biol. Chem., 2001, 276, 47, 44078-44882; Kita, et al., Structure, 2002, 10, 715-724.
  • hydrophobic groove and the ⁇ l2-13 loop may be important features in the interaction of inhibitors with the enzyme.
  • the ⁇ l2-13 loop is a highly flexible and is the least conserved portion of the binding pocket among the members of the PPP superfamily, and together with the hydrophobic groove may be responsible for determining the substrate specificity imparted onto the catalytic subunit by the regulatory subunit MYPTl. It is believed that the compounds described herein may demonstrate selectivity to the specific holoenzyme myosin light chain phosphatase by forming interactions with these two regions of the protein.
  • the inhibitory potency of the thiazole compounds was examined by inhibition of purified myosin light chain phosphatase using 32 P-myosin light chain as a substrate.
  • Myosin light chain phosphatase activity was measured using myosin light chain phosphatase purified from pig aorta smooth muscle, which consists of PPlC and a truncated version of 60-kDa MYPTl fragment. Eto, et al., J. Biochem. (Tokyo), 1995, 118, 1104-1107.
  • PPlC was isolated from rabbit skeletal muscle by acetone treatment, as described by Martin et ah, Protein Expr. Purif., 1994, 5, 211-217. Inhibitors and myosin light chain phosphatase were preincubated for 10 min and then reaction was initiated by addition of 32 P-labeled myosin light chain as a substrate.
  • 32 P- labeled myosin light chain was prepared using chicken gizzard myosin light chain kinase, calmodulin, isolated myosin light chain, and [ ⁇ - 32 P]ATP. After 10 min at 30 0 C, reaction was terminated by addition of 10 % trichloroacetic acid, and released radioactivity of 32 P 1 in supernatant was measured by scintillation counter. The mean value for the duplicate assays was obtained, and myosin light chain phosphatase activity without inhibitor was set as 100 %. IC50 and error values were obtained by a nonlinear regression curve fitting, assuming first order binding, using Kaleidagraph software from the plot of relative activity against inhibitor concentration.
  • FIG. 2 presents a typical inhibition curve of myosin light chain phosphatase activity and summary of IC50 values is given in Table 1.
  • the inhibitory potency appeared to be favoured by providing an extended hydrophobic region.
  • Addition of a guanidine moiety to the aminothiazole also appeared to be beneficial for potency.
  • MYPTl and PPlC The levels of expression of MYPTl and PPlC were determined in both androgen receptor positive prostate cancer cell lines (LNCaP, C4-2 and C4-2B) and androgen receptor negative prostate cancer cell lines (CWR, DU145, PC-3 and PC-3M).
  • lysis buffer (7.4 pH, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovandate, 1 mM phenylmethane sulfonyl fluoride (Sigma), 50 ⁇ L per 5 mL protease cocktail inhibitor (Sigma), and 1% Triton X-100) for one hour at 4 0 C and then spun down at 13,000 rpm for 20 min. The supernatant was removed, and 20-30 ⁇ g of each extract were added to loading sample buffer and boiled for 5 min. This was then loaded onto a precast 4-12% Bis-Tris gel (Invitrogen).
  • Electrophoresis was performed, and proteins were transferred onto a PDVF membrane (Biorad).
  • the membrane was then blocked with a 1% w/v BSA solution (2.5 M NaCl, 1 M Tris HCl, pH 7.4) for one hour prior to incubation with the primary antibody overnight.
  • the membrane was then washed and blocked for 30 min prior to incubation with the species-appropriate HRP-linked secondary antibody (1 :40,000, Jackson Immunoresearch) for one hour.
  • the membrane was then washed and treated with ECL development kit (Perkin-Elmer) and exposed to film.
  • the following antibodies were purchased from Cell Signaling Technology: PPl ⁇ , 1 :1000; phosphol8 PPl ⁇ , 1 :1000; phospho-MLC, 1 :1000.
  • ⁇ - Actin (1 : 10,000) and MLC (1 :500) antibodies were purchased from Sigma.
  • Myosin light chain phosphatase inhibition on a cellular level should cause a direct loss of motility.
  • 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate (17e) on migration, a Boyden chamber experiment in a 96-well plate format was performed using a 96-well Boyden chamber MBA96 (Neuro Probe, Inc., Gaithersburg, MD). Insulin- like growth factor was used as the chemo-attractant since the migration of PC-3 cells is unaffected by serum gradients. The bottom wells were filled with 80 ⁇ l chemoattractant or negative control per well and an 8 ⁇ m porous membrane was placed on top.
  • the cells were plated on top of the membrane at 200,000 cells per well in serum free media plus drug concentration. To the bottom half of the chamber was added IGF-I and the appropriate drug concentration. The chamber was then incubated overnight at 37 0 C with 5% CO2. The chamber was disassembled and the membrane was fixed and stained using the Diff-Quik® Stain Set (Dade Behring, Deerfield, IL) to visualize the cells. The cells that migrated through the membrane to the bottom half of the well were stained and read at 595 nm on a plate reader. Briefly, the membrane was fixed in Diff-Quik® Fixative for 10 minutes and then placed first in Diff-Quik® Solution I for 3 minutes and then into Diff-Quik® Solution II for 3 minutes. The membrane was then washed with water three times and the cells on the upper surface were gently scraped off. The stained membrane was read directly in the spectrophotometer at 595 nm using a 96-well format.
  • PC-3 cells were plated at 200,000 cells per well in 6 well plates and incubated overnight. The media was then removed and replaced with serum free media and incubated for 24 h. Cells were then treated with media containing 1 ⁇ M 4-(2-guanidinothiazol-4-yl)phenyl
  • the following antibodies were purchased from Cell Signaling Technology: PP l ⁇ , 1 :1000; phosphol8 PPl ⁇ , 1 :1000; phospho-MLC, 1 :1000; phospho-Akt substrate, 1 :1000; phospho- PKC substrate, 1 :500; phospho-PKA substrate, 1 :5000; phospho-MAPK/CDK substrate, 1 :5000; phospho-BAD, 1 :2000; phospho-CDK substrate, 1 :1000; phospho-p70S6 Thr 421/Ser 424, 1 :1000; phospho-p70S6 Thr 389, 1 :1000.
  • ⁇ -actin (1 :10,000) and MLC (1 :500) antibodies were purchased from Sigma.
  • Acetylated tubulin (1 :1000) was purchased from Zymed.
  • Phospho-BAD is a known PPl substrate and its dephosphorylation induces apoptosis, whereas 4-(2-guanidinothiazol-4- yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate induces G2/M arrest without affecting phosphorylation of BAD.
  • LNCAP cells were plated onto a 96 well plate at 20,000 cells per well and incubated overnight in RPMI medium with 10% fetal bovine serum, 1% L-glutamine, 1% pen/strep and 0.1% DHT. The medium was removed and replaced with serum free medium plus the test compound (5 wells per concentration), then incubation continued for 48 h. 10 ⁇ L of WST-8 reagent solution was added to each well and incubation continued for 2 h. The plate was read using 450 nm as the measurement wavelength and 655 nm as the reference wavelength. The value for the initially plated cells was subtracted and the percent growth compared to the control was determined.
  • the medium used was RPMI with 10% FBS, 1% L-glutamine and 1% pen/strep.
  • CWR cells were plated at 20,000 cells per well.
  • DU145 and PC-3 were all plated at 5,000 cells per well. The amount growth of the cells was determined as a percentage of that observed for the control wells (no test compound).
  • the results of the growth inhibition experiment are shown in Table 2.
  • the GIso for each of the cell lines demonstrate that the androgen receptor positive, androgen sensitive cell line LNCAP was significantly less sensitive to treatment with 4-(2-guanidinothiazol-4- yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate, having a GIso of 2 ⁇ M.
  • the androgen independent cell lines all showed GIsos of about 0.5 ⁇ M.
  • BrdU uptake was determined by using the FITC kit (BDPharmagen 51-2354AK) and following kit instructions. Cells were plated onto a 6 well plate and incubated overnight (500,000 per well for LNCAP, 300,000 per well for CWR and 200,000 per well for DU145 and PC-3). The medium was removed and replaced with serum free medium with 1 ⁇ M 4-(2- guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate and incubation was continued for 24 h. 10 ⁇ L of the BrdU solution was added to each treated well and incubated for one hour.
  • the cells were trypsinized and washed twice with PBS and collected by centrifugation.
  • the cell pellets were fixed with 4% formaldehyde for 10 min, permeablized and treated with an FTIC-tagged anti-BrdU antibody for 1 h at 25 °C and analyzed by flow cytometry.
  • novel myosin light chain phosphatase inhibitor 4-(2- guanidinothiazol-4-yl)phenyl 5 -(dimethylamino)naphthalene-l -sulfonate is that it is fluorescent, which enabled the distribution of the compound in cells to be observed through fluorescence microscopy.
  • PC-3 and LNCAP cells were plated onto sterilized microscope slides and incubated overnight.
  • the medium was removed and replaced with complete medium with 20 ⁇ M 4-(2-guanidinothiazol-4-yl)phenyl 5 -(dimethylamino)naphthalene-l -sulfonate and incubated for 60 min.
  • the medium was removed, and the slides were washed four times with PBS.
  • the cells were then fixed using 4% formaldehyde for 10 min.
  • actin staining the cells were permeablized for 30 min with 0.1% Triton XlOO followed by treatment with Alexa fluor 680 conjugated phalloidin for 10 min. Slides were then washed four times with PBS.
  • the nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) using mounting medium containing DAPI.
  • DAPI 4',6-diamidino-2-phenylindole
  • 4-(2-Guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l- sulfonate was imaged using a 360 nm excitation and a 560 nm emission.
  • Figure 8a shows untreated PC-3 cells stained with phalloidin to mark actin filaments and DAPI as a nuclear stain.
  • Figure 8b shows untreated LNCaP cells stained with phalloidin to mark actin filaments and DAPI as a nuclear stain.
  • Figure 8c shows PC-3 cells treated with 10 ⁇ M 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (Example 28, 17e) (showing cytoplasmic staining) showing the disruption in the actin cytoskeleton stained by phalloidin.
  • Figure 8d shows LNCaP cells treated with 10 ⁇ M 2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (Example 28, 17e) (showing cytoplasmic staining) showing the disruption in the actin cytoskeleton stained by phalloidin.
  • Figure 8e shows PC- 3 cells treated with 10 ⁇ M 2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l- sulfonate (Example 28, 17e) (showing cytoplamic staining) and DAPI nuclear staining.
  • Figure 8f shows PC-3 cells treated with 10 ⁇ M 2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (Example 28, 17e) only showing cytoplasmic staining.
  • Actin filaments stained with phalloidine-Alexa Fuor 688 were disrupted in cells treated with 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate compared to control cells (Fig. 8a-d), indicating inhibition of myosin light chain phosphatase and reorganization of the actin cytoskeleton within cells. Indeed clumping of actin filaments and loss of their filamentous structure were evident in cells treated with 4-(2- guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate (Fig. 8c-d).
  • PtK2 cells provide an excellent model to study internal cellular structural networks because they provide clear and precise images of the cytoskeleton.
  • the PtK2 cells were obtained from the American Type Tissue Collection and grown in the medium recommended by the supplier at 37 0 C in a humidified 5% CO2 atmosphere. The cells were grown to confluence, disrupted by trypsinization, and seeded at about 10% confluence in a Lab-Tek II chamber slide obtained from Nalge Nunc International. The cells were grown for 2-3 days prior to drug treatment (final dimethyl sulfoxide concentration, 1% (v/v)) and for an additional 16 h following addition of drugs. Cells were washed twice with PBS, fixed with methanol at -20 0 C for 10 min, treated with -20 0 C acetone for 1 min, and washed twice with PBS.
  • the cells were treated for 1 h at 22 0 C in the dark with PBS containing 1.0 ⁇ g/mL DAPI, an FITC-conjugated anti- ⁇ -actin murine monoclonal antibody (Sigma product F 3022) at a 1 :250 dilution, and a Cy3 conjugated anti- ⁇ -tubulin antibody (clone TUB 2.2, Sigma product C 4585) at a 1 :100 dilution.
  • the chamber slide was washed twice with PBS and air-dried. A coverslip was applied with antifade mounting solution.
  • the cells were examined with a Nikon Model Eclipse E800 microscope equiped with epifluorescence and appropriate filters. Images were captured with a Spot digital camera, model 2.3.0, using version 3.0.2 software (Diagnostic Instruments). All images shown were obtained with a 4OX oil objective (N.A. 1.30).
  • PtK2 cells are shown stained for microtubules (red fluorecscence in the cytoplasm in figures (a), (c), (e), (h), (j) and (I)) and microfilaments (green fluorescence in the cytoplasm in figures (b), (d), (f), (i), (k), and (m)).
  • the nucleus is also stained (DAPI - blue).
  • Figures (a) and (b) show control cells stained for (a) microtubules and (b) microfilaments.
  • Figures (c) and (d) show cells treated with 0.2 ⁇ M colchinine stained for (c) microtubules and (d) microfilaments, showing disruption of microtubules.
  • Figures (e) and (f) show cells treated with 0.2 ⁇ M jasplakinolide stained for (e) microtubules and (f) microfilaments, showing disruption of microfilaments.
  • Figures (h)-(m) show cells treated with 4-(2-guanidinothiazol- 4-yl)phenyl 5 -(dimethylamino)naphthalene-l -sulfonate at 1 ⁇ M ((h),(i)), 5 ⁇ M ((j),(k)), and 10 ⁇ M ((l),(m)), and stained for microtutules ((h),(j),(l)) and microfilaments ((i),(k),(m)), showing that the compound disrupts both microfilaments and microtubules.
  • Cells were treated for 16 h with each drug.
  • inhibitors of the cytoskeleton fall into one of two categories: inhibitors of tubulin or of actin.
  • Colchicine and paclitaxel are two classic inhibitors of tubulin function, colchicine causing destabilization, while paclitaxel stabilizes microtubules. While these two compounds cause disruption of normal microtubule distribution, they have minimal effects on cellular actin microfilaments.
  • anti-actin compounds like jasplakinolide, disrupt the microfilament system of the cell but leave the microtubules intact (Fig. 9a- f).
  • Samples of prostate cancer tissue embedded in paraffin were sliced. After slicing, the tissue samples were de-waxed, rehydrated and epitope retrieval was conducted for 20 minutes at 100 0 C. Samples were then cooled at room temperature for 20 minutes. Samples were incubated with 10% goat serum for 10 minutes at room temperature to block non-specific binding sites. Blocking buffer was removed and primary antibody (MYPTl) was added to the samples at a dilution 1 :50 in tris-buffered saline and tween (TBST), supplemented with 5% goat serum for 2.5 hours at room temperature.
  • MYPTl primary antibody
  • the samples were incubated with goat-anti-rabbit-biotin antibody diluted at 1 :200 in TBST, for 30 minutes at room temperature. After the incubation, the antibody solution was removed and streptavidin conjugated Cy3 antibody added at a dilution of 1 : 100 in TBST for 10 minutes at room temperature. The samples were then exposed to compound 17e (100 ⁇ M) diluted in deionized water for 5 minutes at room temperature and then washed twice with deionized water. The slides were mounted in VectorShield mounting medium containing 4',6- diamidino-2-phenylindole (DAPI), and imaged.
  • DAPI VectorShield mounting medium containing 4',6- diamidino-2-phenylindole
  • Figure 11 shows images of human prostate cancer tissue slices stained with 100 ⁇ M 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate (17e).
  • Figure HA shows a bright field (DIC) image of the tissue
  • Figure HB shows the tissue stained with compound 17e (green fluorescence)
  • Figure HC shows a merger of images HA and HB. The compound is shown localized in the cytoplasm of the cells.
  • Figure 12 shows control images with human prostate cancer tissue slices stained with 100 ⁇ M 1- dimethylamino-5-sulfamoylnaphthaline ("dansylamide").
  • Figure 12A is a bright field (DIC) image of the tissue
  • Figure 12B shows tissue stained with l-dimethylamino-5- sulfamoylnaphthaline (very faint fluorescence)
  • Figure 12C shows a merger of images 12A and 12B.
  • DIC dark field
  • Figures 13 to 17 show the relative localization of 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate (17e) and MYPTl in human prostate cancer tissue.
  • Figure 13 shows prostate cancer tissue slices stained with anti-MYPTl antibody and 4-(2-guanidinothiazol-4-yl)phenyl 5 -(dimethylamino)naphthalene- 1 -sulfonate (17e) .
  • Figures 13 A to 13C show expression of MYPTl in prostate cancer cells.
  • Figure 13 A shows human prostate cancer tissue slices with MYPTl distribution visualized by immunofluorescence using MYPTl antibody (red fluorescence)
  • Figure 13B shows a bright field (DIC) image of the same cells
  • Figure 13C shows a merger of images 13A and 13B.
  • the figures demonstrate that MYPTl is expressed in the highly undifferentiated cancerous cells in the tissue.
  • Figures 13E to 13H show a comparison of the distribution of 4-(2-guanidinothiazol- 4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate (17e) and MYPTl protein.
  • Figure 13E shows human prostate cancer tissue slices stained with with MYPTl distribution visualized by immunofluorescence using MYPTl antibody
  • Figure 13F shows a bright field (DIC) image
  • Figure 13G shows the tissue stained with 100 ⁇ M of 4-(2-guanidinothiazol-4- yl)phenyl 5 -(dimethylamino)naphthalene-l -sulfonate (17e) (green fluorescence)
  • Figure 13H is a merger of images 13E, 13F and 13G.
  • the figures demonstrate that compound 17e binds to the areas of the tissue where MYPTl is expressed.
  • Figures 14 and 15 both show co-localization of 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (17e) and MYPTl in prostate cancer cells.
  • the cancer shown in Figure 15 is less aggressive (more differentiated cells) than that shown in Figure 14.
  • Figures 14A and 15A show immunofluorescence with a MYPTl antibody (red fluorescence) (A).
  • Figures 14B and 15B show bright field (DIC) images
  • Figures 14C and 15C show staining with compound 17e (green fluorescence)
  • Figures 14D and 15D show a merger of images 14A, 14B, and 14C (14D) and of images 15A, 15B, and 15C (15D) respectively.
  • Figures 16, 17 and 18 show distribution of 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (17e) and MYPTl immunofluorescence in prostate cancer tissue and cells. Shown are merged images of cells stained with compound 17e (green fluorescence), MYPTl immunofluorescence (red fluorescence), and 4',6-diamidino-2- phenylindole (DAPI) with the brightest regions (purple fluorescence) indicating co- localization of MYPTl and compound 17e.
  • Figure 16 shows low grade prostate cancer cells showing co-localization of MYPTl and compound 17e.
  • Figure 17 shows moderately invasive prostate cancer cells showing co-localization of MYPTl and compound 17e.
  • Figure 18 shows highly invasive prostate cancer cells showing co-localization of MYPTl and compound 17e. Based on the intensity of the fluorescence it appears that the expression of MYPTl as well as the amount of compound 17e is increased in more aggressive cancers.

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Abstract

Novel myosin light chain phosphatase inhibitors, compositions containing them, methods of making and using them, and methods of using fluorescent myosin light chain phosphatase inhibitors are described.

Description

MYOSIN LIGHT CHAIN PHOSPHATASE INHIBITORS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of and priority to U.S. Provisional Application No. 61/041,026, filed March 31, 2008, the entire disclosure of which is hereby incorporated by reference.
TECHNICAL FIELD
This disclosure relates to myosin light chain phosphatase inhibitors including in particular fluorescent myosin light chain phosphatase inhibitors, compositions including the same, and methods for preparing and using the same.
BACKGROUND
Cancers are among the most common causes of death in developed countries. Despite continuing advances, the existing treatments exhibit undesirable side effects and limited efficacy. Identifying new effective cancer drugs is a continuing focus of medical research.
Cell motility is an important property in cancer progression, metastasis and proliferation, potentially representing a significant target for new cancer therapies. The aggressiveness of cancer is directly related to the ability of cancer cells to migrate and invade surrounding tissues. Migration is achieved through a dynamic cycle of extension and contraction of the cell body controlled by the cytoskeleton. Lontay, et al., Cell. Sig., 2005, 17, 1265-1275; Somolyo, et al., Physiol. Rev., 2003, 83, 1325-1358; Xia, et al., Exper. Cell Res., 2005, 304, 506-517.
One of the major messengers in this pathway is myosin light chain (MLC), phosphorylation of which is controlled by a delicate balance between kinase and phosphatase activities. In vivo, myosin light chain phosphorylation is regulated not only through the activation of myosin light chain kinase, but also through the inhibition of myosin light chain phosphatase activity. Phosphorylation of MLC by myosin light chain kinase (MLCK) enhances motor activity of myosin as well as stability of myosin filaments, so that the dephosphorylation by myosin light chain phosphatase (MLCP) produces a relaxation and reorganization of actomyosin filaments. Lontay, et al, Cell. Sig., 2005, 17, 1265-1275 ; Somolyo, et al, Physiol. Rev., 2003, 83, 1325-1358; Xia, et al, Exper. Cell Res., 2005, 304, 506-517. Myosin light chain phosphatase is a serine/threonine phosphatase and consists of a 130 kDa regulatory subunit that binds myosin (MYPTl), a 37 kDa catalytic subunit protein phosphatase 1C (PPlC) and a 21 kDa subunit (M-21) of undetermined function. Takizawa, et al., Am. J. Physiol. Cell. Physiol., 2003, 284, C250-C262. There are several pathways that cause the direct phosphorylation of myosin light chain phosphatase, functionally inactivating the phosphatase activity of the enzyme. Kawano, et al., J. Cell Biol., 1999, 147, 1023-1037. The Rho-kinase pathway is the main route that phosphorylates myosin light chain phosphatase, but other mechanisms also exist. Dimpoulos, et al., Circ. Res., 2007, 100, 121- 129.
PPlC is the catalytic subunit of myosin light chain phosphatase, is a member of the PPP superfamily of phosphatases. There are three known natural product inhibitor classes of PPlC — microcystin, okadaic acid and calyculin, which also inhibit PP2A, another member of the PPP family. Maynes, et al., J. Biol. Chem. 2001, 276, 44078-44882; Kita, A., et al., Structure, 2002, 10, 715-724; Evans, et al, J. Am. Chem. Soc, 1992, 114, 9434-9453; Humphrey, et al, J. Am. Chem. Soc, 1996, 118, 11759-11770; Forsyth, et al, J. Am. Chem. Soc. 1997, 119, 8381-8382. Microcystin, okadaic acid and cantharidin are all extremely toxic. Microcystin covalently binds to protein phosphatases and causes hepatotoxicity. Okadaic acid causes diarrhetic shellfish poisoning. Cantharidin is a very promiscuous phosphatase inhibitor and has a LD50 of approximately 0.5 mg/kg and can be lethal at doses as low as 10 mg. The natural product inhibitors of PP1C/PP2A are also tumor promoting.
The need continues to exist for new effective cancer treatments.
SUMMARY
This disclosure is directed to compounds that function as myosin light chain phosphatase inhibitors. Such compounds are useful in the treatment of a variety of diseases or disorders. The compositions and methods can be used in treating diseases or disorders associated with aberrant myosin light chain phosphatase activity or levels, including, for example, cancer.
The disclosure is also directed to the finding that certain of the myosin light chain phosphatase inhibitors described herein are fluorescent and can be used as theragnostic agents, i.e. an agent having combined therapeutic and diagnostic properties, to detect and/or treat a variety of disorders associated with aberrant (e.g., increased) myosin light chain phosphatase activity or levels. For example, in some cases, a fluorescent myosin light chain phosphatase inhibitor is used as a diagnostic agent (e.g. , to diagnose diseases or conditions in which aberrant levels or activity of myosin light chain phosphatase, or of myosin light chain phosphorylation levels are associated); as a combined diagnostic/therapeutic agent (e.g., to indicate the presence of one or more cells exhibiting aberrant myosin light chain phosphatase activity and/or levels and to treat such cells); and as a tracking/therapeutic agent (e.g., to determine the location of the fluorescent inhibitor in order to help direct and/or potentiate an additional therapy such as surgery or radiation).
Accordingly, in one aspect, provided herein is a compound according to formula I:
Figure imgf000004_0001
I or a salt form thereof; wherein:
Ar1 is selected from the group consisting of unsubstituted or substituted aryl and unsubstituted or substituted heteroaryl, wherein the substituents of the aryl or heteroaryl are selected from the group consisting of -R1; -(Ci-C3)alkylene-Ar2; (C2-C6)alkenyl; (C2-C6)alkynyl; halogen; -C≡N; -NO2; -C(=O)R3; -C(=O)OR3; -C(=O)NR4 2; -C(=NR3)NR4 2;-OR2; -OC(=O)(d-C6)alkyl;
-OC(=O)(Ci-C6)alkylene-R5; -OC(=O)O(Ci-C6)alkyl; -OC(=O)NR4 2; -NR4 2; -NR4C(=O)R3; -NR4C(=O)Ar2; -NR4C(=O)O(Ci-C6)alkyl; -NR4C(=O)NR4 2; -NR4SO2R3; -NR4SO2Ar2; -SR2; -S(O)R2; -SO2R2; -OSO2(C i-C6)alkyl; -OSO2Ar2; and -SO2NR4 2;
A is selected from the group consisting of -C(=0)- and -SO2-;
B is selected from the group consisting of a bond, -0-, -NR3-, and -N(-A- Ar1)-;
D is selected from the group consisting of -H and -C(=NH)-NH2; each R1 is independently unsubstituted (Ci-Ce)alkyl or (Ci-Ce)alkyl substituted with up to five halogen atoms and up to two substituents selected from the group consisting of -C≡N; -C(=O)R3; -C(=O)OR3; -C(=O)NR4 2; -OR3; -OC(=O)(Ci-C6)alkyl; -OC(=O)O(Ci-C6)alkyl; -OC(=O)NR4 2; -NR4 2; -NR3C(=O)R3; -NR3C(=O)NR4 2; -S(Ci-C6)alkyl; -S(O)(Ci-C6)alkyl; and -SO2(Ci-C6)alkyl; each R2 is independently selected from the group consisting of hydrogen, R1, Ar2 and (Ci-C3)alkylene-Ar2; each R is independently hydrogen or (Ci-Ce)alkyl; each R4 is independently hydrogen; (Ci-Ce)alkyl; -(C2-Ce)alkylene-OR3; -(Ci-C6)alkylene-C(=O)OR3; -(Ci-C6)alkylene-OC(=O)R3; -(C2-C6)alkylene-NR6 2; -(Ci-C6)alkylene-C(=O)NR6 2; -(Ci-C6)alkylene-NR3C(=O)R3;
-(Ci-C6)alkylene-NR3C(=O)NR6 2; Ar2, or -(Ci-C3)-alkyleneAr2; or, optionally, within any occurrence of NR4 2, independently of any other occurrence of NR4 2, the two R4 groups in combination are -(CH2)a- or -(CH2)bE(CH2)2-; each R5 is independently Ar2 or 1 ,4-benzoquinon-2-yl optionally substituted with 0, 1, 2, or 3 alkyl groups; each R6 is independently hydrogen; (Ci-Ce)alkyl; -(C2-Ce)alkylene-OR3; -(Ci-C6)alkylene-C(=O)OR3; -(d-C6)alkylene-OC(=O)R3; (C2-C6)alkylene-NR3 2; -(Ci-C6)alkylene-C(=O)NR3 2; -(Ci-C6)alkylene-NR3C(=O)R3;
-(Ci-C6)alkyleneNR3C(=O)NR3 2; -Ar2, or -(Ci-C3)alkylene-Ar2; or, optionally, within any occurrence of NR6 2, independently of any other occurrence of NR6 2, the two R6 groups in combination are -(CH2)a- or -(CH2)bE(CH2)2-; each Ra is independently hydrogen; (d-C6)alkyl; (C2-C6)alkenyl; (C2-C6)alkynyl; halogen; -C≡N; -NO2; -C(=O)R3; -C(=O)OR3; -C(=O)NR3 2; -C(=NR3)NR3 2; -OR3; -OC(=O)(d-C6)alkyl; -OC(=O)O(Ci-C6)alkyl; -OC(=O)NR3 2; -NR3 2; -NR3C(=O)R3; -NR3C(=O)O(d-C6)alkyl; -NR3C(=O)NR3 2; -S(d-C6)alkyl; -S(O)(Ci-C6)alkyl; and -SO2(d-C6)alkyl; and -SO2NR3 2; each Rb is independently hydrogen; (Ci-Ce)alkyl; (C2-Ce)alkenyl; (C2-C6)alkynyl; halogen; -C≡N; -NO2; -C(=O)R3; -C(=O)OR3; -C(=O)NR3 2; -C(=NR3)NR3 2; -OR3; -OC(=O)(d-C6)alkyl; -OC(=O)O(d-C6)alkyl; -OC(=O)NR3 2; -NR3 2; -NR3C(=O)R3; -NR3C(=O)O(d-C6)alkyl; -NR3C(=O)NR3 2; -S(d-C6)alkyl; -S(O)(Ci-C6)alkyl; and -SO2(d-C6)alkyl; -SO2NR3 2; each Rc is independently hydrogen; (Ci-Ce)alkyl; (C2-Ce)alkenyl; (C2-C6)alkynyl; halogen; -C≡N; -NO2; -C(=O)R3; -C(=O)OR3; -C(=O)NR3 2; -C(=NR3)NR3 2; -OR3; -OC(=O)(d-C6)alkyl; -OC(=O)O(d-C6)alkyl; -OC(=O)NR3 2; -NR3 2; -NR3C(=O)R3; -NR3C(=O)O(d-C6)alkyl; -NR3C(=O)NR3 2; -S(Ci-C6)alkyl; -S(O)(Ci-C6)alkyl; and -SO2(Ci-C6)alkyl; -SO2NR3 2; each Rd is independently hydrogen; (Ci-Ce)alkyl; (C2-Ce)alkenyl; (C2-C6)alkynyl; halogen; -C≡N; -NO2; -C(=O)R3; -C(=O)OR3; -C(=O)NR3 2; -C(=NR3)NR3 2; -OR3; -OC(=O)(d-C6)alkyl; -OC(=O)O(Ci-C6)alkyl; -OC(=O)NR3 2; -NR3 2; -NR3C(=O)R3; -NR3C(=O)O(d-C6)alkyl; -NR3C(=O)NR3 2; -S(Ci-C6)alkyl; -S(O)(Ci-C6)alkyl; and -SO2(Ci-C6)alkyl; -SO2NR3 2; each a is independently selected from the group consisting of 4, 5, and 6; each b is independently selected from the group consisting of 2 and 3; each E is independently selected from the group consisting of O, S, NR3; NC(=0)R3; NSO2R3; N(C2-C6)alkylene-OR3; N(Ci-C6)alkylene-C(=O)OR3; N(Ci-C6)alkylene-OC(=O)R3; N(C2-C6)alkylene-NR3 2;
N(Ci-C6)alkylene-C(=O)NR3 2; N(Ci-C6)alkylene-NR3C(=O)R3;
N(Ci-C6)alkylene-NR3C(=O)NR3 2; NAr2; N(Ci-C3)alkylene-Ar2; and NC(=O)Ar2; and each Ar2 is independently selected from the group consisting of unsubstituted aryl, unsubstituted heteroaryl, and aryl or heteroaryl substituted with one or more substitutents independently selected from the group consisting of (Ci-Ce)alkyl; (C2-C6)alkenyl; (C2-C6)alkynyl; halogen; -C≡N; -NO2; -C(=O)R3; -C(=O)OR3; -C(=O)NR3 2; -C(=NR3)NR3 2; -OR3; -OC(=O)(d-C6)alkyl; -OC(=O)O(d-C6)alkyl; -OC(=O)NR3 2; -NR3 2; -NR3C(=O)R3; -NR3C(=O)O(d-C6)alkyl; -NR3C(=O)NR3 2; -S(Ci-C6)alkyl; -S(O)(d-C6)alkyl; and -SO2(d-C6)alkyl; -SO2NR3 2; and (C i -C3)perfluoroalkyl.
In another aspect, there is provided a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound according to formula I, or a pharmaceutically acceptable salt form thereof.
Also provided is a therapeutic method which comprises administering a compound according to formula I, or a pharmaceutically acceptable salt form thereof, to an individual.
There is also provided a method of inhibiting myosin light chain phosphatase comprising contacting an effective amount of a compound according to formula I, or a salt form thereof, with myosin light chain phosphatase. As another aspect, there is provided a method of inhibiting protein phosphatase 1C comprising contacting an effective amount of a compound according to formula I, or a salt form thereof, with protein phosphatase 1C.
As another aspect, methods of increasing the level of phosphorylation of myosin light chain or Ras-1 in a cell are provided comprising contacting an effective amount of a compound according to formula I, or a salt form thereof, with the cell.
A method is also provided of increasing the level of phosphorylation of Ras-1 in a cell, wherein the method comprises contacting an effective amount of a compound according to formula I, or a salt form thereof, with the cell.
Also provided are methods of inhibiting actin and/or tubulin polymerization in a cell, comprising contacting an effective amount of a compound according to formula I, or a salt form thereof, with the cell.
There is also provided a method of treating a disease or condition associated with myosin light chain phosphatase activity comprising causing an effective amount of a compound according to formula I, or a salt form thereof, to be present in an individual in need of such treatment.
There is further provided a method of inducing cell-cycle arrest and/or apoptosis of a cell comprising contacting the cell with a myosin light chain phosphatase inhibitor, for example a compound according to formula I, or a salt form thereof.
As another aspect, a method is provided for treating cancer, comprising administering an effective amount of a myosin light chain phosphatase inhibitor, for example a compound according to formula I, or a salt form thereof, to an individual in need of such treatment.
In a further aspect, a method is provided of killing a tumor cell comprising: contacting the tumor cell with an effective amount of a myosin light chain phosphatase inhibitor, for example a compound according to formula I, or a salt form thereof; and irradiating the tumor cell with an effective amount of ionizing radiation and/or contacting the tumor cell with an effective amount of at least one further chemotherapeutic agent.
Also provided is a method of treating a tumor in an individual, comprising: causing an effective amount of a myosin light chain phosphatase inhibitor, for example a compound according to formula I, or a salt form thereof, to be present in the individual; and irradiating the tumor with an effective amount of ionizing radiation. In a further aspect, a method is provided of detecting the presence of an elevated amount of myosin light chain phosphatase in a subject cell comprising: providing a fluorescent myosin light chain phosphatase inhibitor; contacting the myosin light chain phosphatase inhibitor with the subject cell and with a control cell; observing fluorescence of the cells after the contacting; wherein an elevated level of fluorescence of the subject cells relative to the level of fluorescence of the control cells is indicative of an elevated amount of myosin light chain phosphatase in the subject cell as compared to the control cell.
In another aspect, a method is provided of detecting diseased cells, wherein the diseased cells comprise elevated amounts of myosin light chain phosphatase comprising: providing a fluorescent myosin light chain phosphatase inhibitor; contacting the fluorescent myosin light chain phosphatase inhibitor with tissue; observing for fluorescence of the cells of the tissue after the contacting; wherein an elevated level of fluorescence of the some of the cells relative to others in the tissue or relative to control non-diseased cells that have been contacted with the fluorescent myosin light chain phosphatase inhibitor is indicative that the fluorescent cells may be diseased cells comprising elevated amounts of myosin light chain phosphatase.
There is also provided a method of detecting diseased cells in a tissue of an individual comprising: providing a fluorescent myosin light chain phosphatase inhibitor; contacting the fluorescent myosin light chain phosphatase inhibitor with the tissue; observing for fluorescence of at least some cells of the tissue after the contacting; wherein an elevated level of fluorescence of the some of the cells relative to others in the tissue or relative to control non-diseased cells that have been contacted with the fluorescent myosin light chain phosphatase inhibitor is indicative that the fluorescent cells may be diseased cells comprising elevated amounts of myosin light chain phosphatase.
In another aspect, a method is provided of radiotherapy of tumors, wherein the tumors comprise cells comprise an elevated amount of myosin light chain phosphatase relative to non-tumor cells, comprising: providing a fluorescent myosin light chain phosphatase inhibitor; causing the fluorescent myosin light chain phosphatase inhibitor to be present in the tumor cells in an effective amount to inhibit myosin light chain phosphatase and for fluorescence to be observable; observing the fluorescence; and directing an effective amount of ionizing radiation to the fluorescent tumor cells. Also described is a method of surgery to remove tumor tissue from an individual comprising: providing a fluorescent myosin light chain phosphatase inhibitor; causing the fluorescent myosin light chain phosphatase inhibitor to be present in at least some cells of the tumor tissue in an effective amount for fluorescence of at least some of the tumor tissue to be observable; observing the fluorescence; and surgically removing at least some of the fluorescent tumor tissue, whereby at least a portion of the tumor that comprises fluorescent tumor cells is removed.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the results of molecular modeling studies with 4-(2-guanidinothiazol- 4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate (Example 28, 17e) docked into the crystal structure of PPlC the guanidine group forming hydrogen bonds with a backbone carbonyl of Arg 221 in the hydrophobic groove and the dansyl portion of the molecule aligned in the βl2-13 loop region of the catalytic pocket.
Figure 2 presents a typical inhibition curve of myosin light chain phosphatase activity as a function of inhibitor concentration 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (Example 28, 17e) (D), 2-guanidinothiazol-4- yl)phenyl 3'-methoxybiphenyl-4-carboxylate (Example 1, 10a) ( A ) and amidinothiourea (H2NC(=NH)NHC(=S)NH2) (2-imino-4-thiobiuret) (#).
Figure 3 shows a Western blot analysis of relative levels of MYPT, PPl, MLC and β- actin in androgen receptor positive prostate cancer cell lines (LNCaP, C4-2 and C4-2B) and androgen receptor negative prostate cancer cell lines (CWR, DU145, PC-3 and PC-3M).
Figure 4 shows the effect of the compound of 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (Example 28, 17e) on the chemotaxis of PC-3 cells. The absorbance represents the amount of stained cells that migrated through the 8 μM membrane using insulin-like growth factor- 1 as a chemoattractant.
Figure 5 shows the effect of the compound of 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (Example 28, 17e) on the cell-cycle distribution of PC-3 cells. In (a) the distribution is shown after PC-3 cells were treated with the compound (or control - untreated) at 6, 24, and 48 hours. In (b) the sub-Gl populations of PC-3 cells treated with compound for 48h as compared to control (untreated) is shown. Figure 6 shows levels of phosphorylated substrates in PC-3 cells treated with 1 μM A- (2-guanidinothiazol-4-yl)phenyl 5 -(dimethylamino)naphthalene-l -sulfonate (17e) or 10O nM nocodazole . 4-(2-guanidinothiazol-4-yl)phenyl 5 -(dimethylamino)naphthalene- 1 -sulfonate (17e) is seen to be a selective myosin light chain phosphatase inhibitor since only phospho- MLC changes compared to control upon treatment with the compound. Phospho-BAD is a substrate of another PPlC containing holoenzyme, but not myosin light chain phosphatase. P70S6 is a substrate of PP2A holoenzymes.
Figure 7 shows the BrdU uptake of PC-3 cells treated with 1 μM 5- (dimethylamino)naphthalene-l -sulfonate (17e) for 24 hours as compared to control (no compound treatment). Data points represent pooling of three separate wells.
Figure 8 shows fluorescence imaging of PC-3 and LnCap cells treated with 1 μM A- (2-guanidinothiazol-4-yl)phenyl 5 -(dimethylamino)naphthalene-l -sulfonate (17e) for 60 min. Figures (a) and (b) show (a) PC-3, and (b) LNCap cells stained with 4',6-diamidino-2- phenylindole (DAPI) (the nuclei of the cells show blue fluorescence), and phalloidin (the microfilaments in the cytoplasm of the cells show red fluorescenece. Figures (c) and (d) show (c) PC-3, and (d) LNCap cells treated with 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (shown fluorescing (yellow fluorescence)) and stained with phalloidin (red fluorescence) and showing microfilament destabilization. Figures (e) and (f) show PC-3 cells stained with (e) 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (yellow fluorescence - seen in the cytoplasm only) and DAPI (blue staining of the nucleus) and (f) 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (17e) (yellow fluorescence - seen in the cytoplasm only, with the nuclei seen to be dark).
Figure 9 shows the results of imaging studies of PtK2 cells stained for microtubules (red fluorescence in the cytoplasm in figures (a), (c), (e), (h), Q) and (I)) and microfilaments (green fluorescence in the cytoplasm in figures (b), (d), (f), (i), (k), and (m)). In each figure the nucleus is also stained (DAPI - blue). Figures (a) and (b) show control cells stained for (a) microtubules and (b) microfilaments. Figures (c) and (d) show cells treated with 0.2 μM colchinine stained for (c) microtubules and (d) microfilaments, showing disruption of microtubules. Figures (e) and (f) show cells treated with 0.2 μM jasplakinolide stained for (e) microtubules and (f) microfilaments, showing disruption of microfilaments. Figures (h)- (m) show cells treated with 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (17e) at 1 μM ((h),(i)), 5 μM (Q), (k)), and 10 μM ((l),(m)), and stained for microtutules ((h),(j),(l)) and microfilaments ((i),(k),(m)), showing that the compound demonstrates disruption of both microfilaments and microtubules. Cells were treated for 16 h with each drug.
Figure 10 shows Giemsa- stained CA46 Burkitt's lymphoma cells: (a) untreated controls; and cells treated with (b) 0.2 μM colchinine; or (c) and (d) 4-(2-guanidinothiazol-4- yl)phenyl 5 -(dimethylamino)naphthalene-l -sulfonate (17e) at (c) 5 μM and (d) 0.8 μM.
Figure 11 shows two sets of images of human prostate cancer tissue slices stained with 100 μM 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate (17e). Figure HA shows a bright field (DIC) image of the tissue, Figure HB shows the tissue stained with compound 17e (green fluorescence) while Figure HC shows a merger of images 1 IA and 1 IB.
Figure 12 shows control images of human prostate cancer tissue slices stained with 100 μM l-dimethylamino-5-sulfamoylnaphthaline ("dansylamide"). Figure 12A is a bright field (DIC) image of the tissue, Figure 12B shows tissue stained with l-dimethylamino-5- sulfamoylnaphthaline (very faint fluorescence) while Figure 12C shows a merger of images A and B.
Figure 13 shows the localization of 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (17e) and MYPTl in human prostate cancer tissue. Figures 13A to 13C show expression of MYPTl in prostate cancer cells. Figure 13A shows human prostate cancer tissue slices with MYPTl distribution imaged by immunofluorescence with a MYPTl antibody (red fluorescence), Figure 13B shows a bright field (DIC) image of the same cells, while Figure 13C shows a merger of images 13A and 13B. Figures 13E to 13H show a comparison of the distribution of compound 17e and MYPTl protein. Figure 13E shows human prostate cancer tissue slices imaged by immunofluorescence using a MYPTl antibody, Figure 13F shows a bright field (DIC) image, Figure 13G shows the tissue stained with 100 μM of 17e (green fluorescence) and Figure 13H is a merger of images 13E, 13F and 13G.
Figures 14 and 15 both show co-localization of 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (17e) and MYPTl in prostate cancer cells. The cancer shown in Figure 15 is less aggressive (more differentiated cells). Figures 14A and 15A show immunofluorescence using a MYPTl antibody (red fluorescence). Figures 14B and 15B show a bright field (DIC) image, Figures 14C and 15C shows staining with compound 17e (green fluorescence), while Figures 14D and 15D show a merger of images 14A, 14B, and 14C (14D) and of images 15A, 15B, and 15C (15D) respectively.
Figures 16, 17 and 18 show distribution of 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (17e) and MYPTl immunofluorescence in prostate cancer tissue and cells. Shown are merged images of cells stained with compound 17e (green fluorescence), MYPTl immunofluorescence (red fluorescence), and 4',6-diamidino-2- phenylindole (DAPI) with the brightest regions (purple fluorescence) indicating co- localization of MYPTl and compound 17e. Figure 16 shows low grade prostate cancer cells showing co-localization of MYPTl and compound 17e. Figure 17 shows moderately invasive prostate cancer cells showing co-localization of MYPTl and compound 17e. Figure 18 shows highly invasive prostate cancer cells showing co-localization of MYPTl and compound 17e.
DETAILED DESCRIPTION
Inhibition of myosin light chain phosphatase causes an increase of phosphorylation of MLC in smooth muscle, without kinase activation. Somolyo, et al., Physiol. Rev., 2003, 83, 1325-1358; Xia, et al., Exper. Cell Res., 2005, 304, 506-517. An increased level of phosphorylated myosin light chain, the substrate of myosin light chain phosphatase, can alone initiate apoptotic cell death. Mills, et al., J. Cell. Biol. 1998, 140, 627-636.
Provided herein are novel compounds that are active as selective myosin light chain phosphatase inhibitors. Certain of the novel compounds are fluorescent inhibitors of myosin light chain phosphatases. Also provided are novel methods of using the provided compounds, including methods of treatment and screening methods. Also provided are novel methods related to inhibition of myosin light chain phosphatase, using fluorescent myosin light chain phosphatase inhibitors.
I. Definitions A. General
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. The term "contacting" means bringing at least two moieties together, whether in an in vitro system or an in vivo system.
The expression "effective amount", when used to describe an amount of compound or radiation applied in a method, refers to the amount of a compound that achieves the desired pharmacological effect or other effect, for example an amount that inhibits the abnormal growth or proliferation, or induces apoptosis of cancer cells, resulting in a useful effect.
The terms "treating" and "treatment" mean causing a therapeutically beneficial effect, such as ameliorating existing symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, postponing or preventing the further development of a disorder and/or reducing the severity of symptoms that will or are expected to develop.
As used herein, "individual" (as in the subject of the treatment) means both mammals and non-mammals. Mammals include, for example, humans; non-human primates, e.g. apes and monkeys; cattle; horses; sheep; dogs; cats; dogs; rats; mice; pigs; and goats. Non-mammals include, for example, fish and birds.
B. Chemical
In the following paragraphs some of the definitions include examples. The examples are intended to be illustrative, and not limiting.
The term "(Cx-Cy)alkyl" (wherein x and y are integers) by itself or as part of another substituent means, unless otherwise stated, an alkyl group containing between x and y carbon atoms. An alkyl group formally corresponds to an alkane or cycloalkane with one C-H bond replaced by the point of attachment of the alkyl group to the remainder of the compound. An alkyl group may be straight-chained or branched. Alkyl groups having 3 or more carbon atoms may be cyclic. Cyclic alkyl groups having 7 or more carbon atoms may contain more than one ring and be polycyclic. Examples of straight-chained alkyl groups include methyl, ethyl, n-propyl, n-butyl, and n-octyl. Examples of branched alkyl groups include /-propyl, t- butyl, and 2,2-dimethylethyl. Examples of cyclic alkyl groups include cyclopentyl, cyclohexyl, cyclohexylmethyl, and 4-methylcyclohexyl. Examples of polycyclic alkyl groups include bicyclo[2.2.1]heptanyl, norbornyl, and adamantyl. Preferred (Cx-Cy)alkyl groups are (Ci-C6)alkyl. More preferred are (Ci-C3)alkyl. Most preferred are methyl and ethyl. The term "(Cx-Cy)alkylene" (wherein x and y are integers) refers to an alkylene group containing between x and y carbon atoms. An alkylene group formally corresponds to an alkane with two C-H bond replaced by points of attachment of the alkylene group to the remainder of the compound. Preferred are a divalent straight hydrocarbon group consisting of methylene groups, such as, -CH2-, -CH2CH2-, -CH2CH2CH2-. Preferred (Cx-Cy)alkylene are (C1- Ce)alkylene. More preferred are (Ci-C3)alkylene.
The term "(Cx-Cy) alkenyl" (wherein x and y are integers) denotes a radical containing x to y carbons, wherein at least one carbon-carbon double bond is present (therefore x must be at least 2). Some embodiments are 2 to 4 carbons, some embodiments are 2 to 3 carbons, and some embodiments have 2 carbons. Both E and Z isomers are embraced by the term "alkenyl." Furthermore, the term "alkenyl" includes di- and tri-alkenyls. Accordingly, if more than one double bond is present then the bonds may be all E or Z or a mixtures of E and Z Examples of an alkenyl include vinyl, allyl, 2-butenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexanyl, 2,4-hexadienyl and the like.
The term "(Cx-Cy) alkynyl" (wherein x and y are integers) denotes a radical containing 2 to 6 carbons and at least one carbon-carbon triple bond, some embodiments are 2 to 4 carbons, some embodiments are 2 to 3 carbons, and some embodiments have 2 carbons. Examples of an alkynyl include ethynyl, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2- butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3- hexynyl, 4-hexynyl, 5-hexynyl and the like. The term "alkynyl" includes di- and tri-ynes.
The term "(Cx-Cy) alkoxy" (wherein x and y are integers) employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined above, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs and isomers. Preferred are (Ci-C3)alkoxy, particularly ethoxy and methoxy.
The terms "halo" or "halogen" by themselves or as part of another substituent mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine, chlorine, or bromine, more preferably, fluorine or chlorine.
The term "aromatic" refers to a carbocycle or heterocycle having one or more polyunsaturated rings having aromatic character (i.e. having (4n + 2) delocalized π (pi) electrons where n is an integer). The term "aryl", employed alone or in combination with other terms, means, unless otherwise stated, a carbocyclic aromatic system containing one or more rings (typically one, two or three rings), wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene. Examples include phenyl; anthracyl; and naphthyl. Preferred are phenyl and naphthyl, most preferred is phenyl.
The term "heterocycle" or "heterocyclyl" or "heterocyclic" by itself or as part of another substituent means, unless otherwise stated, an unsubstituted or substituted, stable, mono- or multi-cyclic heterocyclic ring system which consists of carbon atoms and at least one heteroatom selected from the group consisting of N, O, and S, and, wherein the nitrogen and sulfur heteroatoms may be optionally oxidized, and the nitrogen atom may be optionally quaternized. The heterocyclic system may be attached, unless otherwise stated, at any heteroatom or carbon atom which affords a stable structure.
The term "heteroaryl" or "heteroaromatic" refers to a heterocycle having aromatic character. A polycyclic heteroaryl may include one or more rings which are partially saturated. Examples include tetrahydroquinoline and 2,3-dihydrobenzofuryl. For compounds of formula I, the attachment point on ring Ar1 or Ar2 is understood to be on an atom which is part of an aromatic monocyclic ring or a ring component of a polycyclic aromatic which is itself an aromatic ring.
Examples of non-aromatic heterocycles include monocyclic groups such as: aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, imidazoline, pyrazolidine, dioxolane, sulfolane, 2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydropyridine, 1 ,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran, 1,4-dioxane, 1,3-dioxane, homopiperazine, homopiperidine, 1,3-dioxepane, 4,7-dihydro-l,3-dioxepin and hexamethy leneoxide .
Examples of heteroaryl groups include: pyridyl, pyrazinyl, pyrimidinyl, particularly 2- and 4-pyrimidinyl, pyridazinyl, thienyl, furyl, pyrrolyl, particularly 2-pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl, particularly 3- and 5-pyrazolyl, isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl.
Examples of polycyclic heterocycles include: indolyl, particularly 3-, A-, 5-, 6- and 7-indolyl, indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl, particularly 1- and 5-isoquinolyl, 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl, particularly 2- and 5-quinoxalinyl, quinazolinyl, phthalazinyl, 1,5-naphthyridinyl, 1,8-naphthyridinyl, 1 ,4-benzodioxanyl, coumarin, dihydrocoumarin, benzofuryl, particularly 3-, A-, 5-, 6- and 7-benzofuryl, 2,3-dihydrobenzofuryl, 1 ,2-benzisoxazolyl, benzothienyl, particularly 3-, A-, 5-, 6-, and 7-benzothienyl, benzoxazolyl, benzthiazolyl, particularly 2-benzothiazolyl and 5-benzothiazolyl, purinyl, benzimidazolyl, particularly 2-benzimidazolyl, benztriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, and quinolizidinyl.
The aforementioned listing of heterocyclyl and heteroaryl moieties is intended to be representative and not limiting.
The term "substituted" means that an atom or group of atoms formally replaces hydrogen as a "substituent" attached to another group. For aryl and heteroaryl groups, the term "substituted", unless otherwise indicated, refers to any level of substitution, namely mono-, di-, tri-, terra-, or penta-substitution, where such substitution is permitted. The substituents are independently selected, and substitution may be at any chemically accessible position.
The "valency" of a chemical group refers to the number of bonds by which it is attached to other groups of the molecule.
II. Novel Compounds
In one aspect, provided herein is a compound according to formula I:
Figure imgf000016_0001
I or a salt form thereof; wherein:
Ar1 is selected from the group consisting of unsubstituted or substituted aryl and unsubstituted or substituted heteroaryl, wherein the substituents of the aryl or heteroaryl are selected from the group consisting of -R1; -(Ci-C3)alkylene-Ar2; (C2-C6)alkenyl; (C2-C6)alkynyl; halogen; -C≡N; -NO2; -C(=O)R3; -C(=O)OR3; -C(=O)NR4 2; -C(=NR3)NR4 2;-OR2; -OC(=O)(d-C6)alkyl;
-OC(=O)(Ci-C6)alkylene-R5; -OC(=O)O(Ci-C6)alkyl; -OC(=O)NR4 2; -NR4 2; -NR4C(=O)R3; -NR4C(=O)Ar2; -NR4C(=O)O(Ci-C6)alkyl; -NR4C(=O)NR4 2; -NR4SO2R3; -NR4SO2Ar2; -SR2; -S(O)R2; -SO2R2; -OSO2(C i-C6)alkyl; -OSO2Ar2; and -SO2NR4 2;
A is selected from the group consisting of -C(=0)- and -SO2-;
B is selected from the group consisting of a bond, -0-, -NR3-, and -N(-A- Ar1)-;
D is selected from the group consisting of -H and -C(=NH)-NH2; each R1 is independently unsubstituted (Ci-Ce)alkyl or (Ci-Ce)alkyl substituted with up to five halogen atoms and up to two substituents selected from the group consisting of -C≡N; -C(=0)R3; -C(=0)0R3; -C(=O)NR4 2; -OR3; -OC(=O)(Ci-C6)alkyl; -OC(=O)O(Ci-C6)alkyl; -OC(=O)NR4 2; -NR4 2; -NR3C(=O)R3; -NR3C(=O)NR4 2; -S(Ci-C6)alkyl; -S(O)(Ci-C6)alkyl; and -SO2(Ci-C6)alkyl; each R2 is independently selected from the group consisting of hydrogen, R1, Ar2 and (d-C3)alkylene-Ar2; each R3 is independently hydrogen or (Ci-Ce)alkyl; each R4 is independently hydrogen; (Ci-Ce)alkyl; -(C2-Ce)alkylene-OR3; -(Ci-C6)alkylene-C(=O)OR3; -(Ci-C6)alkylene-OC(=O)R3; -(C2-C6)alkylene-NR6 2; -(Ci-C6)alkylene-C(=O)NR6 2; -(Ci-C6)alkylene-NR3C(=O)R3;
-(Ci-C6)alkylene-NR3C(=O)NR6 2; Ar2, or -(Ci-C3)-alkyleneAr2; or, optionally, within any occurrence of NR4 2, independently of any other occurrence of NR4 2, the two R4 groups in combination are -(CH2)a- or -(CH2)bE(CH2)2-; each R5 is independently Ar2 or 1 ,4-benzoquinon-2-yl optionally substituted with O, 1, 2, or 3 alkyl groups; each R6 is independently hydrogen; (Ci-Ce)alkyl; -(C2-Ce)alkylene-OR3; -(Ci-C6)alkylene-C(=O)OR3; -(Ci-C6)alkylene-OC(=O)R3; (C2-C6)alkylene-NR3 2; -(Ci-C6)alkylene-C(=O)NR3 2; -(Ci-C6)alkylene-NR3C(=O)R3;
-(Ci-C6)alkyleneNR3C(=O)NR3 2; -Ar2, or -(d-C3)alkylene-Ar2; or, optionally, within any occurrence of NR6 2, independently of any other occurrence of NR6 2, the two R6 groups in combination are -(CH2)a- or -(CH2)bE(CH2)2-; each Ra is independently hydrogen; (Ci-Ce)alkyl; (C2-Ce)alkenyl; (C2-C6)alkynyl; halogen; -C≡N; -NO2; -C(=0)R3; -C(=0)0R3; -C(=O)NR3 2; -C(=NR3)NR3 2; -OR3; -OC(=O)(d-C6)alkyl; -OC(=O)O(d-C6)alkyl; -OC(=O)NR3 2; -NR3 2; -NR3C(=O)R3; -NR3C(=O)O(d-C6)alkyl; -NR3C(=O)NR3 2; -S(Ci-C6)alkyl; -S(O)(Ci-C6)alkyl; and -SO2(Ci-C6)alkyl; and -SO2NR3 2; each Rb is independently hydrogen; (Ci-Ce)alkyl; (C2-Ce)alkenyl; (C2-C6)alkynyl; halogen; -C≡N; -NO2; -C(=O)R3; -C(=O)OR3; -C(=O)NR3 2; -C(=NR3)NR3 2; -OR3; -OC(=O)(d-C6)alkyl; -OC(=O)O(Ci-C6)alkyl; -OC(=O)NR3 2; -NR3 2; -NR3C(=O)R3; -NR3C(=O)O(d-C6)alkyl; -NR3C(=O)NR3 2; -S(Ci-C6)alkyl; -S(O)(Ci-C6)alkyl; and -SO2(Ci-C6)alkyl; -SO2NR3 2; each Rc is independently hydrogen; (Ci-Ce)alkyl; (C2-Ce)alkenyl; (C2-C6)alkynyl; halogen; -C≡N; -NO2; -C(=O)R3; -C(=O)OR3; -C(=O)NR3 2; -C(=NR3)NR3 2; -OR3; -OC(=O)(d-C6)alkyl; -OC(=O)O(d-C6)alkyl; -OC(=O)NR3 2; -NR3 2; -NR3C(=O)R3; -NR3C(=O)O(d-C6)alkyl; -NR3C(=O)NR3 2; -S(d-C6)alkyl; -S(O)(Ci-C6)alkyl; and -SO2(d-C6)alkyl; -SO2NR3 2; each Rd is independently hydrogen; (Ci-Ce)alkyl; (C2-Ce)alkenyl; (C2-C6)alkynyl; halogen; -C≡N; -NO2; -C(=O)R3; -C(=O)OR3; -C(=O)NR3 2; -C(=NR3)NR3 2; -OR3; -OC(=O)(d-C6)alkyl; -OC(=O)O(d-C6)alkyl; -OC(=O)NR3 2; -NR3 2; -NR3C(=O)R3; -NR3C(=O)O(d-C6)alkyl; -NR3C(=O)NR3 2; -S(d-C6)alkyl; -S(O)(Ci-C6)alkyl; and -SO2(d-C6)alkyl; -SO2NR3 2; each a is independently selected from the group consisting of 4, 5, and 6; each b is independently selected from the group consisting of 2 and 3; each E is independently selected from the group consisting of O, S, NR3; NC(=0)R3; NSO2R3; N(C2-C6)alkylene-OR3; N(d-C6)alkylene-C(=O)OR3; N(Ci-C6)alkylene-OC(=O)R3; N(C2-C6)alkylene-NR3 2;
N(Ci-C6)alkylene-C(=O)NR3 2; N(d-C6)alkylene-NR3C(=O)R3;
N(Ci-C6)alkylene-NR3C(=O)NR3 2; NAr2; N(d-C3)alkylene-Ar2; and NC(=0)Ar2; and each Ar2 is independently selected from the group consisting of unsubstituted aryl, unsubstituted heteroaryl, and aryl or heteroaryl substituted with one or more substitutents independently selected from the group consisting of (Ci-d)alkyl; (C2-C6)alkenyl; (C2-C6)alkynyl; halogen; -C≡N; -NO2; -C(=0)R3; -C(=0)0R3; -C(=O)NR3 2; -C(=NR3)NR3 2; -OR3; -OC(=O)(d-C6)alkyl; -OC(=O)O(d-C6)alkyl; -OC(=O)NR3 2; -NR3 2; -NR3C(=O)R3; -NR3C(=O)O(d-C6)alkyl; -NR3C(=O)NR3 2; -S(Ci-C6)alkyl; -S(O)(d-C6)alkyl; and -SO2(d-C6)alkyl; -SO2NR3 2; and (C i -C3)perfluoroalkyl. In some of the compounds according to Formula I, A is -C(=O)-.
In some of the compounds according to Formula I, A is -SO2-.
In some of the compounds according to Formula I, B is -O-.
In some of the compounds according to Formula I, B is -NR -, for example NH.
In some the compounds according to Formula I, D is -H.
In some of the compounds according to Formula I, D is -Q=NH)-NH2.
In some of the compounds according to Formula I, each of Ra, Rb, Rc, and Rd is hydrogen.
In some of the compounds according to Formula I, Ar1 is unsubstituted or substituted phenyl. In some sub-embodiments thereof, Ar1 is substituted phenyl substituted in at least the 4-position. In some sub-embodiments thereof, Ar1 is monosubstituted phenyl substituted in the 4-position.
In some embodiments of the compounds according to Formula I, Ar1 is unsubstituted or substituted biphenyl-4-yl. In some sub-embodiments thereof, Ar1 is unsubstituted or substituted biphenyl-4-yl which is unsubstituted in at least the phenylene ring thereof (i.e. the ring which has the attachment point of the of the biphenyl group to the remainder of the molecule).
In some embodiments of the compounds according to Formula I, Ar1 is unsubstituted or substituted naphthyl. Sub-embodiments thereof include those wherein the naphthyl is unsubstituted. Other sub-embodiments are those wherein the naphthyl is substituted. Sub- embodiments thereof also include those wherein the naphthyl is a 1 -naphthyl, which may be substituted or unsubstituted, sub-embodiments being those wherein the 1 -naphthyl is substituted. In some sub-embodiments the 1 -naphthyl is monosubstituted.
In embodiments of the compounds according to Formula I, Ar1 is 1 -naphthyl, sub- embodiments include those wherein the 5 -position of the 1 -naphthyl is substituted by a substituent selected from the group consisting of -OR2; -OC(=O)(Ci-C6)alkyl; -OC(=O)(Ci-C6)alkylene-R5; -OC(=O)O(Ci-C6)alkyl; -OC(=O)NR4 2; -NR4 2; -NR4C(=O)R3; -NR4C(=O)Ar2; -NR4C(=O)O(Ci-C6)alkyl; -NR4C(=O)NR4 2; -NR4SO2R3; -NR4SO2Ar2; -SR2; -OSO2(C i-C6)alkyl; and -OSO2Ar2, for example, a substituent selected from the group consisting of -OR2 and -NR4 2, for example, -OH, O(Ci-C6)alkyl, -NH2, -NH(Ci-C6)alkyl, and -N((Ci-Ce)alkyl)2, for example, -N(CHs)2. Sub-embodiments thereof are those wherein the 1- naphthyl is monosubstituted, i.e. having one substituent with other positions of the 1-naphthyl substituted by hydrogen.
Embodiments of the compounds according to Formula I include those wherein Ar1 is 5-dimethylamino- 1 -naphthyl.
Embodiments of the compounds according to Formula I include those wherein A is -C(=O)-, B is -O-, D is -C(=NH)-NH2, and each of Ra, Rb, Rc and Rd is hydrogen.
Embodiments of the compounds according to Formula I include those wherein A is -C(=O)-, B is -NH-, D is -C(=NH)-NH2, and each of Ra, Rb, Rc and Rd is hydrogen.
Embodiments of the compounds according to Formula I include those wherein A is -C(=O)-, B is -O-, D is -C(=NH)-NH2, each of Ra, Rb, Rc and Rd is hydrogen and Ar1 is phenyl, for example 4-substituted phenyl, for example 4-monosubstituted phenyl. Examples thereof include:
4-(2-guanidinothiazol-4-yl)phenyl 4-t-butylbenzoate;
4-(2-guanidinothiazol-4-yl)phenyl 4-n-butylbenzoate;
4-(2-guanidinothiazol-4-yl)phenyl 4-cyclopropylbenzoate; and salt forms thereof.
Embodiments of the compounds according to Formula I include those wherein A is -C(=O)-, B is -O-, D is -C(=NH)-NH2, each of Ra, Rb, Rc and Rd is hydrogen, and Ar1 is biphenyl, for example biphenyl-4-yl. Examples thereof include:
4-(2-guanidinothiazol-4-yl)phenyl biphenyl-4-carboxylate;
4-(2-guanidinothiazol-4-yl)phenyl 3'-methoxybiphenyl-4-carboxylate;
4-(2-guanidinothiazol-4-yl)phenyl 4'-methoxybiphenyl-4-carboxylate; and salt forms thereof.
Embodiments of the compounds according to Formula I include those wherein A is -C(=O)-, B is -O-, D is -C(=NH)-NH2, each of Ra, Rb, Rc and Rd is hydrogen, and Ar1 is naphthyl. Examples thereof include:
4-(2-guanidinothiazol-4-yl)phenyl 1 -naphthoate;
4-(2-guanidinothiazol-4-yl)phenyl 2-naphthoate; and salt forms of any thereof.
Embodiments of the compounds according to Formula I include those wherein A is -C(=O)-, B is -NH-, D is -C(=NH)-NH2, each of Ra, Rb, Rc and Rd is hydrogen, and Ar1 is phenyl, for example 4-substituted phenyl, for example 4-monosubstituted phenyl. Examples thereof include:
4-n-butyl-Λ/-(4-(2-guanidinothiazol-4-yl)phenyl)benzamide;
4-£-butyl-Λ/-(4-(2-guanidinothiazol-4-yl)phenyl)benzamide; and salt forms of any thereof.
Embodiments of the compounds according to Formula I include those wherein A is -C(=O)-, B is -NH-, D is -CC=NH)-NH2, each of Ra, Rb, Rc and Rd is hydrogen, and Ar1 is biphenyl, for example, biphenyl-4-yl. Examples thereof include:
Λ/-(4-(2-guanidinothiazol-4-yl)phenyl)biphenyl-4-carboxamide; and salt forms thereof.
Embodiments of the compounds according to Formula I include those wherein A is -C(=O)-, B is -NH-, D is -C(=NH)-NH2, each of Ra, Rb, Rc and Rd is hydrogen, and Ar1 is naphthyl. Examples thereof include:
Λ/-(4-(2-guanidinothiazol-4-yl)phenyl)- 1 -naphthamide;
Λ/-(4-(2-guanidinothiazol-4-yl)phenyl)-2-naphthamide; and salt forms thereof.
Embodiments of the compounds according to Formula I include those wherein A is -SO2-, B is -O-, D is -C(=NH)-NH2, each of Ra, Rb, Rc and Rd is hydrogen, and Ar1 is phenyl, for example 4-substituted phenyl, for example 4-monosubstituted phenyl. Examples thereof include:
4-(2-guanidinothiazol-4-yl)phenyl 4-(trifluoromethyl)benzenesulfonate;
4-(2-guanidinothiazol-4-yl)phenyl 4-methylbenzenesulfonate;
4-(2-guanidinothiazol-4-yl)phenyl 4-t-butylbenzenesulfonate; and salt forms thereof.
Embodiments of the compounds according to Formula I include those wherein A is -SO2-, B is -O-, D is -C(=NH)-NH2, each of Ra, Rb, Rc and Rd is hydrogen, and Ar1 is biphenyl-4-yl. Examples thereof include:
4-(2-guanidinothiazol-4-yl)phenyl biphenyl-4-sulfonate; and salt forms thereof.
Embodiments of the compounds according to Formula I include those wherein A is -SO2-, B is -O-, D is -C(=NH)-NH2, each of Ra, Rb, Rc and Rd is hydrogen, and Ar1 is naphthyl. Examples thereof include: 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene- 1 -sulfonate; and salt forms thereof.
Embodiments of the compounds according to Formula I include those wherein D is hydrogen and each of Ra, Rb, Rc and Rd is hydrogen. Examples thereof include:
4-(2-aminothiazol-4-yl)phenyl biphenyl-4-carboxylate;
4-(2-aminothiazol-4-yl)phenyl 4-t-butylbenzoate;
4-£-butyl-Λ/-(4-(2-aminothiazol-4-yl)phenyl)benzamide;
4-(2-aminothiazol-4-yl)phenyl 4-n-butylbenzoate;
4-/?-butyl-Λ/-(4-(2-aminothiazol-4-yl)phenyl)benzamide;
4-(2-aminothiazol-4-yl)phenyl 4-cyclopropylbenzoate;
(4-(2-aminothiazol-4-yl)phenyl)(phenyl)methanone;
Λ/-(4-(2-aminothiazol-4-yl)phenyl)- 1 -naphthamide;
Λ/-(4-(2-aminothiazol-4-yl)phenyl)-2-naphthamide;
4-(2-aminothiazol-4-yl)phenyl 1 -naphthoate;
4-(2-aminothiazol-4-yl)phenyl 2-naphthoate; and salt forms thereof.
It is to be understood that other embodiments of the compounds according to formula I will combine the features explicitly described above, and that embodiments of the compounds according to formula I combining such features are therefore contemplated even if the specific combinations are not expressly set forth.
III. Salt Forms
The compounds according to formula I, any of the embodiments thereof, as well as intermediates used in making compounds according to formula I may take the form of salts. The term "salts" embraces addition salts of free acids or free bases which are compounds described herein. The term "pharmaceutically-acceptable salt" refers to salts which possess toxicity profiles within a range that affords utility in pharmaceutical applications. Pharmaceutically unacceptable salts may nonetheless possess properties such as high crystallinity, which may render them useful, for example, in processes of synthesis, purification or formulation of compounds described herein. In general the useful properties of the compounds described herein do not depend on whether the compound is or is not in a salt form, so unless clearly indicated otherwise (such as specifying that the compound should be in "free base" or "free acid" form), reference in the specification to compounds of formula I should be understood as including salt forms of the compound, whether or not this is explicitly stated.
Suitable pharmaceutically-acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include hydrochloric, hydrobromic, hydriodic, nitric, carbonic, sulfuric, and phosphoric acids. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, alginic, β-hydroxybutyric, salicylic, galactaric and galacturonic acid. Examples of pharmaceutically unacceptable acid addition salts include, for example, perchlorates and tetrafluoroborates.
Suitable pharmaceutically acceptable base addition salts include, for example, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, Λ^-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (7V-methylglucamine) and procaine. Examples of pharmaceutically unacceptable base addition salts include lithium salts and cyanate salts.
All of these salts may be prepared by conventional means from the corresponding compound according to formula I by reacting, for example, the appropriate acid or base with the compound according to formula I. Optionally, the salts are in crystalline form, which may be prepared by crystallization of the salt from a suitable solvent. Preparaition and selection of suitable salt forms is described in Handbook of Pharmaceutical Salts: Properties, Selection, and Use By P. H. Stahl and C. G. Wermuth (Wiley- VCH 2002).
IV. Solvate Forms
The compounds according to formula I, and salts thereof as well as intermediates used in making compounds according to formula I, and salts thereof may take the form of solvates, including hydrates. In general, the useful properties of the compounds described herein do not depend on whether the compound or salt thereof is or is not in the form of a solvate, so unless clearly indicated otherwise reference in the specification to compounds of formula I should be understood as encompassing solvate forms of the compound, whether or not this is explicitly stated.
V. Prodrugs
The compounds according to formula I, and salts thereof as well as intermediates used in making compounds according to formula I, and salts thereof, may be administered in the form of prodrugs. By "prodrug" is meant, for example, any compound (whether itself active or inactive) that is converted chemically in vivo into a biologically active compound of the formula I following administration of the prodrug to a subject.
Generally a "prodrug" is a covalently bonded carrier which releases the active parent drug when administered to a subject. Prodrugs can be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compounds. Prodrugs include compounds wherein hydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxyl, amino, sulfhydryl, or carboxyl group respectively. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups in the compounds according to formula I.
The suitability and techniques involved in making and using prodrugs are discussed in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of the ACS Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are hereby incorporated by reference in their entirety.
VI. Stereochemistry, Tautomerism, and Conformational Isomerism
The compounds provided for by formula I may encompass various stereochemical forms and tautomers. The formula also encompasses diastereomers as well as optical isomers, e.g. mixtures of enantiomers including racemic mixtures, as well as individual enantiomers and diastereomers, which arise as a consequence of structural asymmetry in certain compounds of formula I. Separation of the individual isomers or selective synthesis of the individual isomers is accomplished by application of various methods which are well known to practitioners in the art. A. Geometrical Isomerism
Certain compounds of formula I possess an olefϊnic double bond. The stereochemistry of compounds possessing an olefϊnic double bond is designated using the nomenclature using E and Z designations. The compounds are named according to the Cahn-Ingold-Prelog system, described in the IUPAC 1974 Recommendations, Section E: Stereochemistry, in Nomenclature of Organic Chemistry, John Wiley & Sons, Inc., New York, NY, 4th ed., 1992, pp. 127-38, the entire content of which is incorporated herein by reference.
B. Optical Isomerism
Certain compounds of formula I may contain one or more chiral centers, and may exist in, and may be isolated as pure enantiomeric or diastereomeric forms or as racemic mixtures. Formula I therefore encompasses any possible enantiomers, diastereomers, racemates or mixtures thereof which are biologically active in the inhibiting myosin light chain phosphatase.
The isomers resulting from the presence of a chiral center comprise a pair of non-superimposable isomers that are called "enantiomers." Single enantiomers of a pure compound are optically active, i.e., they are capable of rotating the plane of plane polarized light. Single enantiomers are designated according to the Cahn-Ingold-Prelog system.
Formula I encompasses diastereomers as well as their racemic and resolved, diastereomerically and enantiomerically pure forms and salts thereof. Diastereomeric pairs may be resolved by known separation techniques including normal and reverse phase chromatography, and crystallization.
"Isolated optical isomer" means a compound which has been substantially purified from the corresponding optical isomer(s) of the same formula. Preferably, the isolated isomer is at least about 80%, more preferably at least 90% pure, even more preferably at least 98% pure, most preferably at least about 99% pure, by weight.
Isolated optical isomers may be purified from racemic mixtures by well-known chiral separation techniques. According to one such method, a racemic mixture of a compound having the structure of formula I, or a chiral intermediate thereof, is separated into 99% wt.% pure optical isomers by HPLC using a suitable chiral column, such as a member of the series of DAICEL® CHIRALP AK® family of columns (Daicel Chemical Industries, Ltd., Tokyo, Japan). The column is operated according to the manufacturer's instructions. C. Conformational Isomerism
Due to chemical properties such as resonance lending some double bond character to a C-N bond, it is possible that individual conformers of certain compounds of formula I may be observable and even separable under certain circumstances. Formula I therefore includes any possible stable rotamers of formula I which are biologically active in inhibiting myosin light chain phosphatase.
D. Tautomerism
Certain compounds may exist in tautomeric forms, which differ by the location of a hydrogen atom and typically are in rapid equilibrium. In such circumstances, molecular formulae drawn will typically only represent one of the possible tautomers even though equilibration of these tautomeric forms will occur in equilibrium in the compound. Examples include keto-enol tautomerism and amide-imidic acid tautomerism. Tautomerism is frequently also seen in heterocyclic compounds. All tautomeric forms of the compounds according to formula I are to be understood as being included within the scope of the formula.
VII. Pharmaceutical Compositions
The compounds of formula I may be administered in the form of a pharmaceutical composition, in combination with a pharmaceutically acceptable carrier. The active ingredient in such formulations may comprise from 0.1 to 99.99 weight percent. "Pharmaceutically acceptable carrier" means any carrier, diluent or excipient which is compatible with the other ingredients of the formulation and not deleterious to the recipient.
The active agent may be administered with a pharmaceutically acceptable carrier selected on the basis of the selected route of administration and standard pharmaceutical practice. The active agent may be formulated into dosage forms according to standard practices in the field of pharmaceutical preparations. See Alphonso Gennaro, ed., Remington: The Science and Practice of Pharmacy, (20th Edition, Mack Publishing Co., Easton, PA, 2003). Suitable dosage forms may comprise, for example, tablets, capsules, solutions, parenteral solutions, troches, suppositories, or suspensions.
For parenteral administration, the active agent may be mixed with a suitable carrier or diluent such as water, an oil (particularly a vegetable oil), ethanol, saline solution, aqueous dextrose (glucose) and related sugar solutions, glycerol, or a glycol such as propylene glycol or polyethylene glycol. Solutions for parenteral administration preferably contain a water soluble salt of the active agent. Stabilizing agents, antioxidant agents and preservatives may also be added. Suitable antioxidant agents include sulfite, ascorbic acid, citric acid and its salts, and sodium EDTA. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and chlorbutanol. The composition for parenteral administration may take the form of an aqueous or non-aqueous solution, dispersion, suspension or emulsion.
For oral administration, the active agent may be combined with one or more solid inactive ingredients for the preparation of tablets, capsules, pills, powders, granules or other suitable oral dosage forms. For example, the active agent may be combined with at least one excipient such as fillers, binders, humectants, disintegrating agents, solution retarders, absorption accelerators, wetting agents absorbents or lubricating agents. The active agent may be combined with carboxymethylcellulose calcium, magnesium stearate, mannitol and starch, and then formed into tablets by conventional tableting methods.
The specific dose of a compound according to formula I required to obtain therapeutic benefit in the methods of treatment described herein will, of course, be determined by the particular circumstances of the individual patient including the size, weight, age and sex of the patient, the nature and stage of the disease being treated, the aggressiveness of the disease disorder, and the route of administration of the compound.
For example, a daily dosage from about 0.05 to about 50 mg/kg/day may be utilized, for example, a dosage from about 0.1 to about 10 mg/kg/day. Higher or lower doses are also contemplated as it may be necessary to use dosages outside these ranges in some cases. The daily dosage may be divided, such as being divided equally into two to four times per day daily dosing. The compositions may be formulated in a unit dosage form, each dosage containing from about 1 to about 500mg, more typically, about 10 to about lOOmg of active agent per unit dosage. The term "unit dosage form" refers to physically discrete units suitable as a unitary dosage for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
The pharmaceutical compositions described herein may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydropropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes and/or microspheres.
In general, a controlled-release preparation is a pharmaceutical composition capable of releasing the active ingredient at the required rate to maintain constant pharmacological activity for a desirable period of time. Such dosage forms provide a supply of a drug to the body during a predetermined period of time and thus maintain drug levels in the therapeutic range for longer periods of time than conventional non-controlled formulations.
U.S. Patent No. 5,674,533 discloses controlled-release pharmaceutical compositions in liquid dosage forms for the administration of moguisteine, a potent peripheral antitussive. U.S. Patent No. 5,059,595 describes the controlled-release of active agents by the use of a gastro-resistant tablet for the therapy of organic mental disturbances. U.S. Patent No. 5,591,767 describes a liquid reservoir transdermal patch for the controlled administration of ketorolac, a non-steroidal anti-inflammatory agent with potent analgesic properties. U.S. Patent No. 5,120,548 discloses a controlled-release drug delivery device comprised of swellable polymers. U.S. Patent No. 5,073,543 describes controlled-release formulations containing a trophic factor entrapped by a ganglioside-liposome vehicle. U.S. Patent No. 5,639,476 discloses a stable solid controlled-release formulation having a coating derived from an aqueous dispersion of a hydrophobic acrylic polymer. Biodegradable microparticles are known for use in controlled-release formulations. U.S. Patent No. 5,354,566 discloses a controlled-release powder that contains the active ingredient. U.S. Patent No. 5,733,566 describes the use of polymeric microparticles that release antiparasitic compositions.
The controlled-release of the active ingredient may be stimulated by various inducers, for example pH, temperature, enzymes, water, or other physiological conditions or compounds. Various mechanisms of drug release exist. For example, the controlled-release component may swell and form porous openings large enough to release the active ingredient after administration to a patient. The term "controlled-release component" means a compound or compounds, such as polymers, polymer matrices, gels, permeable membranes, liposomes and/or microspheres that facilitate the controlled-release of the active ingredient in the pharmaceutical composition. Optionally, the controlled-release component is biodegradable, induced by exposure to the aqueous environment, pH, temperature, or enzymes in the body. Furthermore, sol-gels may be used, wherein the active ingredient is incorporated into a sol-gel matrix that is a solid at room temperature. This matrix is implanted into a patient, preferably a mammal, having a body temperature high enough to induce gel formation of the sol-gel matrix, thereby releasing the active ingredient into the patient.
The components used to formulate the pharmaceutical compositions are of high purity and are substantially free of potentially harmful contaminants (e.g., at least National Food grade, generally at least analytical grade, and more typically at least pharmaceutical grade). Particularly for human consumption, the composition is preferably manufactured or formulated under Good Manufacturing Practice standards as defined in the applicable regulations of the U.S. Food and Drug Administration. For example, suitable formulations may be sterile and/or substantially isotonic and/or in full compliance with all Good Manufacturing Practice regulations of the U.S. Food and Drug Administration.
VIII. Methods of Synthesis and Useful Intermediates
Processes for preparing compounds according to formula I, intermediates that are useful in the preparation of such compounds, and processes for preparing such intermediates are also provided herein.
In the text, formulae and schemes that follow, unless otherwise indicated Ar1, A, B, D, Ra, Rb, Rc, and Rd are as defined above for formula I.
Compounds of formula I may be prepared from compounds of formula II, wherein L is a leaving group, by reaction with a compound of formula III, as illustrated in Scheme 1.
Figure imgf000029_0001
II III
Scheme 1
Suitable leaving groups L include: halogen, OSO2AIlCyI, OSO2Aryl, and OSO2CF3. Preferred is halogen. A procedure involves treatment of a compound of formula II with a compound of formula III at about 0-120 0C in a suitable solvent. The preferred solvents are those which will promote the nucleophilic displacement and cyclization reactions and will not engage in competing displacement reactions. Suitable solvents include polar solvents, including protic solvents such as alcohols, for example ethanol, and aprotic solvents such as ketones, for example acetone. Other suitable solvents for the reaction include amide solvents such as N,N-dimethylformamide and N-methylpyrrolidone, dimethylsulfoxide, ether solvents such as tetrahydrofuran, and halogenated hydrocarbons such as chloroform. The preferred solvent is ethanol or acetone. In order to facilitate the cyclization reaction, the presence of an acid or base catalyst may be beneficial. In addition, it may also be beneficial to perform the reaction under conditions in which water formed as a result of the cyclization is removed, for example by including a dehydrating agent in the reaction mixture, or removing water by azeotropic distillation. The reaction is preferably performed at elevated temperature, for example the from about 20 0C, 30 0C, or 50 0C to about 100 0C or 120 0C, for example at about the reflux temperature of the solvent.
Compounds of formula II wherein L is halogen (Hal) may be prepared from compounds of formula IV, by reaction with a halogenating agent, as illustrated in Scheme 2.
Figure imgf000030_0001
IV II (L=halogen)
Scheme 2
Suitable halogenating agents include elemental halogens and N-halogen compounds, for example N-haloimides, for example N-halosuccinimides. The preferred halogen is bromine, and the preferred halogenating agents are elemental bromine, and N- bromosuccinimide. In order to limit the halogenation to monohalogenation, the reaction can be performed under neutral or acidic conditions. The reaction may be carried out, for example, at a temperature in the range from about -20 0C to about 80 0C, about -10 0C to about 35 0C, or about 0 0C to about 25 0C. Suitable solvents for conducting the reaction include halogenated solvents, for example chloroform or dichloromethane, carboxylic acids, for example acetic acid. It may be advantageous for the halogenation to be performed upon a derivative of the ketone such as an enolate, enol ether, enol silane, or enol ester.
Compounds of formula II wherein L is OSO2Alkyl, OSO2Aryl, and OSO2CF3 may be prepared from a compound of formula II wherein L is OH by reaction with a suitable sulfonyl chloride. Compounds of formula II wherein L is OH may be prepared from compounds of formula IV by oxidation.
Compounds of formula IV may be prepared by an acylation or sulfonylation reaction between a compound of formula V, wherein X is a suitable leaving group with a phenolic or aminoaromatic compound according to formula VI as illustrated in Scheme 3.
Figure imgf000031_0001
V VI IV
Scheme 3
When A is -SO2-, the reaction is a sulfonylation reaction and the leaving group X is preferably halogen, preferably chlorine. The coupling between a sulfonamide and an aromatic amine or phenol is typically performed using a basic catalyst or reagent in a suitable solvent at a suitable temperature. Suitable bases include tertiary amines such as triethylamine or JV,jV-diisopropylethylamine, or pyridine. Typically, at least one equivalent of base would be used because hydrogen chloride is formed in the reaction. Suitable solvents include pyridine or dichloromethane. The reactions are typically carried out at a temperature between 0 0C and the reflux temperature of the solvent, which is typically about 100 0C. The reaction is preferably carried out at about between 0 0C and about 10 0C. For example, in a typical procedure, the reactions would be conducted by adding the sulfonyl chloride to a solution containing the aromatic amine or phenol and triethylamine in dichloromethane at about 10 0C.
When A is a carbonyl group (CO), the reaction is an acylation reaction and the leaving group X can include: OH, halogen, OAlkyl, OAryl, OC(=O)Alkyl, OC(=O)Aryl. A suitable acylation procedure involves treatment of a compound of formula V (wherein A=CO) with a compound of formula VI at about 0-120 0C in a suitable solvent. The presence of a base, or, when X=OH, a coupling agent, may also be necessary for the reaction to occur. Suitable bases for the reaction include: 4-(Λ/,Λ/-dimethylamino)pyridine, pyridine, triethylamine, Λ/,jV-diisopropylethylamine. The preferred base is dimethylaminopyridine. Suitable coupling agents when X=OH include: carbodiimides, for example 1,3- dicyclohexylcarbodiimide or l-(3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride; phosphonium reagents, for example benzotriazol-l-yloxytris(dimethylamino)phosphonium hexafluorophosphate or benzotriazol- 1 -yloxytripyrrolidinophosphonium hexafluorophosphate; and uronium reagents, for example O-benzotriazol-l-yl-ΛWΛf'.jV'- tetramethyluronium tetrafluoroborate. The preferred coupling agents are carbodiimides, for example 1,3-dicyclohexylcarbodiimide. Suitable solvents for the reaction include amide solvents such as Λ/,N-dimethylformamide, dimethylsulfoxide, ether solvents such as tetrahydrofuran, or halogenated hydrocarbons such as chloroform. The preferred solvent is Λ/,Λ/-dimethylformamide. The reaction is preferably performed at a temperature of 0-50 0C, and most preferably at a temperature of 20-30 0C.
The compounds according to formula III are either commercially available, known in the art or may be prepared by a variety of methods which are known to the person skilled in the art. Thiurea (D=H) and amidinothiourea (D=C(=NH)NH2, 2-imino-4-thiobiuret) are both commercially available from Aldrich Chemical Company.
The compounds according to formula V are likewise either commercially available, known in the art or may be prepared by a variety of methods which are known to the person skilled in the art. For example, sulfonyl chlorides may be prepared by the chlorosulfonation of aromatic compounds, or by the chlorination of various aromatic derivatives (e.g. Johnson, Proc. Natl. Acad. ScL USA, 1939, 25(9): 448-452). For a discussion of the synthesis of sulfonyl chlorides, see, for example G. Hilgetag and A. Martini, Preparative Organic Chemistry (J. Wiley and Sons, 1972) p.670, U.S. Patents 5,387,681, and 6,140,505 and the references cited therein. Similarly, methods of forming aromatic carboxylic acids and carbonyl derivatives are known in the art. For example, suitable methods include reaction of organometallic compounds with carbon dioxide, oxidation of formyl derivatives (which are available through reaction of organometallic compounds with formamides, or electrophilic formylation, e.g. Vilsmeier-Haack, Gattermann-Koch, Gattermann formylation), or organometallically-catalysed formylation reaction, oxidation of aromatic alkyl groups, and hydrolysis of aromatic nitriles (which may be formed, for example, through nucleophilic substitution reactions with nitrile or the reaction of diazonium salts with copper (I) cyanide).
Similarly, the compounds according to formula VI are either commercially available, known in the art or may be prepared by methods known to the person skilled in the art. For example, the methyl ketone group may be introduced via Friedel Crafts acylation of the aromatic ring starting from an arene compound of formula VII as shown in Scheme 4. The reaction is typically formed in the by reacting with acetyl chloride in the presence of a Lewis acid catalyst, for example aluminium trichloride. Typically, the aromatic amine or phenolic group would be protected with a protecting group (PG) for such a reaction (for example an amine protected as an amide, e.g. acetamido, and a phenol protected as alkoxy). The ortho- para directing effect of the oxygen or nitrogen-containing group represented by B renders the selective formation of the compound with the desired regiochemistry possible.
Figure imgf000033_0001
Scheme 4
The synthesis scheme described above represents a convergent strategy whereby the compound according to formula I is constructed by the assembly of simpler compounds (e.g. the compounds of formula III, V, and VII as starting materials) which may be regarded as "building blocks" for the synthesis. By reference to the "building block" analogy, it will be understood, and appreciated by the skilled artisan, that variations of the synthesis scheme described above are feasible. In particular, it will be appreciated that the order of the steps may be varied (e.g. the reaction to form the A-B bond might in some cases be performed after formation of the thiazole ring), and that other variations of the synthesis scheme described are feasible.
The above-described reactions, unless otherwise noted, are usually conducted at a pressure of about one to about three atmospheres, preferably at ambient pressure (about one atmosphere).
The description of the compounds according to formula I and salt forms thereof is intended to include isolated compounds. The expression "isolated compound" refers to a preparation of a compound of formula I, or a mixture of compounds according to formula I, wherein the isolated compound has been separated from the reagents used, and/or byproducts formed, in the synthesis of the compound or compounds. "Isolated" does not mean that the preparation is technically pure (homogeneous), but it is sufficiently pure to compound in a form in which it can be used therapeutically. Preferably an "isolated compound" refers to a preparation of a compound of formula I or a mixture of compounds according to formula I, which contains the named compound or mixture of compounds according to formula I in an amount of at least 10 percent by weight of the total weight. Preferably the preparation contains the named compound or mixture of compounds in an amount of at least 50 percent by weight of the total weight; more preferably at least 80 percent by weight of the total weight; and most preferably at least 90 percent, at least 95 percent or at least 98 percent by weight of the total weight of the preparation.
The compounds according to formula I and intermediates may be isolated from their reaction mixtures and purified by standard techniques such as filtration, liquid-liquid extraction, solid phase extraction, distillation, recrystallization or chromatography, including flash column chromatography, or HPLC. The preferred method for purification of the compounds according to formula I or salts thereof comprises crystallizing the compound or salt from a solvent to form, preferably, a crystalline form of the compounds or salts thereof. Following crystallization, the crystallization solvent is removed by a process other than evaporation, for example filtration or decanting, and the crystals are then preferably washed using pure solvent (or a mixture of pure solvents). Preferred solvents for crystallization include water, alcohols, particularly alcohols containing up to four carbon atoms such as methanol, ethanol, isopropanol, and butan-1-ol, butan-2-ol, and 2-methyl-2-propanol, ethers, for example diethyl ether, diisopropyl ether, t-butyl methyl ether, 1,2-dimethoxyethane, tetrahydrofuran and 1,4-dioxane, carboxylic acids, for example formic acid and acetic acid, and hydrocarbon solvents, for example pentane, hexane, toluene, and mixtures thereof, particularly aqueous mixtures such as aqueous ethanol. Pure solvents, preferably at least analytical grade, and more preferably pharmaceutical grade are preferably used. In a preferred embodiment of the processes of the invention, the products are so isolated. In the compounds according to formula I or salt thereof, and pharmaceutical compositions thereof, the compound according to formula I or salt thereof is preferably in or prepared from a crystalline form, preferably prepared according to such a process.
It will be appreciated by one skilled in the art that certain aromatic substituents in the compounds of the invention, intermediates used in the processes described above, or precursors thereto, may be introduced by employing aromatic substitution reactions to introduce or replace a substituent, or by using functional group transformations to modify an existing substituent, or a combination thereof. Such reactions may be effected either prior to or immediately following the processes mentioned above. The reagents and reaction conditions for such procedures are known in the art. Specific examples of procedures which may be employed include, but are not limited to, electrophilic functionalization of an aromatic ring, for example via nitration, halogenation, or acylation; transformation of a nitro group to an amino group, for example via reduction, such as by catalytic hydrogenation; acylation, alkylation, or sulfonylation of an amino or hydroxyl group; replacement of an amino group by another functional group via conversion to an intermediate diazonium salt followed by nucleophilic or free radical substitution of the diazonium salt; or replacement of a halogen by another group, for example via nucleophilic or organometallically-catalyzed substitution reactions.
Additionally, in the aforesaid processes, certain functional groups which would be sensitive to the reaction conditions may be protected by protecting groups. A protecting group is a derivative of a chemical functional group which would otherwise be incompatible with the conditions required to perform a particular reaction which, after the reaction has been carried out, can be removed to re-generate the original functional group, which is thereby considered to have been "protected". Any chemical functionality that is a structural component of any of the reagents used to synthesize compounds of this invention may be optionally protected with a chemical protecting group if such a protecting group is useful in the synthesis of compounds of this invention. The person skilled in the art knows when protecting groups are indicated, how to select such groups, and processes that can be used for selectively introducing and selectively removing them, because methods of selecting and using protecting groups have been extensively documented in the chemical literature. Techniques for selecting, incorporating and removing chemical protecting groups may be found, for example, in Protective Groups in Organic Synthesis by Theodora W. Greene, Peter G. M. Wuts, John Wiley & Sons Ltd., the entire disclosure of which is incorporated herein by reference.
In addition to use of a protecting group, sensitive functional groups may be introduced as synthetic precursors to the functional group desired in the intermediate or final product. An example of this is an aromatic nitro (-NO2) group. The aromatic nitro group goes not undergo any of the nucleophilic reactions of an aromatic amino group. However, the nitro group can serve as the equivalent of a protected amino group because it is readily reduced to the amino group under mild conditions that are selective for the nitro group over most other functional groups.
It will be appreciated by one skilled in the art that the processes described are not the exclusive means by which compounds of the invention may be synthesized and that an extremely broad repertoire of synthetic organic reactions is available to be potentially employed in synthesizing compounds of the invention. The person skilled in the art knows how to select and implement appropriate synthetic routes. Suitable synthetic methods may be identified by reference to the literature, including reference sources such as Comprehensive Organic Synthesis, Ed. B. M. Trost and I. Fleming (Pergamon Press, 1991), Comprehensive Organic Functional Group Transformations, Ed. A. R. Katritzky, O. Meth-Cohn, and C. W. Rees (Pergamon Press, 1996), Comprehensive Organic Functional Group Transformations II, Ed. A. R. Katritzky and R. J. K. Taylor (Editor) (Elsevier, 2nd Edition, 2004), Comprehensive Heterocyclic Chemistry, Ed. A. R. Katritzky and C. W. Rees (Pergamon Press, 1984), and Comprehensive Heterocyclic Chemistry II, Ed. A. R. Katritzky, C. W. Rees, and E. F. V. Scriven (Pergamon Press, 1996).
IX. Methods of Using Myosin Light Chain Phosphatase Inhibitors and Compounds According to Formula I
Described herein are methods of using the novel compounds according to formula I, and its variants, including fluorescent compounds, according to formula I.
Compounds according to formula I are therapeutically useful. There are therefore provided uses of the compounds according to formula I in therapy and diagnostics, and therapeutic and diagnostic methods comprising administering a compound according to formula I, or a pharmaceutically acceptable salt form thereof, to an individual.
Compounds according to formula I are effective as myosin light chain phosphatase inhibitors. Therefore, also provided is a method of inhibiting a myosin light chain phosphatase, comprising contacting an effective amount of a compound according to formula I, or a salt form thereof, with a myosin light chain phosphatase. The method of inhibiting a myosin light chain phosphatase may be performed by contacting the myosin light chain phosphatase with a compound according to formula I, or a salt form thereof, in vitro, thereby inhibiting myosin light chain phosphatase in vitro. The contacting may be performed in the presence of cells, wherein, optionally, the myosin light chain phosphatase is present within the cells, or alternatively may be performed in a cell free medium. Uses of such an in vitro method of inhibiting a myosin light chain phosphatase include, but are not limited to use in a screening assay (for example, wherein the compound according to formula I is used as a positive control or standard compared to compounds of unknown activity or potency in inhibiting myosin light chain phosphatase). The method of inhibiting a myosin light chain phosphatase may be performed by contacting the myosin light chain phosphatase with a compound according to formula I, or a salt form thereof, in vivo, thereby inhibiting the myosin light chain phosphatase in vivo. The contacting is achieved by causing the compound according to formula I, or a salt form thereof, to be present in the individual in an effective amount to achieve inhibition of the myosin light chain phosphatase. This may be achieved, for example, by administering an effective amount of the compound according to formula I, or a pharmaceutically acceptable salt form thereof, to the individual, or by administering a prodrug of the compound according to formula I, or a pharmaceutically acceptable salt form thereof. Uses of such an in vivo method of inhibiting a myosin light chain phosphatase include, but are not limited to use in methods of treating a disease or condition, wherein inhibiting myosin light chain phosphatase is beneficial, or treating or preventing diseases, wherein myosin light chain phosphatase activity, for example aberrant myosin light chain phosphatase activity, or a deficient level of phosphorylated myosin light chain contributes to the pathology and/or symptomology of the disease, as described in greater detail below.
In another aspect, there is provided a method of inhibiting protein phosphatase 1C comprising contacting an effective amount of a compound according to formula I, or a salt form thereof, with protein phosphatase 1C. The method may be performed by contacting protein phosphatase 1C with the compound according to formula I, or a salt form thereof, in vitro or in vivo. The in vitro method may be performed in the presence of cells, wherein, optionally, the protein phosphatase 1C is present within the cells, or alternatively may be performed in a cell free medium. Uses of such an in vitro method include, but are not limited to use in a screening assay (for example, wherein the compound according to formula I is used as a positive control or standard compared to compounds of unknown activity or potency in inhibiting protein phosphatase 1C). The in vivo method may be performed by causing the compound according to formula I, or a salt form thereof, to be present in the individual in an effective amount to achieve inhibition of the protein phosphatase 1C, for example, by administering an effective amount of the compound according to formula I, or a pharmaceutically acceptable salt form thereof, to the individual, or administering a prodrug of the compound according to formula I, or a pharmaceutically acceptable salt form thereof. Uses of such an in vivo method of inhibiting a protein phosphatase 1C include use in methods of treating a disease or condition, wherein inhibiting protein phosphatase 1C is beneficial, or treating or preventing diseases, wherein protein phosphatase 1C activity contributes to the pathology and/or symptomology of the disease.
As a consequence of the inhibition of myosin light chain phosphatase activity, the compounds according to formula I are useful for increasing the level of phosphorylation of myosin light chain in a cell (or inhibiting the dephosphorylation of myosin light chain). Accordingly, there is also provide a method of increasing the level of phosphorylation of myosin light chain in a cell comprising contacting an effective amount of a compound according to formula I, or a salt form thereof, with the cell. The method may be performed by contacting the cell with a compound according to formula I, or a salt form thereof, in vitro, thereby increasing the amount of myosin light chain phosphorylation in vitro. Uses of such an in vitro method of increasing the amount of myosin light chain phosphorylation include, but are not limited to use in a screening assay (for example, wherein the compound according to formula I is used as a positive control or standard compared to compounds of unknown activity or potency in increasing myosin light chain phosphorylation).
The method of increasing the amount of myosin light chain phosphorylation may also be performed by contacting the cell with a compound according to formula I, or a salt form thereof, in vivo, thereby increasing the amount of myosin light chain phosphorylation in vivo. The contacting is achieved by causing the compound according to formula I, or a salt form thereof, to be present in the individual in an effective amount to achieve an increase in the amount of myosin light chain phosphorylation. This may be achieved, for example, by administering an effective amount of the compound according to formula I, or a pharmaceutically acceptable salt form thereof, to the individual, or by administering a prodrug of the compound according to formula I, or a pharmaceutically acceptable salt form thereof. Uses of such an in vivo method of increasing the amount of myosin light chain phosphorylation include, but are not limited to use in methods of treating a disease or condition, wherein increasing the amount of myosin light chain phosphorylation is beneficial, or treating or preventing diseases, wherein myosin light chain dephosphorylation contributes to the pathology and/or symptomology of the disease, as described in greater detail below.
Also provided is a method of increasing the level of phosphorylation of Ras-1 in a cell, comprising contacting an effective amount of a compound according to formula I, or a salt form thereof, with the cell. The method may be performed by contacting the cell with a compound according to formula I, or a salt form thereof, in vitro, thereby increasing the amount of phosphorylation of Ras-1 in vitro. Uses of such an in vitro method of increasing the amount of phosphorylation of Ras-1 include, but are not limited to use in a screening assay (for example, wherein the compound according to formula I is used as a positive control or standard compared to compounds of unknown activity or potency in increasing phosphorylation of Ras-1).
The method of increasing the amount of phosphorylation of Ras-1 may also be performed by contacting the cell with a compound according to formula I, or a salt form thereof, in vivo, thereby increasing the amount of phosphorylation of Ras-1 in vivo. The contacting is achieved by causing the compound according to formula I, or a salt form thereof, to be present in the individual in an effective amount to achieve an increase in the amount of phosphorylation of Ras-1. This may be achieved, for example, by administering an effective amount of the compound according to formula I, or a pharmaceutically acceptable salt form thereof, to the individual, or by administering a prodrug of the compound according to formula I, or a pharmaceutically acceptable salt form thereof. Uses of such an in vivo method of increasing the amount of phosphorylation of Ras-1 include, but are not limited to use in methods of treating a disease or condition, wherein increasing the amount of phosphorylation of Ras-1 is beneficial, or treating or preventing diseases, wherein Ras-1 dephosphorylation contributes to the pathology and/or symptomology of the disease.
Also provided is a method of inhibiting actin polymerization (or promoting actin depolymerization) in a cell, comprising contacting an effective amount of a compound according to formula I, or a salt form thereof, with the cell. The method may be performed by contacting the cell with a compound according to formula I, or a salt form thereof, in vitro, thereby decreasing the amount of phosphorylation of actin polymerization in vitro. Uses of such an in vitro method of inhibiting actin polymerization of Ras-1 include, but are not limited to use in a screening assay (for example, wherein the compound according to formula I is used as a positive control or standard compared to compounds of unknown activity or potency in inhibiting actin polymerization).
The method of inhibiting actin polymerization may also be performed by contacting the cell with a compound according to formula I, or a salt form thereof, in vivo, thereby inhibiting microfilament formation in vivo. The contacting is achieved by causing the compound according to formula I, or a salt form thereof, to be present in the individual in an effective amount to achieve inhibition of actin polymerization. This may be achieved, for example, by administering an effective amount of the compound according to formula I, or a pharmaceutically acceptable salt form thereof, to the individual, or by administering a prodrug of the compound according to formula I, or a pharmaceutically acceptable salt form thereof. Uses of such an in vivo method of inhibiting actin polymerization include, but are not limited to use in methods of treating a disease or condition, wherein inhibiting actin polymerization is beneficial, or treating or preventing diseases, wherein actin polymerization contributes to the pathology and/or symptomology of the disease.
In another aspect, method of inhibiting tubulin polymerization (or promoting tubulin depolymerization) in a cell is provided, comprising contacting an effective amount of a compound according to formula I, or a salt form thereof, with the cell. The method may be performed by contacting the cell with a compound according to formula I, or a salt form thereof, in vitro, thereby decreasing the amount of phosphorylation of tubulin polymerization in vitro. Uses of such an in vitro method of inhibiting tubulin polymerization of Ras-1 include, but are not limited to use in a screening assay (for example, wherein the compound according to formula I is used as a positive control or standard compared to compounds of unknown activity or potency in inhibiting tubulin polymerization).
The method of inhibiting tubulin polymerization may also be performed by contacting the cell with a compound according to formula I, or a salt form thereof, in vivo, thereby inhibiting microtubule formation in vivo. The contacting is achieved by causing the compound according to formula I, or a salt form thereof, to be present in the individual in an effective amount to achieve inhibition of tubulin polymerization. This may be achieved, for example, by administering an effective amount of the compound according to formula I, or a pharmaceutically acceptable salt form thereof, to the individual, or by administering a prodrug of the compound according to formula I, or a pharmaceutically acceptable salt form thereof. Uses of such an in vivo method of inhibiting tubulin polymerization include, but are not limited to use in methods of treating a disease or condition, wherein inhibiting tubulin polymerization is beneficial, or treating or preventing diseases, wherein tubulin polymerization contributes to the pathology and/or symptomology of the disease.
The compounds according to formula I are effective to treat or prevent diseases or conditions associated with myosin light chain phosphatase activity, for example aberrant myosin light chain phosphatase activity. There is therefore provided a method of treating or prophylaxis of a disease or condition associated with myosin light chain phosphatase activity comprising causing an effective amount of a compound according to formula I, or a salt form thereof, to be present in an individual in need of such treatment. This may be achieved, for example, by administering an effective amount of the compound according to formula I, or a pharmaceutically acceptable salt form thereof, to the individual, or by administering a prodrug of the compound according to formula I, or a pharmaceutically acceptable salt form thereof. A "disease or condition associated with myosin light chain phosphatase activity" (or, equivalently, a "myosin light chain phosphatase-associated disease or condition" is a disease or condition, wherein a myosin light chain phosphatase possesses activity that contributes to the pathology and/or symptomology of the disease or condition or, wherein inhibition of a myosin light chain phosphatase produces an effect which is therapeutically beneficial. In embodiments thereof, the disease or condition is a cancer. In other embodiments thereof, the compound according to formula I or salt thereof used is an embodiment of the compounds according formula I, or a salt thereof, as described above. In some embodiments, a fluorescent compound according to formula I is used.
Myosin light chain phosphatase inhibitors are effective to induce cell cycle arrest and/or apoptosis of a cell. There is therefore also provided a method of inducing cell-cycle arrest and/or apoptosis of a cell comprising contacting the cell with a myosin light chain phosphatase inhibitor. Suitable myosin light chain phosphatase inhibitors include the compound according formula I, or a salt form thereof. The method of inducing cell-cycle arrest and/or apoptosis of a cell may be performed by contacting the cell with a myosin light chain phosphatase inhibitor such as the compound according to formula I, or a salt form thereof, in vitro, thereby inducing cell-cycle arrest and/or apoptosis of a cell in vitro. Uses of such an in vitro method of inducing cell-cycle arrest and/or apoptosis include, but are not limited to use in a screening assay (for example, wherein a known myosin light chain phosphatase inhibitor is used as a positive control or standard compared to compounds of unknown activity or potency in inducing cell-cycle arrest and/or apoptosis). The cell-cycle arrest and/or apoptosis may be induced in a cancer cell. The compound according to formula I or salt thereof used may be an embodiment of the compounds according formula I, or a salt thereof, as described above, for example a fluorescent compound according to formula I.
The method of inducing cell-cycle arrest and/or apoptosis of a cell may be performed by contacting the myosin light chain phosphatase with a myosin light chain phosphatase inhibitor, such as a compound according to formula I, in vivo, thereby inducing cell-cycle arrest and/or apoptosis in an individual in vivo. The contacting is achieved by causing the myosin light chain phosphatase inhibitor, such as the compound according to formula I, or a salt form thereof, to be present in the individual in an amount effective to achieve inhibition of cell-cycle arrest and/or apoptosis. This may be achieved, for example, by administering an effective amount of the myosin light chain phosphatase inhibitor, such as the compound according to formula I, or a pharmaceutically acceptable salt form thereof, to the individual, or by administering a prodrug of the myosin light chain phosphatase inhibitor, such as a compound according to formula I, or a pharmaceutically acceptable salt form thereof. Uses of such an in vitro method of inducing cell-cycle arrest and/or apoptosis include, but are not limited to use in methods of treating a disease or condition wherein inducing cell-cycle arrest and/or apoptosis is beneficial. The cell-cycle arrest and/or apoptosis may be induced in a cancer cell, for example in a patient suffering from cancer. The method may be performed by administering an effective amount of the myosin light chain phosphatase inhibitor, such as the compound according to formula I, a prodrug of a compound according to formula I, or salt form of either, to an individual who is suffering from cancer. The compound according to formula I or salt thereof used may be an embodiment of the compounds according to formula I, or a salt thereof, as described above such as a fluorescent compound according to formula I.
Administration of a myosin light chain phosphatase inhibitor, such as compounds according to formula I, or a salt form thereof, is also effective to treat cancer. There is therefore provided a method for treating cancer comprising causing an effective amount of a myosin light chain phosphatase inhibitor, such as a compound according to formula I, or a salt form thereof, to be present in an individual. The causing may be achieved by administering an effective amount of a myosin light chain phosphatase inhibitor, such as a compound according to formula I, or a salt thereof, to an individual in need of such treatment, or administering a prodrug of such a compound.
A myosin light chain phosphatase inhibitor, such as a compound according to formula I is can be used to treat a broad range of cancers and tumor types, including, but not limited to, bladder cancer, brain cancer, breast cancer, colorectal cancer, cervical cancer, gastrointestinal cancer, genitourinary cancer, head and neck cancer, lung cancer, ovarian cancer, prostate cancer, renal cancer, skin cancer, and testicular cancer. Prostate cancer treated may include androgen-independent prostate cancer.
More particularly, cancers that may be treated by myosin light chain phosphatase inhibitors, such as the compounds, compositions and methods described herein include, but are not limited to, the following: cardiac cancers, including, for example sarcoma, e.g., angiosarcoma, fibrosarcoma, rhabdomyosarcoma, and liposarcoma; myxoma; rhabdomyoma; fibroma; lipoma and teratoma; lung cancers, including, for example, bronchogenic carcinoma, e.g., squamous cell, undifferentiated small cell, undifferentiated large cell, and adenocarcinoma; alveolar and bronchiolar carcinoma; bronchial adenoma; sarcoma; lymphoma; chondromatous hamartoma; and mesothelioma; gastrointestinal cancer, including, for example, cancers of the esophagus, e.g., squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, and lymphoma; cancers of the stomach, e.g., carcinoma, lymphoma, and leiomyosarcoma; cancers of the pancreas, e.g., ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, and vipoma; cancers of the small bowel, e.g., adenocarcinoma, lymphoma, carcinoid tumors, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, and fibroma; cancers of the large bowel, e.g., adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, and leiomyoma; genitourinary tract cancers, including, for example, cancers of the kidney, e.g., adenocarcinoma, Wilm's tumor (nephroblastoma), lymphoma, and leukemia; cancers of the bladder and urethra, e.g., squamous cell carcinoma, transitional cell carcinoma, and adenocarcinoma; cancers of the prostate, e.g., adenocarcinoma, and sarcoma; cancer of the testis, e.g., seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, and lipoma; liver cancers, including, for example, hepatoma, e.g., hepatocellular carcinoma; cholangiocarcinoma; hepatoblastoma; angiosarcoma; hepatocellular adenoma; and hemangioma; bone cancers, including, for example, osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochrondroma (osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors; nervous system cancers, including, for example, cancers of the skull, e.g., osteoma, hemangioma, granuloma, xanthoma, and osteitis deformans; cancers of the meninges, e.g., meningioma, meningiosarcoma, and ; cancers of the brain, e.g., astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma multiform, oligodendroglioma, retinoblastoma, congenital tumors and gliomatosis; cancers of the peripheral nerves, for example schwannoma, and cancers of the spinal cord, e.g., neurofibroma, meningioma, glioma, and sarcoma; gynecological cancers, including, for example, cancers of the uterus, e.g., endometrial carcinoma; cancers of the cervix, e.g., cervical carcinoma, and pre tumor cervical dysplasia; cancers of the ovaries, e.g., ovarian carcinoma, including serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma, granulosa thecal cell tumors, Sertoli Leydig cell tumors, dysgerminoma, and malignant teratoma; cancers of the vulva, e.g., squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, and melanoma; cancers of the vagina, e.g., clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma, and embryonal rhabdomyosarcoma; and cancers of the fallopian tubes, e.g., carcinoma; hematologic cancers, including, for example, cancers of the blood, e.g., acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, and myelodysplastic syndrome, Hodgkin's lymphoma, non Hodgkin's lymphoma (malignant lymphoma) and Waldenstrom's macroglobulinemia; skin cancers, including, for example, malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis; and adrenal gland cancers, including, for example, neuroblastoma.
Cancers may be solid tumors that may or may not be metastatic. Cancers may also occur, as in leukemia, as a diffuse tissue. Thus, the term "tumor cell", as provided herein, includes a cell with any one of the above identified disorders.
The myosin light chain phosphatase inhibitor, such as a compound according to formula I can also be administered in combination with existing methods of treating disorders such as cancers, for example by chemotherapy, irradiation, or surgery. Thus, there is further provided a method of treating cancer comprising administering an effective amount of a myosin light chain phosphatase inhibitor, such as a compound according to formula I, or a salt thereof, to an individual in need of such treatment, wherein an effective amount of at least one further cancer chemotherapeutic agent is administered to the individual. Examples of suitable chemotherapeutic agents include any of: abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, anastrozole, arsenic trioxide, asparaginase, azacitidine, bevacizumab, bexarotene, bleomycin, bortezombi, bortezomib, busulfan intravenous, busulfan oral, calusterone, capecitabine, carboplatin, carmustine, cetuximab, chlorambucil, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, dalteparin sodium, dasatinib, daunorubicin, decitabine, denileukin, denileukin diftitox, dexrazoxane, docetaxel, doxorubicin, dromostanolone propionate, eculizumab, epirubicin, erlotinib, estramustine, etoposide phosphate, etoposide, exemestane, fentanyl citrate, filgrastim, floxuridine, fludarabine, fluorouracil, fulvestrant, gefϊtinib, gemcitabine, gemtuzumab ozogamicin, goserelin acetate, histrelin acetate, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib mesylate, interferon alfa 2a, irinotecan, lapatinib ditosylate, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole, lomustine, meclorethamine, megestrol acetate, melphalan, mercaptopurine, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone phenpropionate, nelarabine, nofetumomab, oxaliplatin, paclitaxel, pamidronate, panitumumab, pegaspargase, pegfϊlgrastim, pemetrexed disodium, pentostatin, pipobroman, plicamycin, procarbazine, quinacrine, rasburicase, rituximab, sorafenib, streptozocin, sunitinib, sunitinib maleate, tamoxifen, temozolomide, teniposide, testolactone, thalidomide, thioguanine, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, vorinostat, and zoledronate.
Also provided is a method of treating cancer comprising administering an effective amount of a myosin light chain phosphatase inhibitor, such as a compound according to formula I, or a salt thereof, to an individual in need of such treatment, wherein an effective amount of ionizing radiation is administered to the individual. In these methods, the further cancer therapeutic agent and/or the ionizing radiation may be administered concomitantly and/or non-concomitantly, e.g. sequentially, with the myosin light chain phosphatase inhibitor, such as a compound according to formula I, or a salt form thereof.
The myosin light chain phosphatase inhibitor, such as a compound according to formula I, can also be administered to an individual in combination with surgical methods to treat cancers, e.g., resection of tumors. The compounds can be administered to the individual prior to, during, or after the surgery. The compounds can be administered parenterally or injected into the tumor or surrounding area after tumor removal, e.g., to minimize metastases or to treat residual tumor cells present. When the compound is fluorescent, the compound may be used to detect the presence of the tumor and to guide surgical resection. Such fluorescent compounds can further therapeutically treat the cancer through their myosin light chain phosphatase inhibitory properties. Accordingly, there is provided a method of guided surgery to remove at least a portion of a tumor from an individual comprising providing a fluorescent myosin light chain phosphatase inhibitor; causing the fluorescent myosin light chain phosphatase inhibitor to be present in at least some tumor cells in an effective amount to inhibit a myosin light chain phosphatase and for fluorescence to be observable; observing the fluorescence; and performing surgery on the individual to remove at least a portion of the tumor that comprises fluorescent tumor cells. Causing the fluorescent myosin light chain phosphatase inhibitor to be present can occur by administering a compound according to formula I, or a prodrug or salt form thereof, to an individual.
In a further aspect there is provided a method of killing a tumor cell comprising contacting the tumor cell with an effective amount of a myosin light chain phosphatase inhibitor, such as a compound according to formula I, or a salt form thereof; and irradiating the tumor cell with an effective amount of ionizing radiation.
In a further aspect there is provided a method of killing a tumor cell comprising contacting the tumor cell with an effective amount of a myosin light chain phosphatase inhibitor, such as a compound according to formula I, or a salt form thereof; and contacting the tumor cell with an effective amount of at least one further chemotherapeutic agent.
Causing an effective amount of the compound a myosin light chain phosphatase inhibitor, such as according to formula I, or a salt form thereof, may be achieved, for example, by administering an effective amount of a myosin light chain phosphatase inhibitor, such as the compound according to formula I, a prodrug of a compound according to formula I, or a pharmaceutically acceptable salt form thereof, to the individual. In the aforementioned methods of killing tumor cells, the tumor cell may be a prostate cancer cell, for example an androgen-independent prostate cancer cell.
In the methods of treatment described herein, the compounds according to formula I may be administered to individuals (mammals, including animals and humans) afflicted with a disease such as such as cancer. In particular embodiments, the individual treated is a human.
The compounds may be administered by any route, including oral, rectal, sublingual, and parenteral administration. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, intravaginal, intravesical (e.g., to the bladder), intradermal, transdermal, topical or subcutaneous administration. Also contemplated is the instillation of a drug in the body of the patient in a controlled formulation, with systemic or local release of the drug to occur at a later time. For example, the drug may be localized in a depot for controlled release to the circulation, or for release to a local site of tumor growth. The compounds may be administered in the form of a pharmaceutical composition.
One or more compounds useful in the practice of the methods described herein may be administered simultaneously, by the same or different routes, or at different times during treatment. The compounds may be administered before, along with, or after other medications, including other compounds.
The treatment using methods of treatment described herein may be carried out for as long a period as necessary, either in a single, uninterrupted session, or in discrete sessions. The treating physician can increase, decrease, or interrupt treatment based on patient response. Treatment may be carried out, for example, for from about four to about sixteen weeks. The treatment schedule may be repeated as required.
X. Methods of Using Fluorescent Myosin Light Chain Phosphatase Inhibitors, Including Fluorescent Compounds According to Formula I
Fluorescent compounds according to formula I provide added diagnostic and tracking functionalities to the therapeutic functionality of compounds according to formula I. Thus, there are provided methods of using fluorescent myosin light chain phosphatase inhibitors, including fluorescent compounds according to formula I, and salt forms thereof.
One such method provided is a method of detecting the presence of an elevated amount of a myosin light chain phosphatase in a subject cell, comprising: providing a fluorescent myosin light chain phosphatase inhibitor; contacting the fluorescent myosin light chain phosphatase inhibitor with the subject cell and with a control cell; observing fluorescence of the subject and control cells after the contacting; wherein an elevated level of fluorescence of the subject cell relative to the level of fluorescence of the control cell is indicative of an elevated amount of the myosin light chain phosphatase in the subject cell as compared to the control cell.
Fluorescence may be observed, for example, of the cytoplasm of the cells, whereby an elevated amount of the myosin light chain phosphatase in the cytoplasm of the subject cell as compared to the control cell is detected.
Uses of the provided method of detecting elevated myosin light chain phosphatase include, but are not limited to, detecting the presence of disease conditions associated with an elevated level of myosin light chain phosphatase activity and/or amount.
Accordingly, there is also provided a method of detecting diseased (e.g., cancerous) cells in an individual, comprising: providing a fluorescent myosin light chain phosphatase inhibitor; contacting the fluorescent myosin light chain phosphatase inhibitor with a tissue of the individual; and observing for fluorescence of the cells of the tissue after the contacting; wherein an elevated level of fluorescence of at least some of the cells in the tissue relative to other cells in the tissue or relative to control non-diseased cells that have been contacted with the fluorescent myosin light chain phosphatase inhibitor is indicative that the fluorescent cells may be diseased cells comprising elevated amounts of a myosin light chain phosphatase.
The contacting may be performed in vitro or in vivo, for example by causing an effective amount of a fluorescent myosin light chain phosphatase inhibitor to be present in the tissue, for example, by administering an effective amount of a fluorescent myosin light chain phosphatase inhibitor or a prodrug thereof to an individual.
There is also provided a method of radiotherapy of tumors comprising: providing a fluorescent myosin light chain phosphatase inhibitor; causing the fluorescent myosin light chain phosphatase inhibitor to be present in the tumor cells in an effective amount to inhibit myosin light chain phosphatase and for fluorescence to be observable; observing the fluorescence; and directing an effective amount of ionizing radiation to the fluorescent tumor cells. An advantage of the provided method of radiotherapy of tumors using fluorescent myosin light chain phosphatase inhibitors is that the fluorescent myosin light chain phosphatase inhibitor simultaneously renders the tumor cells visible while arresting growth of the cells and possibly sensitizing the cells to the effect of the ionizing radiation. Since the tumor cells are visible, the irradiation can be directed to the tumor tissue, avoiding unnecessary damage to undiseased tissue. At the same time, the applied radiation may be more effective since the tumor cells may be sensitized to its effect. Further, by directing the radiation selectively to the tumor cells, the amount of radiation applied to the tumor tissue can, if desirable, be maximized since the radiation applied can be focused upon the tumor tissue made visible though its fluorescence.
There is also provided a method of guided surgery or resection of at least a portion of a tumor, comprising providing a fluorescent myosin light chain phosphatase inhibitor, causing the fluorescent myosin light chain phosphatase inhibitor to be present in at least some cells of the tumor tissue in an effective amount for fluorescence of the tumor tissue to be observable, observing the fluorescence, and surgically removing at least some of the fluorescent tumor tissue, whereby at least a portion of the tumor that comprises fluorescent tumor cells is removed. Causing the fluorescent myosin light chain phosphatase inhibitor to be present can occur by administering a compound according to formula I, or a prodrug or salt form thereof, to an individual. An advantage of the methods is that the fluorescent compounds simultaneously render the tumor cells visible while potentially also therapeutically treating the cancer through their myosin light chain phosphatase inhibitory properties. Since the tumor cells are visible, the surgery can be focused on the tumor tissue, avoiding unnecessary damage to undiseased tissue, while also minimizing the opportunity for some tumor to be inadvertently left behind.
In some embodiments of each of the aforementioned methods of using fluorescent myosin light chain phosphatase inhibitors, the fluorescent myosin light chain phosphatase inhibitor is a compound according to formula I, or a salt form thereof, or any of the embodiments thereof as described above. An example is 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate, or a salt form thereof.
EXAMPLES
The following non-limiting examples are provided to illustrate the invention. General Experimental Methods.
NMR spectra were recorded using a Varian-400 spectrometer for 1H (400 MHz) and 13C (100 MHz). Chemical shifts (δ) are given in ppm downfield from tetramethylsilane, an internal standard, and coupling constants (J-values) are in hertz (Hz). Purifications were performed by flash chromatography.
Examples 1-30. Exemplary Compounds of Formula I.
Scheme 5 below illustrates the structures and methods of synthesis of exemplary compounds according to formula I.
Figure imgf000050_0001
15a-e 16a-e 17a-e
11a R,X= Ph,O; 11b R.X= tBu:O; 11c R,X= tBu:N; 11d R1X= nBu,Q: 11e R.X= nBu.N; 11f R.X= c- propyiO; Hg R1X= BeRZOyLO; 11h RnX= 2-naph.N; 11i R1X= 2-naph.O; 11j RnX= 1-naph.N 15a R= 4- CF3Phn 15b R=4-MePh, 15c R= 4-tBuPh, 15d R= Biphenyl, 15e R= dansyl
Scheme 5 Example 1. 4-(2-Guanidinothiazol-4-yl)phenyl 3'-methoxybiphenyl-4-carboxylate (10a)
Figure imgf000051_0001
(a) 3'-Methoxybiphenyl-4-carboxylic acid (7a).
Figure imgf000051_0002
A solution of 3-methoxyphenyl boronic acid (1 g, 6.58 mmol), 4-iodobenzoic acid (1.63 g, 6.58 mmol) and cesium carbonate (5.36 g, 16.45 mmol) in 3:1 1,2- dimethoxyethane/water was deoxygenated with nitrogen for 15 minutes. Pd(PPlIs)4 (380 mg, 0.329 mmol) was then added and the solution was heated to 80° C for 6h. The reaction was allowed to cool and acidified with 2M HCl which caused a precipitate to form. The precipitate was filtered and the filtrate was extracted twice with dichloromethane. The organic layer was then dried over magnesium sulfate, filtered through diatomaceous earth filter aid (CELITE®) and evaporated under reduced pressure to a white solid which was purified via column chromatography (4:1 hexane/ethyl acetate w/ 1% acetic acid) and was combined with the precipitate to yield 1.04 g (69%). 1HNMR (DMSO): δ 8.01 (d, J=8.1 Hz, 2H), 7.80 (d, J=8.1 Hz, 2H), 7.41 (t, J=7.8 Hz, IH), 7.30-7.25 (m, 3H), 6.99 (d, J=8.1 Hz, IH), 3.83 (s, 3H). MS, ESI, m/e 229 (MH+).
(b) 4-Acetylphenyl 3'-methoxybiphenyl-4-carboxylate (8a).
Figure imgf000051_0003
3'-Methoxybiphenyl-4-carboxylic acid (500 mg, 2.03 mmol) was dissolved in thionyl chloride (25 mL) and allowed to stir overnight. The thionyl chloride was then pumped off and the acid chloride was carried on crude. The acid chloride was dissolved in dichloromethane and 4-hydroxyacetophenone (276 mg, 2.03 mmol) was added to the solution. Sodium hydride (60% in mineral oil, 81 mg, 2.03 mmol) was then added slowly to the reaction and allowed to stir for 5h. The reaction was then quenched with saturated aqueous sodium bicarbonate and the solution was extracted twice with dichloromethane. The combined organic layers were then dried over magnesium sulfate, filtered and evaporated under reduced pressure to a solid which was carried on crude. 1HNMR (DMSO): δ 8.21 (d, J=8.4 Hz, 2H), 8.09 (d, J=8.7 Hz, 2H), 7.94 (d, J=8.4 Hz, 2H), 7.48 (d, J=8.7 Hz, 2H), 7.43 (d, J=7.8 Hz, IH), 7.36-7.32 (m, 2H), 7.03 (dd, 1=12, 1.8 Hz, IH), 3.85 (s, 3H), 2.62 (s, 3H). 13CNMR (DMSO): δ 196.88, 163.94, 159.77, 154.19, 145.36, 140.05, 134.55, 130.46, 130.19, 129.88, 127.42, 127.23, 122.20, 119.30, 114.21, 112.42, 55.17, 26.74.
(c) 4-(2-Bromoacetyl)phenyl 3'-methoxybiphenyl-4-carboxylate (9a).
Figure imgf000052_0001
Bromine (226 mg, 1.66 mmol) was added dropwise to a solution of 4-Acetylphenyl 3'-methoxybiphenyl-4-carboxylate (577 mg, 1.66 mmol) in acetic acid which yielded 700 mg (99%) of the sub-title compound. 1HNMR (DMSO): δ 8.22 (d, J=8.4 Hz, 2H), 8.13 (d, J=8.7 Hz, 2H), 7.94 (d, J=8.4 Hz, 2H), 7.53 (d, J=8.4 Hz, 2H), 7.45 (t, J=7.8 Hz, IH), 7.34 (m, 2H), 7.03 (dd, J=7.5, 2.1 Hz, IH).
(d) 4-(2-Guanidinothiazol-4-yl)phenyl 3'-methoxybiphenyl-4-carboxylate (10a).
2-Imino-4-thiobiuret (186 mg, 1.57 mmol) was added to a solution of 4-(2- bromoacetyl)phenyl 3'-methoxybiphenyl-4-carboxylate (669 mg, 1.57 mmol) in acetone and the solution was heated under reflux to give 490 mg (56%) of the title compound as a hydrobromide salt. 1HNMR (DMSO): δ 8.19 (m, 6H), 8.05 (d, J=8.5 Hz, 2H), 7.92 (d, J=7 Hz, IH), 7.80 (s, IH), 7.68-7.62 (m, IH), 7.43 (t, J=8 Hz, IH), 7.39 (m, 2H), 7.33 (d, J=8 Hz, IH), 7.29 (s, IH), 7.04-6.96 (m, IH). 13CNMR (DMSO): δ 164.27, 159.77, 153.84, 150.47, 145.21, 140.07, 133.84, 130.93, 130.38, 130.19, 129.80, 129.61, 127.16, 122.24, 119.28, 116.58, 114.15, 112.42, 108.20. Example 2. 4-(2-Guanidinothiazol-4-yl)phenyl 4'-methoxybiphenyl-4-carboxylate (10b)
Figure imgf000053_0001
(a) 4'-Methoxybiphenyl-4-carboxylic acid (7b).
Figure imgf000053_0002
Method A.
Coumalic acid (1.06 g, 7.56 mmol) and 4-ethylnyl anisole were dissolved in diglyme and brought to reflux for 36h. The reaction was then allowed to cool and the solvent was removed under reduced pressure. The resultant slurry was then recrystallized from ethanol to yield 754 mg (44%) of a light tan solid.
Method B.
A solution of 4-methoxyphenyl boronic acid (1 g, 6.58 mmol), 4-iodobenzoic acid (1.63 g, 6.58 mmol) and cesium carbonate (5.36 g, 16.45 mmol) in 3:1 1,2- dimethoxyethane/water was deoxygenated with nitrogen for 15 minutes. Pd(PPtLs)4 (380 mg, 0.329 mmol) was then added and the solution was heated to 80° C for 6h. The reaction was allowed to cool and acidified with 2M HCl which caused a precipitate to form. The precipitate was filtered and the filtrate was extracted twice with dichloromethane. The organic layer was then dried over magnesium sulfate, filtered through diatomaceous earth filter aid (CELITE®) and evaporated under reduced pressure to yield a combined 1.5O g (95%). 1HNMR (DMSO): δ 7.99 (d, J=8.4 Hz, 2H), 7.74 (d, J=8.4 Hz, 2H), 7.69 (d, J=8.7 Hz, 2H), 7.04 (d, J=8.7 Hz, 2H), 3.80 (s, 3H). 13CNMR (DMSO): δ 167.17, 159.49, 143.90, 131.16, 129.91, 128.77, 128.09, 126.08, 114.45, 55.17. (b) 4-Acetylphenyl 4'-methoxybiphenyl-4-carboxylate (8b).
Figure imgf000054_0001
4'-Methoxybiphenyl-4-carboxylic acid (400 mg, 1.60 mmol) was dissolved in thionyl chloride (25 rnL) and allowed to stir overnight. The thionyl chloride was then removed under reduced pressure and the acid chloride was carried on crude. The acid chloride was dissolved in dichloromethane and 4-hydroxyacetophenone (218 mg, 1.60 mmol) was added to the solution. Sodium hydride (60% in mineral oil, 64 mg, 1.60 mmol) was then added slowly to the reaction and allowed to stir for 5h. The reaction was then quenched with saturated aqueous sodium bicarbonate and the solution was extracted twice with dichloromethane. The combined organic layers were then dried over magnesium sulfate, filtered and evaporated under reduced pressure to a solid which was carried on crude. 1HNMR (DMSO): δ 8.18 (d, J=5.1 Hz, 2H), 8.08 (d, J=5.1 Hz, 2H), 7.88 (d, J=5.1 Hz, 2H), 7.76 (d, J=5.1 Hz, 2H), 7.48 (d, J=5.1 Hz, 2H), 7.08 (d, J=5.1 Hz, 2H), 3.82 (s, 3H), 2.62 (s, 3H).
(c) 4-(2-Bromoacetyl)phenyl 4'-methoxybiphenyl-4-carboxylate (10b).
Figure imgf000054_0002
Bromine (125 mg, 0.78 mmol) was added to a solution of 4'-Methoxy-biphenyl-4- carboxylic acid 4-acetyl-phenyl ester (266 mg, 0.78 mmol) in acetic acid which yielded 285 mg (86%) of an intermediate α-bromo ketone which was carried on crude.
(d) 4-(2-Guanidinothiazol-4-yl)phenyl 4'-methoxybiphenyl-4-carboxylate (10b).
4-(2-Bromoacetyl)phenyl 4'-methoxybiphenyl-4-carboxylate (280 mg, 0.66 mg) and 2-imino-4-thiobiuret (78 mg, 0.66 mmol) were combined in acetone and brought to reflux yielding 136 mg (40%) of the title compound as a hydrobromide salt. 1HNMR (DMSO): δ 8.18 (d, J=8.1 Hz, 2H), 8.00 (d, J=8.4 Hz, 2H), 7.87 (d, J=8.1 Hz, 2H), 7.74 (d, J=8.7 Hz, 2H), 7.53 (s, br, 4H), 7.35 (d, J=8.4 Hz, 2H), 7.08 (d, J=8.7 Hz, 2H), 3.82 (s, 3H). 113X/ NMR (DMSO): δ 164.36, 159.74, 155.25, 150.12, 148.71, 144.95, 131.69, 130.75, 130.44, 128.21, 126.85, 126.33, 122.13, 114.52, 105.92, 55.22. Example 3. 4-(2-Guanidinothiazol-4-yl)phenyl biphenyl-4-carboxylate (13a)
Figure imgf000055_0001
(a) 4-Acetylphenyl biphenyl-4-carboxylate (Ha).
Figure imgf000055_0002
4-Hydroxyacetophenone (Ig, 7.35 mmol) was added to a slurry of NaH (60% in mineral oil, 294 mg, 7.35 mmol) in tetrahydrofuran and was allowed to stir for 10 min. 4- biphenylcarbonyl chloride (1.59 g, 7.35 mmol) was then added to this mixture dropwise and allowed to stir for 5h. The reaction was quenched with water and the precipitate was filtered and dried in a dessicator. The aqueous layer was then extracted three times with ethyl acetate. The organic layer was dried over magnesium sulfate, filtered and evaporated under reduced pressure to yield 2.08 g (89%) of a white solid. . 1HNMR (DMSO): δ 8.22 (d, J=6.9 Hz, 2H), 8.08 (d, J=7.2 Hz, 2H), 7.93 (d, J=6.1 Hz, 2H), 7.78 (d, J=6 Hz, 2H), 7.49 (m, 5H), 2.61 (s, 3H). 13CNMR (CDCl3): δ 164.75, 154.94, 146.90, 135.03, 131.01, 130.24, 129.23, 128.64, 127.90, 127.54, 122.17, 31.164, 26.86. MS, APCI, m/e 317 (MH+). (b) 4-(2-Bromoacetyl)phenyl biphenyl-4-carboxylate (12a).
Figure imgf000056_0001
Bromine (757 mg, 4.74) was added dropwise to a solution of 4-Acetylphenyl biphenyl-4-carboxylate (1.5 g, 4.74 mmol) in acetic acid. The reaction yielded 1.3 g (70%) of the title compound. 1HNMR (DMSO): δ 8.23 (d, J=7.8 Hz, 2H), 8.14 (d, J=8.1 Hz, 2H), 7.93 (d, J=8.1 Hz, 2H), 7.79 (d, J=6.9 Hz, 2H), 7.54-7.50 (m, 5H). 13CNMR (DMSO): δ 190.64, 163.86, 154.63, 145.49, 138.52, 131.66, 130.49, 129.06, 128.55, 127.06, 122.40, 34.05.
(c) 4-(2-Guanidinothiazol-4-yl)phenyl biphenyl-4-carboxylate (13a).
2-Imino-4-thiobiuret (695 mg, 5.88 mmol) was added to a solution 4-(2- bromoacetyl)phenyl biphenyl-4-carboxylate (1.3 g, 3.30 mmol) and heated under reflux to yield 1.25 g (76%) of the title compound as a hydrobromide salt. 1HNMR (DMSO): δ 8.25 (s, br, 4H), 8.22 (d, J=8.4 Hz, 2H), 8.07 (d, J=8.7 Hz, 2H), 7.92 (d, J=8.4 Hz, 2H), 7.83 (s, IH), 7.78 (d, J=7.2 Hz, 2H), 7.53 (t, J=6.9 Hz, 2H), 7.47 (d, J=7.2 Hz, IH), 7.40 (d, J=8.7 Hz, 2H). 13CNMR (DMSO): δ 164.30, 159.95, 153.73, 150.52, 145.35, 138.59, 130.87, 130.48, 129.11, 128.57, 127.55, 127.20, 127.07, 127.00, 122.27, 108.39. MS, APCI, m/e 415 (MH+).
Example 4. 4-(2-Guanidinothiazol-4-yl)phenyl 4-^-butylbenzoate (13b)
Figure imgf000056_0002
(a) 4-Acetylphenyl 4-*-butylbenzoate (lib).
Figure imgf000057_0001
4-Hydroxyacetophenone (691 mg, 5.08 mmol) was added to a slurry of sodium hydride (60% in mineral oil, 244 mg, 6.10 mmol) in tetrahydrofuran and was allowed to stir for 10 min. 4-t-butyl benzoyl chloride (1 g, 5.08 mmol) was then added to this mixture dropwise and allowed to stir for 5h. The reaction was then quenched with water and extracted three times with ethyl acetate. The organic layer was dried over magnesium sulfate, filtered, and evaporated under reduced pressure to yield 1.36 g (90%) of a white solid. 1HNMR (CDCl3): δ 8.13 (d, J=8.7 Hz, 2H), 8.05 (d, J=8.7 Hz, 2H), 7.54 (d, J=8.7 Hz, 2H), 7.32 (d, J=8.4 Hz, 2H), 2.63 (s, 3H), 1.37 (s, 9H). 13CNMR (CDCl3): δ 197.25, 164.85, 158.04, 155.02, 134.86, 130.38, 130.21, 126.37, 125.88, 122.19, 35.45, 31.28, 26.85. MS, APCI, m/e 297 (MH+).
(b) 4-(2-Bromoacetyl)phenyl 4-^-butylbenzoate (lib).
Figure imgf000057_0002
Bromine (425 mg, 2.66 mmol) was added to a solution of 4-t-butyl-benzoic acid 4-acetyl- phenyl ester (788 mg, 2.66 mmol) in chloroform. The reaction yielded 859 mg (86%) of the α-bromo ketone. 1HNMR (CDCl3): δ 8.12 (d, J=8.4 Hz, 2H), 8.07 (d, J=8.7 Hz, 2H), 7.54 (d, J=8.4 Hz, 2H), 7.35 (d, J=8.7 Hz, 2H), 4.45 (s, 2H), 1.37 (s, 9H). 13CNMR (CDCl3): δ 190.39, 185.77, 158.17, 155.61, 131.78, 130.91, 130.42, 125.93, 122.54, 39.67, 35.48, 31.30, 30.89.
(c) 4-(2-Guanidinothiazol-4-yl)phenyl 4-^-butylbenzoate (13b).
2-Imino-4-thiobiuret (126 mg, 1.065 mmol) was added to a solution of 4-(2- bromoacetyl)phenyl 4-t-butylbenzoate (400 mg, 1.065 mmol) and the solution was heated under reflux 207 mg (41%) of the hydrobromide salt of the title compounds as a while solid. 1HNMR (DMSO): δ 8.23 (s, br, 4H), 8.08 (d, J=8.5 Hz, 2H), 8.06 (d, J=9 Hz, 2H), 7.83 (s, IH), 7.65 (d, J=8 Hz, 2H), 7.35 (d, J=9 Hz, 2H), 1.33 (s, 9H). 13CNMR (DMSO): δ 164.36, 159.97, 157.24, 153.71, 150.52, 149.03, 130.81, 129.72, 127.15, 126.02, 125.76, 122.27, 108.35, 34.93, 30.72.
Example 5. 4-*-Butyl-7V-(4-(2-guanidinothiazol-4-yl)phenyl)benzamide (13c)
Figure imgf000058_0001
(a) 7V-(4-Acetylphenyl)-4-*-butylbenzamide (lie).
Figure imgf000058_0002
4'-£-Butylbenzoyl chloride was added dropwise to a solution of 4-aminoacetophenone and TEA (2mL) in ethyl acetate. The solution was allowed to stir for 4h, was quenched with water and extracted with ethyl acetate three times. The organic layers were then dried over magnesium sulfate, filtered and evaporated under reduced pressure to yield 1.56 g (71%) of the amide. 1HNMR (DMSO): δ 7.965 (d, J=2.7 Hz, 4H), 7.907 (d, J=8.4 Hz, 2H), 7.560 (d, J=8.4 Hz, 2H), 2.547 (s, 3H), 1.322 (s, 9H).
(b) 7V-[4-(2-Bromoacetyl)phenyl]-4-^-butylbenzamide (12c).
Figure imgf000058_0003
Bromine (1.29 g, 5.08 mmol) was added to a solution of JV-(4-acetylphenyl)-4-£- butylbenzamide (1.5 g, 5.08 mmol) in chloroform and reacted to yield 1.90 g (99%) of the title compound. 1HNMR (CDCl3): δ 8.108 (d, J=7.5 Hz, 2H), 7.82 (d, J=7.5 Hz, 4H), 7.511 (d, J=7.5 Hz, 2H), 6.693 (s, IH), 1.351 (s, 9H). (c) 4-*-Butyl-7V-(4-(2-guanidinothiazol-4-yl)phenyl)benzamide (13c).
2-Imino-4-thiobiuret (63.1 mg, 0.534 mmol) was added to a solution of N-(4-(2- bromoacetyl)phenyl)-4-£-butylbenzamide (200 mg, 0.534 mmol) and the solution was heated under reflux to yield 79.2 mg (31%) of the title compound as a hydrobromide salt. 1HNMR (DMSO): δ 8.156 (s, 4H), 7.916 (m, 6H), 7.654 (s, IH), 7.343 (d, J= 8.1 Hz, 2H), 1.351 (s, 9H). 13CNMR (DMSO): δ 170.386, 165.921, 161.012, 153.986, 149.724, 146.143, 139.219, 132.280, 128.303, 128.211, 127.824, 126.204, 120.121, 106.613, 34.717, 30.950
Example 6. 4-(2-Guanidinothiazol-4-yl)phenyl 4-n-butylbenzoate (13d)
Figure imgf000059_0001
(a) 4-Acetylphenyl 4-butylbenzoate (Hd).
Figure imgf000059_0002
4-Hydroxyacetophenone (691 mg, 5.08 mmol) was added to a slurry of sodium hydride (60% in mineral oil, 244 mg, 6.10 mmol) in tetrahydrofuran and was allowed to stir for 10 min. 4-n-Butyl benzoyl chloride (I g, 5.08 mmol) was then added to this mixture dropwise and allowed to stir for 5h. The reaction was then quenched with water and extracted three times with ethyl acetate. The organic layer was dried over magnesium sulfate, filtered, and evaporated under reduced pressure to yield 1.36 g (90%) of a white solid. 1HNMR (CDCl3): δ 8.11 (d, J=8.1 Hz, 2H), 8.04 (d, J=8.7 Hz, 2H), 7.34 (d, J=I.8 Hz, 2H), 7.31 (d, J=2.1 Hz, 2H), 2.71 (t, J=7.5 Hz, 2H), 2.62 (s, 3H), 1.64 (quin, J=7.4 Hz, 2H), 1.37 (sext, J=7.5 Hz, 2H), 0.95 (t, J=7.2 Hz, 3H). 13CNMR (CDCl3): δ 197.26, 164.91, 155.00, 149.99, 134.84, 131.08, 130.53, 130.20, 128.97, 126.58, 122.18, 115.51, 35.99, 33.43, 26.84, 22.50, 14.10. MS, APCI, m/e 297 (MH+). (b) 4-(2-Bromoacetyl)phenyl 4-butylbenzoate (12d).
Figure imgf000060_0001
Bromine (471 mg, 2.95 mmol) was added to a solution of 4-acetylphenyl 4- butylbenzoate (876 mg, 2.95 mmol) in chloroform and reacted to yield 1.06 g (96%) of the α- bromo ketone. 1HNMR (CDCl3): δ 8.13-8.04 (m, 4H), 7.49-7.42 (m, 4H), 4.96 (t, J=I.5 Hz, 2H), 2.69-2.68 (m, 2H), 1.59-1.57 (m, 2H), 1.32-1.28 (m, 2H), 0.92-0.87 (m, 3H). 13CNMR (CDCl3): δ 190.69, 164.08, 154.77, 149.50, 131.67, 130.56, 130.02, 129.90, 128.93, 122.48, 34.86, 34.12, 32.71, 21.70, 13.73.
(c) 4-(2-Guanidinothiazol-4-yl)phenyl 4-n-butylbenzoate (13d).
2-Imino-4-thiobiuret (157 mg, 1.33 mmol) was added to a solution of 4-(2- bromoacetyl)phenyl 4-butylbenzoate (500 mg, 1.33 mmol) in ethanol, and the solution was heated under reflux yielding 335 mg (53%) of the hydrobromide salt of the title compound as a while solid. 1HNMR (DMSO): δ 8.156 (s, 4H), 8.04 (d, J=8 Hz, 2H), 7.831 (d, J=8.8 Hz, 2H), 7.419 (d, J=8.4 Hz, 4H), 7.277 (s, IH), 2.681 (t, J=7.6 Hz, 2H), 1.576 (p, J=7.6 Hz, 2H), 1.298 (sextup, J=7.2 Hz, 2H), 0.889 (t, J=7.2 Hz, 3H). 13CNMR (DMSO): δ 170.121, 164.339, 151.160, 149.310, 138.795, 129.889, 128.874, 127.149, 126.762, 126.081, 122.593, 102.987, 34.779, 32.666, 21.644, 13.691.
Example 7. 4-n-Butyl-7V-(4-(2-guanidinothiazol-4-yl)phenyl)benzamide (13e)
Figure imgf000060_0002
(a) 7V-(4-Acetylphenyl)-4-n-butylbenzamide (lie).
Figure imgf000061_0001
4-Butylbenzoyl chloride was added dropwise to a solution of 4-aminoacetophenone and TEA (2mL) in ethyl acetate. The solution was allowed to stir for 4h, was quenched with water and extracted with ethyl acetate three times. The organic layers were then dried over magnesium sulfate, filtered and evaporated under reduced pressure to yield 1.56 g (71%) of the amide. 1HNMR (DMSO): δ 7.959 (m, 4H), 7.897 (d, J=8.1 Hz, 2H), 7.358 (d, J=8.1 Hz, 2H), 2.658 (t, J=7.8 Hz, 2H), 2.546 (s, 3H), 1.583 (sextup, J=7.2 Hz, 2H), 1.323 (sextup, J=7.5 Hz, 2H), 0.900 (t, J=7.5 Hz, 3H).
(b) 7V-(4-(2-Bromoacetyl)phenyl)-4-n-butylbenzamide (12e).
Figure imgf000061_0002
Bromine (1.099 g, 6.88 mmol) was added dropwise to a solution of N-(4- acetylphenyl)-4-n-butylbenzamide (2.032 g, 6.88 mmol) in methanol to yield 2.43 g (94%) of the title compound. 1HNMR (DMSO): δ 7.876 (d, J=8.1 Hz, 2H), 7.787 (d, J=8.7 Hz, 2H), 7.421 (d, J=8.7 Hz, 2H), 7.347 (d, J=8.1 Hz, 2H), 3.801 (s, 2H), 2.657 (t, J=7.5 Hz, 2H), 1.586 (p, J=7.2 Hz, 2H), 1.314 (sextup, J=7.2 Hz, 2H), 0.905 (t, J=7.2 Hz, 3H).
(c) 4-n-Butyl-7V-(4-(2-guanidinothiazol-4-yl)phenyl)benzamide (13e).
2-Imino-4-thiobiuret (315.7 mg, 2.67 mmol), was added to a solution of N-(4-(2- bromoacetyl)phenyl)-4-/?-butylbenzamide (1 g, 2.67 mmol) in acetone and the solution was heated under reflux to give 577 mg (46%) of the title compound as a hydrobromide salt. 1HNMR (DMSO): δ 8.156 (s, 4H), 7.916 (m, 6H), 7.654 (s, IH), 7.343 (d, J= 8.1 Hz, 2H), 2.65 (t, J=7.5, 2H), 1.577 (p, J=7.2 Hz, 2H), 1.301 (sextup, J=7.5 Hz, 2H), 0.895 (t, J=7.5 Hz, 3H). 13CNMR (DMSO): δ 170.224, 165.407, 160.801, 154.007, 149.551, 146.320, 139.288, 132.180, 128.323, 128.181, 127.679, 126.207, 120.117, 106.534, 59.663, 34.586, 32.803, 21.634, 20.665, 13.994, 13.661. Example 8. 4-(2-Guanidinothiazol-4-yl)phenyl 4-cyclopropylbenzoate (13f)
Figure imgf000062_0001
(a) 4-Cyclopropylbenzoic acid.
Figure imgf000062_0002
Coulmalic acid (2.63 g, 18.74 mmol) and ethynylcyclopropane (1.24 g, 18.74 mmol) were combined in diglyme and brought to reflux for 36h. The reaction was then cooled and the solvent was removed under reduced pressure. The resultant slurry was recyrstallized in EtOH to yield 1.26 g (42%). 1HNMR (DMSO): δ 7.81 (d, J=8.4 Hz, 2H), 7.16 (d, J=8.4 Hz, 2H), 1.97 (sept, J=5.1 Hz, IH), 1.01 (m, 2H), 0.74 (m, 2H). 13CNMR (DMSO): δ 167.24, 149.51, 129.32, 127.65, 125.19, 15.25, 10.37.
(b) 4-Acetylphenyl 4-cyclopropylbenzoate (Hf).
Figure imgf000062_0003
Oxalyl chloride(2.92 g, 22.2 mmol) was added dropwise to a solution of 4- cyclopropyl-benzoic acid (1.20 g, 7.4 mmol) in dichloromethane. The solution was allowed to stir overnight and the solvent was removed under reduced pressure. The acid chloride was then carried on crude.4-Hydroxyacetophenone (1.01 g, 7.4 mmol) was added to a slurry of NaH (60% in mineral oil, 296 mg, 7.4 mmol) in dichloromethane and allowed to stir for 10 min. 4-Cyclopropyl-benzoyl chloride (1.33 g, 7.4 mmol) was then added and the solution was allowed to stir for 3h. The reaction was then quenched with water and extracted three times with dichloromethane. The combined organic layers were then dried over magnesium sulfate, filtered and evaporated under reduced pressure to yield 1.83 g (88% over two steps). 1HNMR (CDCl3): δ 8.07 (t, J=8.7 Hz, 4H), 7.31 (d, J=8.4 Hz, 2H), 7.17 (d, J=8.4 Hz, 2H), 2.62 (s, 3H), 1.98 (sept, J=5.1 Hz, IH), 1.10 (m, 2H), 0.82 (m, 2H). 13CNMR (CDCl3): δ 196.91, 164.60, 154.77, 151.36, 134.61, 130.30, 129.94, 125.91, 125.55, 121.95, 26.62, 15.85, 10.61.
(c) 4-(2-Bromoacetyl)phenyl 4-cyclopropylbenzoate (12f).
Figure imgf000063_0001
Bromine (1.04 g, 6.53 mmol) was added dropwise to a solution of 4-acetylphenyl 4- cyclopropylbenzoate (1.83 g, 6.53 mmol) in chloroform which yielded 2.32 g (99%) of the title compound. 1HNMR (CDCl3): δ 8.07 (d, J=8.7 Hz, 4H), 7.35 (d, J=8.7 Hz, 2H), 7.17 (d, J=8.4 Hz, 2H), 4.45 (s, 2H), 1.99 (sept, J=4.8 Hz, IH), 1.10 (m, 2H), 0.82 (m, 2H). 13CNMR (CDCl3): δ 190.10, 164.44, 155.38, 151.51, 131.36, 130.64, 130.33, 129.94, 125.58, 122.28, 121.64, 30.69, 15.85, 10.62.
(d) 4-(2-Guanidinothiazol-4-yl)phenyl 4-cyclopropylbenzoate (13f).
2-Imino-4-thiobiuret (763 mg, 6.46 mmol), was added to a solution of 4-(2- bromoacetyl)phenyl 4-cyclopropylbenzoate (2.32 g, 6.46 mmol) in acetone and the solution was heated under reflux to give 1.96 g (66%) of the title compound as a hydrobromide salt. 1HNMR (DMSO): δ 8.26 (s, br, 4H), 8.05 (d, J=8.7 Hz, 2H), 8.00 (d, J=8.4 Hz, 2H), 7.81 (s, IH), 7.34 (d, J=8.7, 2H), 7.28 (d, J=8.4 Hz, 2H), 2.05 (sept, J=5.1 Hz, IH), 1.07 (m, 2H), 0.80 (m, 2H). 13CNMR (DMSO): δ 164.38, 159.89, 153.78, 151.13, 150.52, 149.03, 130.75, 129.83, 127.16, 125.56, 122.23, 108.33, 15.39, 10.68. MS, ESI, m/e 379 (MH+).
Example 9. 7V-[4-(4-Benzoylphenyl)-thiazol-2-yl]-guanidine (13g)
Figure imgf000063_0002
(a) l-(4-Benzoylphenyl)ethanone
Figure imgf000064_0001
A solution of 4-benzoyl-benzoic acid (500 mg, 2.21 mmol) in tetrahydrofuran was cooled to -78° C and 1.6 M solution of methyllithium (4.14 niL, 6.63 mmol) was added dropwise. The reaction was then allowed to stir for Ih at -78° C and then another 3h at room temperature. The reaction was then quenched by slowly adding saturated aqueous sodium bicarbonate and extracted with ethyl acetate. The organic layer was then dried over magnesium sulfate, filtered and evaporated under reduced pressure to yield l-(4-benzoyl- phenyl)-ethanone which was used without further purification. 1HNMR (CDCI3): δ 7.88 (d, J=8.1 Hz, 2H), 7.51 (d, J=8.4 Hz, 2H), 7.40 (d, J=7.2 Hz, 2H), 7.31 (t, J=6.6 Hz, 2H), 7.24 (t, J=6.6 Hz, IH), 2.56 (s, 3H). 13CNMR (CDCl3): δ 197.89, 153.34, 147.13, 135.54, 128.29, 128.24, 127.24, 125.92, 125.76, 26.56.
(b) l-(4-Benzoylphenyl)-2-bromoethanone
Figure imgf000064_0002
Bromine (353 mg, 2.21 mmol) was added to a solution of l-(4- benzoylphenyl)ethanone in chloroform and reacted to give the crude α-bromo ketone which was used without further purification after isolation.
(c) 7V-[4-(4-Benzoylphenyl)-thiazol-2-yl]-guanidine (13g)
The crude l-(4-benzoylphenyl)-2-bromoethanone was dissolved in acetone and 2- imino-4-thiobiuret (261 mg, 2.21 mmol) was added and the solution was heated under reflux to yield 152 mg (17% over 3 steps). 1HNMR (DMSO): δ 8.24 (s, br, 4H), 8.01 (d, J=4.8 Hz, 2H), 7.90 (d, J=4.8 Hz, 2H), 7.31 (s, IH), 7.29-7.21 (m, 5H). 13CNMR (DMSO): δ 197.48, 159.77, 153.67, 145.48, 139.34, 129.71, 129.18, 128.54, 128.03, 127.54, 127.31, 126.16, 108.94. MS, ESI, m/e 404 (MH+). Example 10. 7V-(4-(2-Guanidinothiazol-4-yl)phenyl)-2-naphthamide (13h)
Figure imgf000065_0001
(a) 7V-(4-Acetylphenyl)-2-naphthamide (Hh).
Figure imgf000065_0002
2-Naphthoyl chloride (I g, 5.25 mmol) was added to a solution of 4- aminoacetophenone (710 mg, 5.25 mmol) in 1 :1 dichloromethane/pyridine and allowed to stir for 4h. The solvent was then removed under reduced pressure and the oil was extracted with dichloromethane and saturated aqueous copper sulfate. The combined organic layers were then washed with water, dried over magnesium sulfate, filtered and evaporated under reduced pressure to yield 1.46 g (96%) of a white solid. 1HNMR (DMSO): δ 8.62 (s, IH), 8.12-8.09 (m, IH), 8.06 (m, 2H), 8.01 (s, 5H), 7.68-7.61 (m, 2H), 2.56 (s, 3H). 13CNMR (DMSO): δ 196.53, 165.96, 143.62, 134.32, 131.94, 131.79, 129.29, 128.95, 128.22, 128.04, 127.94, 127.64, 126.87, 124.39, 119.38, 26.42.
(b) 7V-(4-(2-Bromoacetyl)phenyl)-2-naphthamide (12h).
Figure imgf000065_0003
Bromine (123 mg, 0.773 mmol) was added dropwise to a solution of N-(4- acetylphenyl)-2-naphthamide (224 mg, 0.773 mmol) in acetic acid to give an α-bromoketone was carried on without further purification or characterization.
(c) 7V-(4-(2-Guanidinothiazol-4-yl)phenyl)-2-naphthamide (13h).
2-Imino-4-thiobiuret (42 mg, 0.353 mmol) was added to a solution of N-(4-(2- Bromoacetyl)phenyl)-2-naphthamide (130 mg, 0.353 mmol) and heated under reflux to yielding 33 mg (20%) of the title compound as a hydrobromide salt. 1HNMR (DMSO): δ 8.62 (s, IH), 8.24 (s, br, 4H), 8.12-7.93 (m, 9H), 7.72 (s, IH), 7.65 (m, 2H). 13CNMR (DMSO): δ 168.281, 160.314, 154.022, 148.829, 139.221, 134.719, 132.955, 130.594, 129.002, 128.626, 128.102, 127.037, 126.323, 126.417, 125.229, 125.016, 119.997, 106.621.
Example 11. 4-(2-Guanidinothiazol-4-yl)phenyl 2-naphthoate (13i)
Figure imgf000066_0001
(a) 4-Acetylphenyl 2-naphthoate (Hi).
Figure imgf000066_0002
4-Hydroxyacetophenone (713 mg, 5.24 mmol) was added to a slurry of sodium hydride (60% in mineral oil, 210 mg, 5.24 mmol) in tetrahydrofuran and was allowed to stir for 10 min. 2-Napthoyl chloride (I g, 5.24 mmol) was then added to this mixture dropwise and allowed to stir for 5h. The reaction was then quenched with water and extracted three times with ethyl acetate. The organic layer was dried over magnesium sulfate, filtered, and evaporated under reduced pressure to yield 952 mg (63%) of a white solid. 1HNMR (DMSO): δ 8.88 (s, IH), 8.22 (d, J=7.8 Hz, IH), 8.16-8.06 (m, 5H) 7.76-7.65 (m, 2H), 7.52 (d, J=8.7 Hz, 2H), 2.63 (s, 3H). 13CNMR (DMSO): δ 197.65, 165.06, 155.03, 136.10, 135.31, 132.76, 132.45, 130.67, 130.26, 129.82, 129.37, 128.48, 127.92, 126.49, 125.72, 123.03, 27.50. MS, APCI, m/e 373 (MH+).
(b) 4-(2-Bromoacetyl)phenyl 2-naphthoate (12i).
Figure imgf000066_0003
Bromine (511 mg, 3.19) was added dropwise to a solution of 4-acetylphenyl 2- naphthoate (928 mg, 3.19 mmol) in acetic acid and reacted to yield the α-bromoketone which was carried on without further purification or characterization. (c) 4-(2-Guanidinothiazol-4-yl)phenyl 2-naphthoate (13i).
2-Imino-4-thiobiuret (128 mg, 1.08 mmol) was added to a solution naphthalene-2- carboxylic acid 4-(2-bromoacetyl)phenyl 2-naphthoate (400 mg, 1.08 mmol) and the solution was heated under reflux yielding 425 mg (84%) of the title compound as a hydrobromide salt. 1HNMR (DMSO): δ 8.88 (s, IH), 8.22, (s, br, 4H), 8.14 (s, 2H), 8.09 (d, J=8.7 Hz, 4H), 7.84, (s, IH), 7.70 (m, 2H), 7.43 (d, J=8.7 Hz, 2H). 13CNMR (DMSO): δ 164.7191, 161.559, 154.923, 149.112, 140.111, 135.226, 133.558, 130.716, 129.221, 128.447, 128.201, 127.357, 126.273, 126.197, 125.219, 125.196, 120.197, 106.121. MS, ESI, m/e 389 (MH+).
Example 12. 7V-(4-(2-Guanidinothiazol-4-yl)phenyl)-l-naphthamide (13j)
Figure imgf000067_0001
(a) Naphthalene-1-carboxylic acid (4-acetyl-phenyl)-amide (Hj).
1-Naphthoyl chloride (2 g, 10.5 mmol) was added to a solution of 4- aminoacetophenone (1.42 g, 10.5 mmol) in 1 :1 dichloromethane/pyridine and allowed to stir for 4h. The solvent was then removed under reduced pressure and the oil was extracted with dichloromethane and saturated aqueous copper sulfate. The combined organic layers were then washed with water, dried over magnesium sulfate, filtered and evaporated under reduced pressure to yield 2.96 g (97%) of a white solid. 1HNMR (DMSO): δ 8.19 (m, IH), 8.11 (d, J=8.1 Hz, IH), 8.00 (m, 5H), 7.81 (d, J=6.9 Hz, IH), 7.63 (m, 3H), 2.57 (s, 3H). 13CNMR (DMSO): δ 196.53, 167.62, 143.57, 134.15, 133.08, 132.00, 130.40, 129.49, 129.39, 128.33, 127.10, 126.39, 125.68, 124.94, 118.98, 26.44.
(b) Naphthalene-1-carboxylic acid [4-(2-bromo-acetyl)-phenyl]-amide (12j).
Bromine (552 mg, 3.46 mmol) was added dropwise to a solution of naphthalene- 1- carboxylic acid (4-acetyl-phenyl)-amide (I g, 3.46 mmol) in chloroform and reacted to yield 1.18 g (93%) of the title compound. 1HNMR (DMSO): δ 8.20-8.17 (m, IH), 8.11 (d, J=8.1 Hz, IH), 8.07-7.97 (m, 5H), 7.81 (d, J=6.6 Hz, IH), 7.63 (m, 3H), 4.90 (s, 2H). (c) Naphthalene-1-carboxylic acid [4-(2-guanidino-thiazol-4-yl)-phenyl]-amide (13j).
2-Imino-4-thiobiuret (161 mg, 1.36 mmol) was added to a solution of naphthalene- 1- carboxylic acid [4-(2-bromo-acetyl)-phenyl] -amide (500 mg, 1.36 mmol) in acetone and the solution was heated under reflux to yield 461 mg (72%) of the title compound as a hydrobromide salt. 1HNMR (DMSO): δ 8.22 (m, br, 5H), 8.09 (d, J=8.5 Hz, IH), 8.04-8.02 (m, IH), 7.99 (d, J=9 Hz, 2H), 7.92 (d, J=8.5 Hz, 2H). 7.79 (d, J=6.5 Hz, IH), 7.71 (s, IH), 7.64-7.58 (m, 3H). 13CNMR (DMSO): δ 167.26, 160.24, 153.89, 149.53, 139.35, 134.48, 133.08, 130.17, 129.56, 128.45, 128.30, 126.99, 126.42, 126.33, 125.53, 124.98, 119.72, 106.88.
Example 13. N-[4-(4-Methoxyphenyl)-thiazol-2-yl]-guanidine (13m).
Figure imgf000068_0001
2-Imino-4-thiobiuret (695 mg, 5.88 mmol) was added to a solution of 2-bromo-l-(4- methoxy-phenyl)-ethanone (1.35 g, 5.88 mmol) and the mixture was heated under reflux to give 1.44 g (74%) of the hydrobromide salt of the title compound as a while solid. 1HNMR (DMSO): δ 8.25 (s, br, 4H), 7.88 (d, J=7.2 Hz, 2H), 7.61 (s, IH), 6.98 (d, J=7.2 Hz), 3.79 (s, 3H). 13CNMR (DMSO): δ 159.60, 159.27, 153.77, 149.60, 127.35, 125.77, 114.03, 106.02, 55.16.
Example 14. 4-(2-Aminothiazol-4-yl)phenyl biphenyl-4-carboxylate (14a)
Figure imgf000068_0002
(a) 4-Acetylphenyl biphenyl-4-carboxylate (Ha).
Figure imgf000069_0001
4-Hydroxyacetophenone (Ig, 7.35 mmol) was added to a slurry of NaH (60% in mineral oil, 294 mg, 7.35 mmol) in tetrahydrofuran and was allowed to stir for 10 min. 4- biphenylcarbonyl chloride (1.59 g, 7.35 mmol) was then added to this mixture dropwise and allowed to stir for 5h. The reaction was quenched with water and the precipitate was filtered and dried in a dessicator. The aqueous layer was then extracted three times with ethyl acetate. The organic layer was dried over magnesium sulfate, filtered and evaporated under reduced pressure to yield 2.08 g (89%) of a white solid. . 1HNMR (DMSO): δ 8.22 (d, J=6.9 Hz, 2H), 8.08 (d, J=7.2 Hz, 2H), 7.93 (d, J=6.1 Hz, 2H), 7.78 (d, J=6 Hz, 2H), 7.49 (m, 5H), 2.61 (s, 3H). 13CNMR (CDCl3): δ 164.75, 154.94, 146.90, 135.03, 131.01, 130.24, 129.23, 128.64, 127.90, 127.54, 122.17, 31.164, 26.86. MS, APCI, m/e 317 (MH+).
(b) 4-(2-Bromoacetyl)phenyl biphenyl-4-carboxylate (12a).
Figure imgf000069_0002
Bromine (757 mg, 4.74) was added dropwise to a solution of 4-acetylphenyl biphenyl- 4-carboxylate (1.5 g, 4.74 mmol) in acetic acid and reacted to yield 1.3 g (70%) of the title compound. 1HNMR (DMSO): δ 8.23 (d, J=7.8 Hz, 2H), 8.14 (d, J=8.1 Hz, 2H), 7.93 (d, J=8.1 Hz, 2H), 7.79 (d, J=6.9 Hz, 2H), 7.54-7.50 (m, 5H). 13CNMR (DMSO): δ 190.64, 163.86, 154.63, 145.49, 138.52, 131.66, 130.49, 129.06, 128.55, 127.06, 122.40, 34.05.
(c) Biphenyl-4-carboxylic acid 4-(2-amino-thiazol-4-yl)-phenyl ester (14a).
Thiourea (336.8 mg, 4.43 mmol) was added to a solution of 4-(2-bromoacetyl)phenyl biphenyl-4-carboxylate (350 mg, 1.11 mmol) in acetone and heated under reflux yielding 131.4 mg (24%) of the title compound as a hydrobromide salt. 1HNMR (DMSO): δ 8.206 (d, J=8.7 Hz, 2H), 7.923 (d, J=8.7 Hz, 2H), 7.805 (m, 4H), 7.492 (m, 5H), 7.263 (s, IH). 13CNMR (DMSO): δ 170.111, 164.309, 151.122, 145.5, 138.643, 130.564, 129.969, 129.195, 128.674, 127.492, 127.285, 127.192, 127.092, 122.625, 122.291, 103.028.
Example 15. 4-(2-Aminothiazol-4-yl)phenyl 4-*-butylbenzoate (14b)
Figure imgf000070_0001
May be prepared by a method analogous to that described in Example 14 starting from 4-hydroxyacetophenone.
Example 16. 4-*-Butyl-7V-(4-(2-aminothiazol-4-yl)phenyl)benzamide (14c)
Figure imgf000070_0002
(a) 7V-(4-Acetyl-phenyl)-4-*-butylbenzamide (lie).
Figure imgf000070_0003
4'-£-Butylbenzoyl chloride was added dropwise to a solution of 4-aminoacetophenone and TEA (2mL) in ethyl acetate. The solution was allowed to stir for 4h, was quenched with water and extracted with ethyl acetate three times. The organic layers were then dried over magnesium sulfate, filtered and evaporated under reduced pressure to yield 1.56 g (71%) of the amide. 1HNMR (DMSO): δ 7.965 (d, J=2.7 Hz, 4H), 7.907 (d, J=8.4 Hz, 2H), 7.560 (d, J=8.4 Hz, 2H), 2.547 (s, 3H), 1.322 (s, 9H). (b) 7V-[4-(2-Bromoacetyl)phenyl]-4-*-butylbenzamide (12c).
Figure imgf000071_0001
Bromine (1.29 g, 5.08 mmol) was added to a solution of JV-(4-acetylphenyl)-4-£- butylbenzamide (1.5 g, 5.08 mmol) in chloroform and reacted to yield 1.90 g (99%). 1HNMR (CDCl3): δ 8.108 (d, J=7.5 Hz, 2H), 7.82 (d, J=7.5 Hz, 4H), 7.511 (d, J=7.5 Hz, 2H), 6.693 (s, IH), 1.351 (s, 9H).
(c) 4-*-Butyl-7V-(4-(2-aminothiazol-4-yl)phenyl)benzamide (14c).
Thiourea (102 mg, 1.34 mmol) was added to a solution of 7V-[4-(2- bromoacetyl)phenyl]-4-£-butylbenzamide (500 mg, 1.34 mmol) in acetone and the solution was heated under reflux yielding 81.4 mg (14%) of the title compound as a hydrobromide salt. 1HNMR (DMSO): δ 7.933 (t, J=8.4 Hz, 4H), 7.613 (d, J=8.7 Hz, 2H), 7.735 (s, IH) 7.536 (d, J=8.7 Hz, 2H), 1.302 (s, 9H). 13CNMR (DMSO): δ 199.806, 169.829, 166.451, 162.089, 155.367, 141.712, 132.533, 130.025, 128.376, 125.862, 120.491, 31.592.
Example 17. 4-(2-Aminothiazol-4-yl)phenyl 4-n-butylbenzoate (14d)
Figure imgf000071_0002
May be prepared by a method analogous to that described in Example 14 starting from 4-hydroxyacetophenone and 4-n-butylbenzoyl chloride.
Example 18. 4-n-Butyl-7V-(4-(2-aminothiazol-4-yl)phenyl)benzamide (14e)
Figure imgf000071_0003
May be prepared by a method analogous to that described in Example 14 starting from 4-aminoacetophenone and 4-n-butylbenzoyl chloride.
Example 19. 4-(2-Aminothiazol-4-yl)phenyl 4-cyclopropylbenzoate (14f)
Figure imgf000072_0001
May be prepared as described in Example 8, except that the bromoketone is reacted with thiourea instead of 2-imino-4-thiobiuret in the last step as described in step (c) of Example 14.
Example 20. (4-(2-Aminothiazol-4-yl)phenyl)(phenyl)methanone (14g)
Figure imgf000072_0002
May be prepared as described in Example 9, except that the bromoketone is reacted with thiourea instead of 2-imino-4-thiobiuret in the last step as described in step (c) of Example 14.
Example 21. 7V-(4-(2-Aminothiazol-4-yl)phenyl)-2-naphthamide (14h)
Figure imgf000072_0003
May be prepared as described in Example 10, except that the bromoketone is reacted with thiourea instead of 2-imino-4-thiobiuret in the last step as described in step (c) of Example 14. Example 22. 4-(2-Aminothiazol-4-yl)phenyl 2-naphthoate (14i)
Figure imgf000073_0001
May be prepared as described in Example 11 , except that the bromoketone is reacted with thiourea instead of 2-imino-4-thiobiuret in the last step as described in step (c) of Example 14.
Example 23. 7V-(4-(2-guanidinothiazol-4-yl)phenyl)-l-naphthamide (14j)
Figure imgf000073_0002
May be prepared as described in Example 12, except that the bromoketone is reacted with thiourea instead of 2-imino-4-thiobiuret in the last step as described in step (c) of Example 14.
Example 24. 4-(2-Guanidinothiazol-4-yl)phenyl 4-(trifluoromethyl)benzenesulfonate (17a)
Figure imgf000073_0003
(a) 4-Acetylphenyl 4-(trifluoromethyl)benzenesulfbnate (15a).
Figure imgf000073_0004
4-(Trifluoromethyl)benzene sulfonyl chloride (I g, 4.09 mmol) was added to a solution of 4-hydroxyacetophenone (557 mg, 4.09 mmol), TEA (1 mL) and N,N-dimethyl-4- aminopyridine (10 mg, cat.) in dichloromethane. The reaction was allowed to stir for 2h and was then quenched with water and washed with IM HCl. The organic layer was then dried over magnesium sulfate, filtered and evaporated under reduced pressure to yield 1.37 g (97%) of a white solid. 1HNMR (DMSO): δ 8.13 (d, J=8.4 Hz, 2H), 8.07 (d, J=8.7 Hz, 2H), 7.99 (d, J=8.7 Hz, 2H), 7.24 (d, J=8.7 Hz, 2H), 2.56 (s, 3H). 13CNMR (DMSO): δ 196.67, 151.79, 135.82, 130.40, 129.30, 127.09, 122.24, 26.72.
(b) 4-(2-Bromoacetyl)phenyl 4-(trifluoromethyl)benzenesulfonate
Figure imgf000074_0001
Bromine (634 mg, 3.97 mmol) was added to a solution of 4-acetylphenyl A- (trifluoromethyl)benzenesulfonate (1.37 g, 3.97 mmol) in chloroform and reacted to give the title α-bromoketone which was carried on crude.
(c) 4-(2-Guanidinothiazol-4-yl)phenyl 4-(trifluoromethyl)benzenesulfonate (17a).
2-Imino-4-thiobiuret (469 mg, 3.97 mmol) was added to a solution of A-(I- bromoacetyl)phenyl 4-(trifluoromethyl)benzenesulfonate (1.68 g, 3.97 mmol) in acetone and brought to reflux to yield 1.31 g (63% over two steps) of the hydrobromide salt of the title compound. 1HNMR (DMSO): δ 8.21 (s, br, 4H), 8.12 (d, J=8.7 Hz, 2H), 8.08 (d, J=8.7 Hz, 2H), 8.00 (d, J=9 Hz, 2H), 7.83 (s, IH), 7.13 (d, J=8.7 Hz, 2H). 13CNMR (DMSO): δ 159.99, 153.63, 148.36, 148.26, 138.01, 132.44, 129.38, 127.72, 127.00, 122.36, 109.48.
Example 25. 4-(2-Guanidinothiazol-4-yl)phenyl 4-methylbenzenesulfonate (17b)
Figure imgf000074_0002
(a) 4-Acetylphenyl 4-methylbenzenesulfonate (15b).
Figure imgf000075_0001
p-Toluenesulfonyl chloride (I g, 5.25 mmol) was added to a solution of 4- hydroxyacetophenone (715 mg, 5.25 mmol), triethylamine (1 mL) and JV,iV-dimethyl-4- aminopyridine (10 mg, cat.) in dichloromethane. The reaction was allowed to stir for 2h and was then quenched with water and washed with IM NaOH. The organic layer was then dried over magnesium sulfate, filtered and evaporated under reduced pressure to yield 1.52 g (100%) of a white solid. 1HNMR (DMSO): δ 7.96 (d, J=8.7 Hz, 2H), 7.76 (d, J=8.4 Hz, 2H), 7.48 (d, J=8.1 Hz, 2H), 7.17 (d, J=8.7 Hz, 2H), 2.55 (s, 3H), 2.42 (s, 3H).
(b) 4-(2-Bromoacetyl)phenyl 4-methylbenzenesulfonate.
Figure imgf000075_0002
Bromine (826 mg, 5.17 mmol) was added to a solution of 4-acetylphenyl 4- methylbenzenesulfonate (1.5 g, 5.17 mmol) in chloroform and reacted to give the title α- bromoketone which was carried on crude.
(c) Synthesis of Toluene-4-sulfonic acid 4-(2-guanidino-thiazol-4-yl)-phenyl ester (17b).
2-Imino-4-thiobiuret (611 mg, 5.17 mmol) was added to a solution of 4-(2- bromoacetyl)phenyl 4-methylbenzenesulfonate (1.91 g, 5.17 mmol) in acetone and heated under reflux to yield 1.71 g (71% over two steps) as a hydrobromide salt. 1HNMR (DMSO): δ 8.20 (s, br, 4H), 7.97 (d, J=8.7 Hz, 2H), 7.81 (s, IH), 7.75 (d, J=8.1 Hz, 2H), 7.47 (d, J=8.4 Hz, 2H), 7.06 (d, J=9 Hz, 2H), 2.42 (s, 3H). 13CNMR (DMSO): δ 159.97, 153.64, 148.65, 148.38, 145.83, 132.08, 131.21, 130.21, 128.23, 127.54, 122.35, 109.26, 21.13. MS, ESI, m/e 389 (MH+). Example 26. 4-(2-Guanidinothiazol-4-yl)phenyl 4-^-butylbenzenesulfonate (17c)
Figure imgf000076_0001
(a) 4-Acetylphenyl 4-^-butylbenzenesulfonate (15c).
Figure imgf000076_0002
4-t-Butylbenzene sulfonyl chloride (I g, 4.29 mmol) was added to a solution of 4- hydroxyacetophenone (585 mg, 4.29 mmol), triethylamine (1 mL) and JV,iV-dimethyl-4- aminopyridine (10 mg, cat.) in dichloromethane. The reaction was allowed to stir for 2h and was then quenched with water and washed with IM NaOH. The organic layer was then dried over magnesium sulfate, filtered and evaporated under reduced pressure to yield 1.40 g (98%) of a white solid. 1HNMR (CDCl3): δ 7.90 (d, J=8.5 Hz, 2H), 7.75 (d, J=9Hz, 2H), 7.53 (d, J=10.5 Hz, 2H), 7.10 (d, J=9 Hz, 2H), 2.57 (s, 3H), 1.34 (s, 9H). 13CNMR (CDCl3): δ 196.90, 158.94, 153.18, 135.87, 132.31, 130.27, 128.53, 126.54, 122.71, 105.86, 35.62, 31.22, 26.87.
(b) 4-(2-bromoacetyl)phenyl 4-^-butylbenzenesulfonate
Figure imgf000076_0003
Bromine (673 mg, 4.21 mmol) was added dropwise to a solution of 4-acetylphenyl A- t-butylbenzenesulfonate (1.40 mg, 4.21 mmol) in chloroform and reacted to yield 1.73 g (100%) of the title α-bromoketone which was carried on crude. (c) 4-*-Butyl-benzenesulfonic acid 4-(2-guanidino-thiazol-4-yl)-phenyl ester (17c).
2-Imino-4-thiobiuret (497 mg, 4.21 mmol) was added to a solution of 4-(2- bromoacetyl)phenyl 4-t-butylbenzenesulfonate (1.73 g, 4.21 mmol) and heated under reflux to give 1.76 g (81% over 2 steps) of the title compound as a hydrobromide salt. 1HNMR (DMSO): δ 8.22 (s, br, 4H), 7.98 (d, J=8.7 Hz, 2H), 7.28-7.80 (m, 3H), 7.69 (d, J=8.7 Hz, 2H), 7.10 (d, J=8.7 Hz, 2H), 1.30 (s, 9H). 13CNMR (DMSO): δ 159.89, 158.23, 153.65, 148.62, 148.37, 132.05, 131.48, 128.02, 127.55, 126.61, 122.22, 109.24, 35.07, 30.56. MS, ESI, m/e 431 (MH+).
Example 27. 4-(2-Guanidinothiazol-4-yl)phenyl biphenyl-4-sulfonate (17d)
Figure imgf000077_0001
(a) 4-Acetylphenyl biphenyl-4-sulfonate (15d).
Figure imgf000077_0002
A solution of phenylboronic acid (172 mg, 1.41 mmol), 4-acetylphenyl 4- bromobenzenesulfonate (500 mg, 1.41 mmol) and cesium carbonate (691 mg, 2.12 mmol) in 3:1 1 ,2-dimethoxyethane/water was deoxygenated for 15 min. Pd(PPh3)4 (82 mg, 0.071 mmol) was then added and the solution was heated to 800C for 6h. The solution was then diluted with dichloromethane, filtered through diatomaceous earth filter aid (CELITE®) and extracted twice with dichloromethane. The organic layer was then dried over magnesium sulfate, filtered and evaporated under reduced pressure to an oil that was purified on a silica gel column (3:1 hexane/ethyl acetate) to yield 258 mg (52%). 1HNMR (CDCl3): δ 7.91 (t, J=9 Hz, 3H), 7.75 (d, J=7.5 Hz, 2H), 7.62 (d, J=8 Hz, 2H), 7.50 (t, J=7.5 Hz, 2H), 7.45 (t, J=6.5 Hz, 2H), 7.14 (d, J=8 Hz, 2H), 2.58 (s, 3H). 113X/ NMR (CDCl3): δ 196.04, 154.10, 152.91, 147.67, 136.00, 133.76, 130.10, 129.16, 128.97, 127.78, 127.36, 122.51, 121.77, 26.63.
(b) 4-(2-Bromoacetyl)phenyl biphenyl-4-sulfonate
Figure imgf000078_0001
Bromine (113 mg, 0.71 mmol) was added to a solution of biphenyl-4-sulfonic acid A- acetyl-phenyl ester (250 mg, 0.71 mmol) in chloroform and the mixture was reacted to give the title α-bromoketone which was carried on crude
(c) 4-(2-Guanidinothiazol-4-yl)phenyl biphenyl-4-sulfonate (17d)
2-Imino-4-thibiuret (84 mg, 0.71 mmol) was added to a solution of 4-(2- bromoacetyl)phenyl biphenyl-4-sulfonate (306 mg, 0.71 mmol) in acetone and the solution was heated under reflux to yield 60 mg (16% over 2 steps) of the title compound as a hydrobromide salt. 1HNMR (DMSO): δ 8.19 (s, br, 4H), 8.01-7.95 (m, 6H), 7.79 (t, J=6.6 Hz, 3H), 7.50 (m, 3H), 7.12 (d, J=8.7 Hz, 2H). 13CNMR (DMSO): δ 153.62, 148.60, 142.74, 137.63, 132.76, 132.167, 129.19, 129.05, 128.91, 127.75, 127.59, 127.16, 122.40, 118.45, 114.76, 109.32.
Example 28. 4-(2-Guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l- sulfonate (17e)
Figure imgf000078_0002
Dansyl chloride (750 mg, 2.78 mmol) was added to a solution of A- hydroxyacetophenone (379 mg, 2.78 mmol), triethylamine (1 mL) and N,N-dimethyl-4- aminopyridine (10 mg, cat.) in dichloromethane. The reaction was allowed to stir for 2h and was then quenched with water and washed with IM NaOH. The organic layer was then dried over magnesium sulfate, filtered and evaporated under reduced pressure to yield a yellow solid which was carried on crude. 1HNMR (CDCl3): δ 8.60 (d, J=8.4 Hz, IH), 8.45 (d, J=8.7 Hz, IH), 8.07 (d, J=7.5 Hz, IH), 7.79 (d, J=8.7 Hz, 2H), 7.68 (t, J=7.8 Hz, IH), 7.44 (t, J=7.5 Hz, IH), 7.25 (d, J=7.5 Hz, IH), 6.99 (d, J=8.7 Hz, 2H), 2.91 (s, 6H), 2.51 (s, 3H). 13CNMR (CDCl3): δ 196.56, 152.99, 151.97, 135.57, 132.28, 131.27, 130.56, 129.94, 129.70, 129.19, 122.90, 122.16, 119.15, 115.69, 45.40, 26.56. Bromine (444 mg, 2.78 mmol) was then added to a solution of the ketone (1.02 g, 2.78 mmol) in chloroform and the mixture was reacted to give an α-bromoketone which was carried on crude. 2-Imino-4-thiobiuret (328.5 mg, 2.78 mmol) was then added to a solution of the α-bromoketone in acetone and heated under reflux to give 203 mg (13% over 3 steps) of the title compound hydrobromide salt as a yellow solid. 1HNMR (DMSO): δ 8.70 (d, J=8.4 Hz, IH), 8.61 (d, J=8.7 Hz, IH), 8.40 (d, J=9 Hz, IH), 8.31 (d, J=8.4 Hz, IH), 8.16 (s, br, 4H), 8.08 (t, J=6.9 Hz, IH), 7.91- 7.86 (m, 2H), 7.75 (d, J=3 Hz, IH), 6.93 (t, J=8.7 Hz, 2H), 3.00 (s, 3H), 2.87 (s, 3H).
The compound exhibited fluorescence with an absorption wavelength of 360 nm and an emission wavelength of 560 nm.
Example 29. 7V-(4-(2-Guanidinothiazol-4-yl)phenyl)-4-methyl-7V- tosylbenzenesulfonamide (17f).
Figure imgf000079_0001
(a) N-(4-Acetylphenyl)-4-methyl-7V-tosylbenzenesulfonamide (15f).
Figure imgf000080_0001
p-Toluenesulfonyl chloride (I g, 5.25 mmol) was added to a solution of 4- aminoacetophenone (710 mg, 5.25 mmol), triethylamine (1 mL) and N,N-dimethyl-4- aminopyridine (10 mg, cat.) in dichloromethane. The reaction was allowed to stir for 2h and was then quenched with water and washed with IM HCl. The organic layer was then dried over magnesium sulfate, filtered and evaporated under reduced pressure to yield 1.04 g (69%) of a white solid. 1HNMR (DMSO): δ 8.00 (d, J=8.4 Hz, 2H), 7.70 (d, J=8.4 Hz, 4H), 7.49 (d, J=8.4 Hz, 4H), 7.16 (d, J=8.4 Hz, 2H), 2.61 (s, 3H), 2.45 (s, 6H). MS, ESI, m/e 444 (MH+).
(b) N-(4-(2-Bromoacetyl)phenyl)-4-methyl-7V-tosylbenzenesulfonamide.
Figure imgf000080_0002
Bromine (553 mg, 3.46 mmol) was added to a solution of bistoluene-4-sulfonic acid 4-acetyl-phenyl amide (I g, 3.46 mmol) in chloroform and the mixture was reacted to give the title compound which was carried on crude.
(c) 7V-(4-(2-Guanidinothiazol-4-yl)phenyl)-4-methyl-7V-tosylbenzenesulfonamide (17f).
2-Imino-4-thiobiuret (409 mg, 3.46 mmol) was added to a solution of bistoluene-4- sulfonic acid 4-(2-bromo-acetyl)-phenyl amide (1.27 g, 3.46 mmol) in acetone and heated under reflux to give 992 mg (46% over two steps) of the title compound as a hydrobromide salt. 1HNMR (DMSO): δ 8.20 (s, br, 4H), 8.02 (d, J=8.4 Hz, 2H), 7.90 (s, IH), 7.71 (d, J=8.4 Hz, 4H), 7.49 (d, J=8.4 Hz, 4H), 7.03 (d, J=8.7 Hz, 2H), 2.45 (s, 6H). 13CNMR (DMSO): δ 160.56, 153.79, 148.33, 145.43, 135.60, 134.73, 133.01, 131.52, 129.95, 127.98, 126.90, 110.21, 21.14. MS, ESI, m/e 542 (MH+).
Example 30. 3-(2-Guanidinothiazol-4-yl)phenyl 4-^-butylbenzenesulfonate (17g).
Figure imgf000081_0001
(a) 3-Acetylphenyl 4-^-butylbenzenesulfonate (15g).
Figure imgf000081_0002
4-t-Butyl-benzenesulfonyl chloride (I g, 4.29 mmol) was added to a solution of 3- aminoacetophenone (585 mg, 4.29 mmol), triethylamine (1 mL) and N,N-dimethyl-4- aminopyridine (10 mg, cat.) in dichloromethane. The reaction was allowed to stir for 2h and was then quenched with water and washed with IM HCl. The organic layer was then dried over magnesium sulfate, filtered and evaporated under reduced pressure to yield 1.37 g (96%) of a white solid. IH-NMR (CDCl3) δ 7.84 (d, IH, J=7.7Hz), 7.75 (d, 2H, J=8.4Hz), 7.54 (d, 2H, J=8.4Hz), 7.44 (s, IH), 7.41 (d, IH, J=7.9Hz), 7.26 (s, IH), 2.49 (s, 2H), 1.34 (s, 9H). 13CNMR (DMSO): δ 196.300, 158.650, 149.707, 138.496, 131.941, 129.914, 128.351, 127.116, 126.757, 126.235, 122.187, 35.351, 30.944, 26.539.
(b) 3-(2-Bromoacetyl)phenyl 4-^-butylbenzenesulfonate.
Figure imgf000081_0003
Bromine (660 mg, 4.12 mmol) was added to a solution of 3-acetylphenyl 4-t- butylbenzenesulfonate (1.37 g, 4.12 mmol) in chloroform and the mixture was reacted to give the title compound which was carried on crude. (c) 3-(2-Guanidinothiazol-4-yl)phenyl 4-^-butylbenzenesulfonate (17g).
2-Imino-4-thiobiuret (472.6 mg, 4.00 mmol) was added to a solution of 4-t-butyl- benzenesulfonic acid 3-(2-bromo-acetyl)-phenyl ester (1.645 g, 4.00 mmol) in acetone and heated under reflux to give 1.383 g (65% over two steps) of the title compound as a hydrobromide salt. 1H-NMR (DMSO) δ 8.23 (s, 4H), 7.90 (d, IH, J=7.8Hz), 7.79 (d, 2H, J=3.5Hz), 7.67 (d, 2H, J=8.6Hz), 7.44 (t, IH, J=8.0Hz), 7.01 (dd, IH, J=2.3Hz, J=8.2Hz), 1.26 (s, 9H). 13CNMR (DMSO): δ 159.938, 158.231, 153.610, 149.348, 134.719, 131.183, 130.296, 128.175, 126.5253, 124.736, 121.514, 119.653, 109.768, 35.033, 33.479.
Molecular Modelling
Modelling of potential myosin light chain kinase inhibitor was performed using PPlC structural data, beginning with calyculin, a potent inhibitor for myosin light chain phosphatase. The crystal structure of PPlC was obtained from the Protein Data Bank (structure 1JK7). The binding pocket was defined by a 15 angstrom sphere from the residue selection (Arg 96, He 130, lie 133, Tyr 134, Trp 206, Arg 221 and Tyr 272). Molecules to be docked into the crystal structure were built in the Sybyl shell and minimized using the conjugate gradient method with 1000 iterations or a stopping point of 0.01 kJ energy differential between conformers. The FlexX suite was then used to dock the ligands into the binding pocket of PPlC with an output of 30 conformers.
Flexible docking of proposed inhibitors onto the X-ray crystal structure of PPlC (PDB # 1JK7) was performed to assess potential binding conformations into the catalytic pocket of myosin light chain phosphatase. The X-ray crystal structure of the PPlC catalytic pocket is comprised of a bimetallic center and three adjoining grooves of which the most important region involved in substrate recognition is the hydrophilic groove Gupta, et al., J. Med. Chem. 1997, 40, 3199-3206; Goldberg, et al., Nature, 1995, 376, 745-753; Maynes, et al.,. J. Biol. Chem., 2001, 276, 47, 44078-44882; Kita, et al., Structure, 2002, 10, 715-724.
These compounds showed promising interactions with the catalytic pocket of PPlC as shown in Figure 1 for the compound of Example 23. Analysis of the flexible docking results suggested two possible binding modes for this class of compounds. The main binding conformation that the inhibitors adopt involves the carbonyl oxygen(s) of the linker group interacting with the bimetallic center of the catalytic site, leaving the hydrophobic tail of the molecule to interact with the βl2-13 loop of PPlC. The guanidino head group of the molecule forms a potential hydrogen bond network with a portion of the hydrophobic groove of the protein. On the other hand, when the linker group of the docked molecule was an amide, the main binding mode of this set of compounds was reversed so that the guanidino head group interacted with the metal center and the hydrophobic tail docked into the hydrophobic groove of the protein.
While not being bound by theory, it is believed that the hydrophobic groove and the βl2-13 loop may be important features in the interaction of inhibitors with the enzyme. The βl2-13 loop is a highly flexible and is the least conserved portion of the binding pocket among the members of the PPP superfamily, and together with the hydrophobic groove may be responsible for determining the substrate specificity imparted onto the catalytic subunit by the regulatory subunit MYPTl. It is believed that the compounds described herein may demonstrate selectivity to the specific holoenzyme myosin light chain phosphatase by forming interactions with these two regions of the protein. Beneficial effects on inhibitory potency are observed in compounds where a guanadino functionality on a thiazole ring is combined with an ester linkage. The guanidine group appears to enhance potency (see data below). Molecular modeling suggests a binding mode in which the electron rich carbonyl oxygen of the linker directly beneficially interacts with the bimetallic center of the catalytic pocket. It is believed that similarly beneficial interaction is achieved with a sulfonic ester linkage.
These favourable interations with the catalytic pocket of PPlC as shown in Figure 1 for 4-(2-guanidinothiazol-4-yl)phenyl 5 -(dimethylamino)naphthalene-l -sulfonate (Example 28, 17e) When docked into PPlC, the compound adopted a conformation that placed the guanidino portion of the molecule so as to form hydrogen bonds with the backbone carbonyl of Arg 221 in the hydrophobic groove and the dansyl portion of the molecule aligned in the βl2-13 loop region of the catalytic pocket.
Biological Evaluation
We first examined inhibition of the myosin light chain phosphatase complex and isolated PPlC with known natural PP1/PP2A inhibitors. These natural inhibitors inhibit both the recombinant PPlC and the myosin light chain phosphatase holoenzyme, but the IC50 at which they block these enzymes can be vastly different. For example, okadaic acid is 10-fold more potent towards myosin light chain phosphatase than PPlC, with an IC50 of 91 nM against the holoenzyme. In contrast, smaller compounds, cantharidin and cantharidic acid, are over 5 times more potent toward PPlC. These results suggest that PPlC is a poor model for assessing the binding interaction of myosin light chain phosphatase inhibitors. To avoid this problem, we used the purified myosin light chain phosphatase holoenzyme for all of our inhibition assays.
1. Myosin Light Chain Phosphatase Inhibition
The inhibitory potency of the thiazole compounds was examined by inhibition of purified myosin light chain phosphatase using 32P-myosin light chain as a substrate.
Myosin light chain phosphatase activity was measured using myosin light chain phosphatase purified from pig aorta smooth muscle, which consists of PPlC and a truncated version of 60-kDa MYPTl fragment. Eto, et al., J. Biochem. (Tokyo), 1995, 118, 1104-1107. PPlC was isolated from rabbit skeletal muscle by acetone treatment, as described by Martin et ah, Protein Expr. Purif., 1994, 5, 211-217. Inhibitors and myosin light chain phosphatase were preincubated for 10 min and then reaction was initiated by addition of 32P-labeled myosin light chain as a substrate. Eto, et al, Methods Enzymol., 2003, 366, 241-260. 32P- labeled myosin light chain was prepared using chicken gizzard myosin light chain kinase, calmodulin, isolated myosin light chain, and [γ-32P]ATP. After 10 min at 30 0C, reaction was terminated by addition of 10 % trichloroacetic acid, and released radioactivity of 32P1 in supernatant was measured by scintillation counter. The mean value for the duplicate assays was obtained, and myosin light chain phosphatase activity without inhibitor was set as 100 %. IC50 and error values were obtained by a nonlinear regression curve fitting, assuming first order binding, using Kaleidagraph software from the plot of relative activity against inhibitor concentration.
Figure 2 presents a typical inhibition curve of myosin light chain phosphatase activity and summary of IC50 values is given in Table 1. In Figure 2, the myosin light chain phosphatase activity is shown as a function of inhibitor concentration for the compound 4-(2- guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate (Example 28, 17e) (D), 2-Guanidinothiazol-4-yl)phenyl 3'-methoxybiphenyl-4-carboxylate (Example 1, 10a) ( A ) and amidinothiourea (H2NC(=NH)NHC(=S)NH2) (2-imino-4-thiobiuret) (#).
As shown in Table 1, the inhibitory potency appeared to be favoured by providing an extended hydrophobic region. Addition of a guanidine moiety to the aminothiazole also appeared to be beneficial for potency. No significant difference between carboxylic acid ester and amide as compared to sulfonic ester activities, suggesting that the nature of the linking group is not critical to activity.
Table 1. Myosin Light Chain Phosphatase Inihibitory Activites of Example Compounds
Figure imgf000085_0001
2. Expression of MCLP in androgen dependent and independent cell lines
To demonstrate the role of myosin light chain phosphatase in cancer cell lines, the expression levels of the components of myosin light chain phosphatase in various prostate cancer cell lines were measured by western blot analysis for MYPTl and PPlC. The levels of expression of MYPTl and PPlC were determined in both androgen receptor positive prostate cancer cell lines (LNCaP, C4-2 and C4-2B) and androgen receptor negative prostate cancer cell lines (CWR, DU145, PC-3 and PC-3M).
Cell pellets were disrupted in lysis buffer (7.4 pH, 5 mM EDTA, 50 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovandate, 1 mM phenylmethane sulfonyl fluoride (Sigma), 50 μL per 5 mL protease cocktail inhibitor (Sigma), and 1% Triton X-100) for one hour at 4 0C and then spun down at 13,000 rpm for 20 min. The supernatant was removed, and 20-30 μg of each extract were added to loading sample buffer and boiled for 5 min. This was then loaded onto a precast 4-12% Bis-Tris gel (Invitrogen). Electrophoresis was performed, and proteins were transferred onto a PDVF membrane (Biorad). The membrane was then blocked with a 1% w/v BSA solution (2.5 M NaCl, 1 M Tris HCl, pH 7.4) for one hour prior to incubation with the primary antibody overnight. The membrane was then washed and blocked for 30 min prior to incubation with the species-appropriate HRP-linked secondary antibody (1 :40,000, Jackson Immunoresearch) for one hour. The membrane was then washed and treated with ECL development kit (Perkin-Elmer) and exposed to film. The following antibodies were purchased from Cell Signaling Technology: PPlα, 1 :1000; phosphol8 PPlα, 1 :1000; phospho-MLC, 1 :1000. β- Actin (1 : 10,000) and MLC (1 :500) antibodies were purchased from Sigma.
The results in Figure 3 showed that levels of both MYPTl and PPlC were upregulated in the androgen receptor negative prostate cancer cell lines (CWR, DU145, PC-3 and PC-3M) as compared to androgen receptor positive prostate cancer cell lines (LNCaP, C4-2 and C4-2B). This is a valuable finding given that 80-90% of prostate cancer patients develop androgen-independent tumors 12-33 months after androgen ablation therapy and poorer outcome. See Hellerstedt, et al, CA Cancer J. CHn., 2002, 52, 154-179. The increased expression of myosin light chain phosphatase presents this enzyme as a strong target for treating these more aggressive types of cancer.
3. Effects of 4-(2-Guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l- sulfonate (Example 28, 17e) on Prostate Cancer Cells
(a) Chemotaxis Activity
Myosin light chain phosphatase inhibition on a cellular level should cause a direct loss of motility. To assess the effects of 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate (17e) on migration, a Boyden chamber experiment in a 96-well plate format was performed using a 96-well Boyden chamber MBA96 (Neuro Probe, Inc., Gaithersburg, MD). Insulin- like growth factor was used as the chemo-attractant since the migration of PC-3 cells is unaffected by serum gradients. The bottom wells were filled with 80μl chemoattractant or negative control per well and an 8μm porous membrane was placed on top. The cells were plated on top of the membrane at 200,000 cells per well in serum free media plus drug concentration. To the bottom half of the chamber was added IGF-I and the appropriate drug concentration. The chamber was then incubated overnight at 37 0C with 5% CO2. The chamber was disassembled and the membrane was fixed and stained using the Diff-Quik® Stain Set (Dade Behring, Deerfield, IL) to visualize the cells. The cells that migrated through the membrane to the bottom half of the well were stained and read at 595 nm on a plate reader. Briefly, the membrane was fixed in Diff-Quik® Fixative for 10 minutes and then placed first in Diff-Quik® Solution I for 3 minutes and then into Diff-Quik® Solution II for 3 minutes. The membrane was then washed with water three times and the cells on the upper surface were gently scraped off. The stained membrane was read directly in the spectrophotometer at 595 nm using a 96-well format.
The results of the experiment are shown in Figure 4. Error is represented as S.E.M., n=5. The absorbance represents the amount of stained cells that migrated through the 8 μM membrane. PC-3 cells did not significantly migrate in the absence of IGF-I. In the presence of IGF-I, 4-(2-guanidinothiazol-4-yl)phenyl 5 -(dimethylamino)naphthalene-l -sulfonate caused a dose dependent decrease in the migration of PC-3 cells through the membrane when compared to the control wells containing only IGF-I.
(b) Effect on Cell Cycle Distribution and Morphology
The effect of 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l- sulfonate (17e) on the cell cycle distribution of PC-3 cells was evaluated. PC-3 cells were plated at 200,000 cells per well in 6 well plates and incubated overnight. The media was then removed and replaced with serum free media and incubated for 24 h. Cells were then treated with media containing 1 μM 4-(2-guanidinothiazol-4-yl)phenyl
5-(dimethylamino)naphthalene-l -sulfonate (17e) for treated wells and serum free media for control wells. Cells were then incubated for 6, 24 and 48h, washed, trypsinized, and ethanol- fixed before submitting for cells cycle analysis.
The results of cell cycle analysis are shown in Figure 5. Shown in (a) is the distribution of PC-3 cells among Gl, S, and G2/M phases at time-points of 6, 24, and 48 hours with or without treatment with 1 μM of 5-(dimethylamino)naphthalene-l -sulfonate (17e) while (b) shows sub Gl populations of PC-3 cells treated for 48 hours as compared to control. Error is represented as S.E.M., n=3. Cell cycle analysis of PC-3 cells treated with A- (2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate (17e) for 8 h showed that the compound treatment resulted in a shift to S and G2/M phases and complete G2/M arrest after 24 h of treatment.
The cell cycle effects of 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (17e) on the cell cycle distribution of PC-3 cells were also reflected in changes in cellular morphology in that the PC-3 cells became more spherical and detached from the plate after 24 h treatment with 1 μM of the compound.
(c) Effect on Phosphorylation Levels of Various Proteins
To evaluate the effect of 4-(2-guanidinothiazol-4-yl)phenyl
5-(dimethylamino)naphthalene-l -sulfonate (17e) on the phosphorylation levels of various proteins, PC-3 cells were treated with 1 μM 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate and the lysates of cells treated for 30 min, 60 min, 2 h and 4 h were subjected to western blot analysis. Nocodazole was used as a positive control to differentiate between the effects of phosphatase inhibition and the effects of G2/M arrest on the protein phosphorylation levels. Additionally the selectivity of the effect of A-(I- guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate for increasing the phosphorylation of myosin light chain relative to other proteins was evaluated by Western blot analysis, using antibodies recognizing phosphorylation sites of Akt, PKC, PKA, MAPK/CDK, and CDK. Western blot analysis was performed as described in 2 above. The following antibodies were purchased from Cell Signaling Technology: PP lα, 1 :1000; phosphol8 PPlα, 1 :1000; phospho-MLC, 1 :1000; phospho-Akt substrate, 1 :1000; phospho- PKC substrate, 1 :500; phospho-PKA substrate, 1 :5000; phospho-MAPK/CDK substrate, 1 :5000; phospho-BAD, 1 :2000; phospho-CDK substrate, 1 :1000; phospho-p70S6 Thr 421/Ser 424, 1 :1000; phospho-p70S6 Thr 389, 1 :1000. β-actin (1 :10,000) and MLC (1 :500) antibodies were purchased from Sigma. Acetylated tubulin (1 :1000) was purchased from Zymed.
The results are shown in Figure 6. PC-3 cells treated with 4-(2-guanidinothiazol-4- yl)phenyl 5 -(dimethylamino)naphthalene-l -sulfonate showed a large increase in the levels of phosphorylated myosin light chain at 30 min, with the maximum effect occurring at 60 min. Increased phosphorylation of myosin light chain slowly decreased to near normal levels after 4 h. The nocodazole treated cells showed no change in phosphorylated myosin light chain levels at either 1 or 4 h. The inhibitory phosphorylation of PPl -alpha at Thr320 was only slightly changed by the treatment suggesting less potency of 17e towards the PPl alpha isoform. The increase in the amount of phosphorylated MLC in relation to the constant level of total MLC clearly demonstrated that 17e causes myosin light chain phosphatase inhibition in PC-3 cells. Further, the finding that another G2/M arresting agent, nocodazole, did not induce an increase in phosphorylated MLC demonstrated that this is not an effect of the cells being arrested in their cell cycle.
However, as is also shown in Figure 6, the effect on phosphorylation levels of proteins in PC-3 cells was selective to myosin light chain. A 4-h treatment of PC-3 cells with of 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate did not affect the staining pattern on the blots for Akt, PKC, PKA, MAPK/CDK, and CDK, indicating that detectable phosphorylation levels were unchanged by the treatment with the compound. In addition, phosphorylation of BAD at Serl l2 and p70S6 at Thr 389 and Thr 421/Ser 424 was unaffected by the compound treatment as well. Phospho-BAD is a known PPl substrate and its dephosphorylation induces apoptosis, whereas 4-(2-guanidinothiazol-4- yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate induces G2/M arrest without affecting phosphorylation of BAD. These results demonstrate that the compound induces an increase in myosin light chain phosphorylation by selectively inhibiting myosin light chain phosphatase.
(d) Effects on the growth of prostate cancer cells
In order to determine the effect of 4-(2-guanidinothiazol-4-yl)phenyl 5 -(dimethylamino)naphthalene-l -sulfonate on prostate cancer growth, a growth inhibition assay was performed against a panel of human prostate cancer cell lines.
LNCAP cells were plated onto a 96 well plate at 20,000 cells per well and incubated overnight in RPMI medium with 10% fetal bovine serum, 1% L-glutamine, 1% pen/strep and 0.1% DHT. The medium was removed and replaced with serum free medium plus the test compound (5 wells per concentration), then incubation continued for 48 h. 10 μL of WST-8 reagent solution was added to each well and incubation continued for 2 h. The plate was read using 450 nm as the measurement wavelength and 655 nm as the reference wavelength. The value for the initially plated cells was subtracted and the percent growth compared to the control was determined. For androgen independent cell lines, the medium used was RPMI with 10% FBS, 1% L-glutamine and 1% pen/strep. CWR cells were plated at 20,000 cells per well. DU145 and PC-3 were all plated at 5,000 cells per well. The amount growth of the cells was determined as a percentage of that observed for the control wells (no test compound).
The results of the growth inhibition experiment are shown in Table 2. The GIso for each of the cell lines demonstrate that the androgen receptor positive, androgen sensitive cell line LNCAP was significantly less sensitive to treatment with 4-(2-guanidinothiazol-4- yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate, having a GIso of 2 μM. The androgen independent cell lines all showed GIsos of about 0.5 μM. This demonstrates that androgen- insensitive prostate cell lines are more sensitive to treatment with 4-(2-guanidinothiazol-4- yl)phenyl 5 -(dimethylamino)naphthalene-l -sulfonate than androgen-sensitive prostate cells, as represented by the lines selected for study. The effects on proliferation correlated well with the rank order profile of MYPTl expression.
Table 2. Growth Inhibition Potency (GI50) of 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l-sulfonate versus Prostate Cancer Cell Lines
Figure imgf000090_0001
In order to rule out differing rates of growth as the source of the sensitivity to 4-(2- guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l-sulfonate, a BrdU uptake assay was performed with both androgen dependent and independent cell lines.
BrdU uptake was determined by using the FITC kit (BDPharmagen 51-2354AK) and following kit instructions. Cells were plated onto a 6 well plate and incubated overnight (500,000 per well for LNCAP, 300,000 per well for CWR and 200,000 per well for DU145 and PC-3). The medium was removed and replaced with serum free medium with 1 μM 4-(2- guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate and incubation was continued for 24 h. 10 μL of the BrdU solution was added to each treated well and incubated for one hour. The cells were trypsinized and washed twice with PBS and collected by centrifugation. The cell pellets were fixed with 4% formaldehyde for 10 min, permeablized and treated with an FTIC-tagged anti-BrdU antibody for 1 h at 25 °C and analyzed by flow cytometry.
The results of the BrdU-uptake assay are shown in Figure 7. When compared to control, the more aggressive cell lines in the dependent and independent progression models showed a greatly reduced level of incorporated BrdU, demonstrating that cell lines with increased expression of myosin light chain phosphatase are more sensitive to myosin light chain phosphatase inhibitors.
4. Immunofluorescence of Prostate Cancer Cells Treated with 4-(2-Guanidinothiazol-4- yl)phenyl 5-(dimethylamino)naphthalene-l-sulfonate (Example 28, 17e)
An advantageous property of the novel myosin light chain phosphatase inhibitor 4-(2- guanidinothiazol-4-yl)phenyl 5 -(dimethylamino)naphthalene-l -sulfonate is that it is fluorescent, which enabled the distribution of the compound in cells to be observed through fluorescence microscopy.
PC-3 and LNCAP cells were plated onto sterilized microscope slides and incubated overnight. The medium was removed and replaced with complete medium with 20 μM 4-(2-guanidinothiazol-4-yl)phenyl 5 -(dimethylamino)naphthalene-l -sulfonate and incubated for 60 min. The medium was removed, and the slides were washed four times with PBS. The cells were then fixed using 4% formaldehyde for 10 min. For actin staining, the cells were permeablized for 30 min with 0.1% Triton XlOO followed by treatment with Alexa fluor 680 conjugated phalloidin for 10 min. Slides were then washed four times with PBS. The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) using mounting medium containing DAPI. 4-(2-Guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l- sulfonate was imaged using a 360 nm excitation and a 560 nm emission.
The results are shown in Figure 8. Figure 8a shows untreated PC-3 cells stained with phalloidin to mark actin filaments and DAPI as a nuclear stain. Figure 8b shows untreated LNCaP cells stained with phalloidin to mark actin filaments and DAPI as a nuclear stain. Figure 8c shows PC-3 cells treated with 10 μM 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (Example 28, 17e) (showing cytoplasmic staining) showing the disruption in the actin cytoskeleton stained by phalloidin. Figure 8d shows LNCaP cells treated with 10 μM 2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (Example 28, 17e) (showing cytoplasmic staining) showing the disruption in the actin cytoskeleton stained by phalloidin. Figure 8e shows PC- 3 cells treated with 10 μM 2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l- sulfonate (Example 28, 17e) (showing cytoplamic staining) and DAPI nuclear staining. Figure 8f shows PC-3 cells treated with 10 μM 2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (Example 28, 17e) only showing cytoplasmic staining.
Actin filaments stained with phalloidine-Alexa Fuor 688 were disrupted in cells treated with 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate compared to control cells (Fig. 8a-d), indicating inhibition of myosin light chain phosphatase and reorganization of the actin cytoskeleton within cells. Indeed clumping of actin filaments and loss of their filamentous structure were evident in cells treated with 4-(2- guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate (Fig. 8c-d). Indeed, increased clumping of the microfilaments and a general loss of their filamentous nature was observed (Fig. 8c-d). This phenotype is consistent to the disruption of microfilaments induced by ectopic expression of CPI-17. Eto, et ah, Cell Motil Cytoskeleton 2000, 46, 222- 234. The fluorescence of 17e also allowed visualization of its entry and compartmentalization in the cells. After 60 min, PC-3 and LNCaP cells both showed high levels of 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate, but only in the cytoplasm of the cell (Fig. 8e-f). The localization of 4-(2-guanidinothiazol-4- yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate remained unchanged after 24 h of treatment in PC-3 cells. The results suggested that the compound rapidly enters the cell and that it does not enter the nucleus.
5. Immunofluorescence Studies of Potorous tridactylis PtK2 Kidney Epithelial Cells Treated with 4-(2-Guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l- sulfonate (Example 28, 17e)
Since we observed the disruption of the microfilament structure of the cells treated with 17e, we began to explore the full effects of this compound on the cellular cytoskeleton. PtK2 cells provide an excellent model to study internal cellular structural networks because they provide clear and precise images of the cytoskeleton.
The PtK2 cells were obtained from the American Type Tissue Collection and grown in the medium recommended by the supplier at 37 0C in a humidified 5% CO2 atmosphere. The cells were grown to confluence, disrupted by trypsinization, and seeded at about 10% confluence in a Lab-Tek II chamber slide obtained from Nalge Nunc International. The cells were grown for 2-3 days prior to drug treatment (final dimethyl sulfoxide concentration, 1% (v/v)) and for an additional 16 h following addition of drugs. Cells were washed twice with PBS, fixed with methanol at -20 0C for 10 min, treated with -20 0C acetone for 1 min, and washed twice with PBS. The cells were treated for 1 h at 22 0C in the dark with PBS containing 1.0 μg/mL DAPI, an FITC-conjugated anti-β-actin murine monoclonal antibody (Sigma product F 3022) at a 1 :250 dilution, and a Cy3 conjugated anti-β-tubulin antibody (clone TUB 2.2, Sigma product C 4585) at a 1 :100 dilution. The chamber slide was washed twice with PBS and air-dried. A coverslip was applied with antifade mounting solution. The cells were examined with a Nikon Model Eclipse E800 microscope equiped with epifluorescence and appropriate filters. Images were captured with a Spot digital camera, model 2.3.0, using version 3.0.2 software (Diagnostic Instruments). All images shown were obtained with a 4OX oil objective (N.A. 1.30).
The results of the imaging studies are shown in Figure 9. PtK2 cells are shown stained for microtubules (red fluorecscence in the cytoplasm in figures (a), (c), (e), (h), (j) and (I)) and microfilaments (green fluorescence in the cytoplasm in figures (b), (d), (f), (i), (k), and (m)). In each figure the nucleus is also stained (DAPI - blue). Figures (a) and (b) show control cells stained for (a) microtubules and (b) microfilaments. Figures (c) and (d) show cells treated with 0.2 μM colchinine stained for (c) microtubules and (d) microfilaments, showing disruption of microtubules. Figures (e) and (f) show cells treated with 0.2 μM jasplakinolide stained for (e) microtubules and (f) microfilaments, showing disruption of microfilaments. Figures (h)-(m) show cells treated with 4-(2-guanidinothiazol- 4-yl)phenyl 5 -(dimethylamino)naphthalene-l -sulfonate at 1 μM ((h),(i)), 5 μM ((j),(k)), and 10 μM ((l),(m)), and stained for microtutules ((h),(j),(l)) and microfilaments ((i),(k),(m)), showing that the compound disrupts both microfilaments and microtubules. Cells were treated for 16 h with each drug.
Most inhibitors of the cytoskeleton fall into one of two categories: inhibitors of tubulin or of actin. Colchicine and paclitaxel are two classic inhibitors of tubulin function, colchicine causing destabilization, while paclitaxel stabilizes microtubules. While these two compounds cause disruption of normal microtubule distribution, they have minimal effects on cellular actin microfilaments. Conversely, anti-actin compounds, like jasplakinolide, disrupt the microfilament system of the cell but leave the microtubules intact (Fig. 9a- f).
Treatment of PtK2 kidney epithelial cells with 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (17e) resulted in the disruption of both the microtubule and microfilament systems (Fig. 9h-m), with the most substantial effects observed with 4-(2-guanidinothiazol-4-yl)phenyl 5 -(dimethylamino)naphthalene-l -sulfonate at 10 μM. Myosin light chain phosphatase is directly involved in the action of actin filaments release from contraction. In addition to dephosphorylation of myosin light chain, the M21 subunit of myosin light chain phosphatase directly binds to tubulin microtubules. Takizawa, et al, Am. J. Physiol Cell Physiol, 2003, 284, C250-C262. These data show that inhibition of myosin light chain phosphatase by 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate causes a disruption in the regulation of both actin filaments and microtubules.
6. Effect on Cellular Morphology of Burkitt's Lymphoma Cells
As noted above, the cell cycle effects of 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (17e) on the cell cycle distribution of PC-3 cells were also reflected in changes in cellular morphology in that the PC-3 cells became more spherical and detached from the plate after 24 h treatment with 1 μM of the compound.
To further examine the effects of 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (17e) on the cell cycle, Burkitt's lymphoma cell line CA46 cells (stained with Giemsa) were assessed in order to determine the mitotic index. The results are shown in Figure 10 which shows: (a) untreated controls; and cells treated with (b) 0.2 μM colchinine; or 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l- sulfonate (17e) at (c) 5 μM and (d) 0.8 μM. Treatment with 4-(2-guanidinothiazol-4- yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate caused a high mitotic index in the CA46 cells and the arrested cells had clearly disrupted chromosomes.
7. Tissue and Cellular Distribution of 4-(2-Guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l-sulfonate (Example 28, 17e) and MYPTl in Prostate Cancer Tissue
Samples of prostate cancer tissue were stained with solutions of 100 μM 4-(2- guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate (Example 28, 17e) and MYPTl distribution in the samples was imaged by immunofluorescence using a MYPTl antibody.
Samples of prostate cancer tissue embedded in paraffin were sliced. After slicing, the tissue samples were de-waxed, rehydrated and epitope retrieval was conducted for 20 minutes at 1000C. Samples were then cooled at room temperature for 20 minutes. Samples were incubated with 10% goat serum for 10 minutes at room temperature to block non-specific binding sites. Blocking buffer was removed and primary antibody (MYPTl) was added to the samples at a dilution 1 :50 in tris-buffered saline and tween (TBST), supplemented with 5% goat serum for 2.5 hours at room temperature. After washing twice in TBST, the samples were incubated with goat-anti-rabbit-biotin antibody diluted at 1 :200 in TBST, for 30 minutes at room temperature. After the incubation, the antibody solution was removed and streptavidin conjugated Cy3 antibody added at a dilution of 1 : 100 in TBST for 10 minutes at room temperature. The samples were then exposed to compound 17e (100 μM) diluted in deionized water for 5 minutes at room temperature and then washed twice with deionized water. The slides were mounted in VectorShield mounting medium containing 4',6- diamidino-2-phenylindole (DAPI), and imaged.
The results of such imaging experiments are shown in Figures 11 to 17.
Figure 11 shows images of human prostate cancer tissue slices stained with 100 μM 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate (17e). Figure HA shows a bright field (DIC) image of the tissue, Figure HB shows the tissue stained with compound 17e (green fluorescence) while Figure HC shows a merger of images HA and HB. The compound is shown localized in the cytoplasm of the cells. Figure 12 shows control images with human prostate cancer tissue slices stained with 100 μM 1- dimethylamino-5-sulfamoylnaphthaline ("dansylamide"). Figure 12A is a bright field (DIC) image of the tissue, Figure 12B shows tissue stained with l-dimethylamino-5- sulfamoylnaphthaline (very faint fluorescence) while Figure 12C shows a merger of images 12A and 12B. In contrast to the strong fluorescence observed with compound 17e, only very faint fluorescence is shown for l-dimethylamino-5-sulfamoylnaphthaline, showing that the staining for compound 17e is due to specific binding.
Figures 13 to 17 show the relative localization of 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate (17e) and MYPTl in human prostate cancer tissue.
Figure 13 shows prostate cancer tissue slices stained with anti-MYPTl antibody and 4-(2-guanidinothiazol-4-yl)phenyl 5 -(dimethylamino)naphthalene- 1 -sulfonate (17e) . Figures 13 A to 13C show expression of MYPTl in prostate cancer cells. Figure 13 A shows human prostate cancer tissue slices with MYPTl distribution visualized by immunofluorescence using MYPTl antibody (red fluorescence), Figure 13B shows a bright field (DIC) image of the same cells, while Figure 13C shows a merger of images 13A and 13B. The figures demonstrate that MYPTl is expressed in the highly undifferentiated cancerous cells in the tissue. Figures 13E to 13H show a comparison of the distribution of 4-(2-guanidinothiazol- 4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate (17e) and MYPTl protein. Figure 13E shows human prostate cancer tissue slices stained with with MYPTl distribution visualized by immunofluorescence using MYPTl antibody, Figure 13F shows a bright field (DIC) image, Figure 13G shows the tissue stained with 100 μM of 4-(2-guanidinothiazol-4- yl)phenyl 5 -(dimethylamino)naphthalene-l -sulfonate (17e) (green fluorescence) and Figure 13H is a merger of images 13E, 13F and 13G. The figures demonstrate that compound 17e binds to the areas of the tissue where MYPTl is expressed.
Figures 14 and 15 both show co-localization of 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (17e) and MYPTl in prostate cancer cells. The cancer shown in Figure 15 is less aggressive (more differentiated cells) than that shown in Figure 14. Figures 14A and 15A show immunofluorescence with a MYPTl antibody (red fluorescence) (A). Figures 14B and 15B show bright field (DIC) images, Figures 14C and 15C show staining with compound 17e (green fluorescence), while Figures 14D and 15D show a merger of images 14A, 14B, and 14C (14D) and of images 15A, 15B, and 15C (15D) respectively.
Figures 16, 17 and 18 show distribution of 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate (17e) and MYPTl immunofluorescence in prostate cancer tissue and cells. Shown are merged images of cells stained with compound 17e (green fluorescence), MYPTl immunofluorescence (red fluorescence), and 4',6-diamidino-2- phenylindole (DAPI) with the brightest regions (purple fluorescence) indicating co- localization of MYPTl and compound 17e. Figure 16 shows low grade prostate cancer cells showing co-localization of MYPTl and compound 17e. Figure 17 shows moderately invasive prostate cancer cells showing co-localization of MYPTl and compound 17e. Figure 18 shows highly invasive prostate cancer cells showing co-localization of MYPTl and compound 17e. Based on the intensity of the fluorescence it appears that the expression of MYPTl as well as the amount of compound 17e is increased in more aggressive cancers.
All references cited herein are incorporated by reference. A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

Claims What is claimed is:
1. A compound according to formula I:
Figure imgf000098_0001
I or a salt form thereof; wherein:
Ar1 is selected from the group consisting of unsubstituted or substituted aryl and unsubstituted or substituted heteroaryl, wherein the substituents of the aryl or heteroaryl are selected from the group consisting of -R1; -(Ci-C3)alkylene-Ar2; (C2-C6)alkenyl; (C2-C6)alkynyl; halogen; -C≡N; -NO2; -C(=O)R3; -C(=O)OR3; -C(=O)NR4 2; -C(=NR3)NR4 2;-OR2; -OC(=O)(d-C6)alkyl;
-OC(=O)(Ci-C6)alkylene-R5; -OC(=O)O(d-C6)alkyl; -OC(=O)NR4 2; -NR4 2; -NR4C(=O)R3; -NR4C(=O)Ar2; -NR4C(=O)O(Ci-C6)alkyl; -NR4C(=O)NR4 2; -NR4SO2R3; -NR4SO2Ar2; -SR2; -S(O)R2; -SO2R2; -OSO2(C i-C6)alkyl; -OSO2Ar2; and -SO2NR4 2;
A is selected from the group consisting of -C(=0)- and -SO2-;
B is selected from the group consisting of a bond, -0-, -NR3-, and -N(-A- Ar1)-;
D is selected from the group consisting of -H and -C(=NH)-NH2; each R1 is independently unsubstituted (Ci-Ce)alkyl or (Ci-Ce)alkyl substituted with up to five halogen atoms and up to two substituents selected from the group consisting of -C≡N; -C(=0)R3; -C(=0)0R3; -C(=O)NR4 2; -OR3; -OC(=O)(Ci-C6)alkyl; -OC(=O)O(Ci-C6)alkyl; -OC(=O)NR4 2; -NR4 2; -NR3C(=O)R3; -NR3C(=O)NR4 2; -S(d-C6)alkyl; -S(O)(d-C6)alkyl; and -SO2(d-C6)alkyl; each R2 is independently selected from the group consisting of hydrogen, R1, Ar2 and (d-C3)alkylene-Ar2; each R is independently hydrogen or (Ci-Ce)alkyl; each R4 is independently hydrogen; (Ci-Ce)alkyl; -(C2-Ce)alkylene-OR3; -(Ci-C6)alkylene-C(=O)OR3; -(Ci-C6)alkylene-OC(=O)R3; -(C2-C6)alkylene-NR6 2; -(Ci-C6)alkylene-C(=O)NR6 2; -(Ci-C6)alkylene-NR3C(=O)R3;
-(Ci-C6)alkylene-NR3C(=O)NR6 2; Ar2, or -(Ci-C3)-alkyleneAr2; or, optionally, within any occurrence of NR4 2, independently of any other occurrence of NR4 2, the two R4 groups in combination are -(CH2)a- or -(CH2)bE(CH2)2-; each R5 is independently Ar2 or 1 ,4-benzoquinon-2-yl optionally substituted with 0, 1, 2, or 3 alkyl groups; each R6 is independently hydrogen; (Ci-Ce)alkyl; -(C2-C6)alkylene-OR3; -(Ci-C6)alkylene-C(=O)OR3; -(Ci-C6)alkylene-OC(=O)R3; (C2-C6)alkylene-NR3 2; -(Ci-C6)alkylene-C(=O)NR3 2; -(Ci-C6)alkylene-NR3C(=O)R3;
-(Ci-C6)alkyleneNR3C(=O)NR3 2; -Ar2, or -(d-C3)alkylene-Ar2; or, optionally, within any occurrence of NR6 2, independently of any other occurrence of NR6 2, the two R6 groups in combination are -(CH2)a- or -(CH2)bE(CH2)2-; each Ra is independently hydrogen; (Ci-Ce)alkyl; (C2-C6)alkenyl; (C2-C6)alkynyl; halogen; -C≡N; -NO2; -C(=O)R3; -C(=O)OR3; -C(=O)NR3 2; -C(=NR3)NR3 2; -OR3; -OC(=O)(d-C6)alkyl; -OC(=O)O(Ci-C6)alkyl; -OC(=O)NR3 2; -NR3 2; -NR3C(=O)R3; -NR3C(=O)O(d-C6)alkyl; -NR3C(=O)NR3 2; -S(Ci-C6)alkyl; -S(O)(Ci-C6)alkyl; and -SO2(Ci-C6)alkyl; and -SO2NR3 2; each Rb is independently hydrogen; (Ci-Ce)alkyl; (C2-C6)alkenyl; (C2-C6)alkynyl; halogen; -C≡N; -NO2; -C(=O)R3; -C(=O)OR3; -C(=O)NR3 2; -C(=NR3)NR3 2; -OR3; -OC(=O)(d-C6)alkyl; -OC(=O)O(d-C6)alkyl; -OC(=O)NR3 2; -NR3 2; -NR3C(=O)R3; -NR3C(=O)O(d-C6)alkyl; -NR3C(=O)NR3 2; -S(d-C6)alkyl; -S(O)(Ci-C6)alkyl; and -SO2(d-C6)alkyl; -SO2NR3 2; each Rc is independently hydrogen; (d-C6)alkyl; (C2-C6)alkenyl; (C2-C6)alkynyl; halogen; -C≡N; -NO2; -C(=O)R3; -C(=O)OR3; -C(=O)NR3 2; -C(=NR3)NR3 2; -OR3; -OC(=O)(d-C6)alkyl; -OC(=O)O(d-C6)alkyl; -OC(=O)NR3 2; -NR3 2; -NR3C(=O)R3; -NR3C(=O)O(d-C6)alkyl; -NR3C(=O)NR3 2; -S(d-C6)alkyl; -S(O)(Ci-C6)alkyl; and -SO2(d-C6)alkyl; -SO2NR3 2; each Rd is independently hydrogen; (Ci-Ce)alkyl; (C2-C6)alkenyl; (C2-C6)alkynyl; halogen; -C≡N; -NO2; -C(=O)R3; -C(=O)OR3; -C(=O)NR3 2; -C(=NR3)NR3 2; -OR3; -OC(=O)(d-C6)alkyl; -OC(=O)O(d-C6)alkyl; -OC(=O)NR3 2; -NR3 2; -NR3C(=O)R3; -NR3C(=O)O(d-C6)alkyl; -NR3C(=O)NR3 2; -S(d-C6)alkyl; -S(O)(Ci-C6)alkyl; and -SO2(d-C6)alkyl; -SO2NR3 2; each a is independently selected from the group consisting of 4, 5, and 6; each b is independently selected from the group consisting of 2 and 3; each E is independently selected from the group consisting of O, S, NR ; NC(=0)R3; NSO2R3; N(C2-C6)alkylene-OR3; N(d-C6)alkylene-C(=O)OR3; N(Ci-C6)alkylene-OC(=O)R3; N(C2-C6)alkylene-NR3 2;
N(Ci-C6)alkylene-C(=O)NR3 2; N(Ci-C6)alkylene-NR3C(=O)R3;
N(Ci-C6)alkylene-NR3C(=O)NR3 2; NAr2; N(Ci-C3)alkylene-Ar2; and NC(=O)Ar2; and each Ar2 is independently selected from the group consisting of unsubstituted aryl, unsubstituted heteroaryl, and aryl or heteroaryl substituted with one or more substitutents independently selected from the group consisting of (Ci-Ce)alkyl; (C2-C6)alkenyl; (C2-C6)alkynyl; halogen; -C≡N; -NO2; -C(=O)R3; -C(=O)OR3; -C(=O)NR3 2; -C(=NR3)NR3 2; -OR3; -OC(=O)(Ci-C6)alkyl; -OC(=O)O(Ci-C6)alkyl; -OC(=O)NR3 2; -NR3 2; -NR3C(=O)R3; -NR3C(=O)O(d-C6)alkyl; -NR3C(=O)NR3 2; -S(Ci-C6)alkyl; -S(O)(Ci-C6)alkyl; and -SO2(Ci-C6)alkyl; -SO2NR3 2; and (Ci-C3)perfluoroalkyL
2. A compound according to claim 1 , or a salt form thereof, wherein A is -C(=0)-.
3. A compound according to claim 1, or a salt form thereof, wherein A is -SO2-.
4. A compound according to any one of claims 1 to 3, or a salt form thereof, wherein B is -0-.
5. A compound according to any one of claims 1 to 3, or a salt form thereof, wherein B is -NR3-.
6. A compound according to any one of claims 1 to 5, or a salt form thereof, wherein D is -H.
7. A compound according to one of claims 1 to 5, or a salt form thereof, wherein D is -C(=NH)-NH2.
8. A compound according to any one of claims 1 to 7, wherein each of Ra, Rb, Rc, and Rd is hydrogen.
9. A compound according to any one of claims 1 to 8, or a salt form thereof, wherein Ar1 is unsubstituted or substituted phenyl.
10. A compound according to any one of claims 9, or a salt form thereof, wherein Ar1 is substituted phenyl substituted in at least the 4-position.
11. A compound according to claim 10, or a salt form thereof, wherein Ar1 is monosubstituted phenyl substituted in the 4-position.
12. A compound according to any one of claims 1 to 8, or a salt form thereof, wherein Ar1 is unsubsituted or substituted biphenyl-4-yl.
13. A compound according to claim 12, or a salt form thereof, wherein Ar1 is unsubsituted or substituted biphenyl-4-yl which is unsubstituted in at least the phenylene ring thereof.
14. A compound according to any one of claims 1 to 8, or a salt form thereof, wherein Ar1 is unsubsituted or substituted naphthyl.
15. A compound according to claim 14, or a salt form thereof, wherein Ar1 is substituted naphthyl.
16. A compound according to claim 14, or a salt form thereof, wherein Ar1 is substituted or unsubstituted 1 -naphthyl.
17. A compound according to claim 16, or a salt form thereof, wherein Ar1 is substituted 1 -naphthyl.
18. A compound according to claim 17, or a salt form thereof, wherein Ar1 is monosubstituted 1 -naphthyl.
19. A compound according to claim 17, or a salt form thereof, wherein Ar1 is substituted 1 -naphthyl, wherein the 5-position of the 1 -naphthyl is substituted by a substituent selected from the group consisting of -OR2; -OC(=O)(Ci-C6)alkyl; -OC(=O)(Ci-C6)alkylene-R5; -OC(=O)O(d-C6)alkyl; -OC(=O)NR4 2; -NR4 2; -NR4C(=O)R3; -NR4C(=O)Ar2; -NR4C(=O)O(Ci-C6)alkyl; -NR4C(=O)NR4 2; -NR4SO2R3; -NR4SO2Ar2; -SR2; -OSO2(Ci-C6)alkyl; and -OSO2Ar2.
20. A compound according to claim 17, or a salt form thereof, wherein Ar1 is substituted 1-naphthyl, wherein the 5-position of the 1-naphthyl is substituted by a substituent selected form the group consisting of -OR2 and -NR4 2.
21. A compound according to claim 17, or a salt form thereof, wherein Ar1 is substituted 1-naphthyl, wherein the 5-position of the 1-naphthyl is substituted by a substituent selected form the group consisting of -OH, O(Ci-C6)alkyl, -NH2, -NH(Ci-C6)alkyl, and -N((Ci-C6)alkyl)2.
22. A compound according to claim 18, or a salt form thereof, wherein Ar1 is monosubstituted 1-naphthyl, wherein the 5-position of the 1-naphthyl is substituted by a substituent selected from the group consisting of -OR2; -OC(=O)(Ci-C6)alkyl; -OC(=O)(Ci-C6)alkylene-R5; -OC(=O)O(Ci-C6)alkyl; -OC(=O)NR4 2; -NR4 2;
Figure imgf000102_0001
-NR4SO2R3; -NR4SO2Ar2; -SR2; -OSO2(Ci-C6)alkyl; and -OSO2Ar2.
23. A compound according to claim 18, or a salt form thereof, wherein Ar1 is monosubstituted 1-naphthyl, wherein the 5-position of the 1-naphthyl is substituted by a substituent selected from the group consisting of -OR2 and -NR4 2.
24. A compound according to claim 18, or a salt form thereof, wherein Ar1 is monosubstituted 1-naphthyl, wherein the 5-position of the 1-naphthyl is substituted by a substituent selected from the group consisting of -OH, O(Ci-Ce)alkyl, -NH2, -NH(Ci-C6)alkyl, and -N((Ci-C6)alkyl)2.
25. A compound according to claim 18, or a salt form thereof, wherein Ar1 is monosubstituted 1-naphthyl, wherein the 5-position of the 1-naphthyl is substituted by -N((Ci-C6)alkyl)2.
26. A compound according to claim 18, or a salt form thereof, wherein Ar1 is monosubstituted 1-naphthyl, wherein the 5-position of the 1-naphthyl is substituted by -N(CHs)2.
27. A compound according to claim 1, or a salt form thereof, wherein A is -C(=0)-, B is -0-, D is -C(=NH)-NH2, and each of Ra, Rb, Rc and Rd is hydrogen.
28. A compound according to claim 1, or a salt form thereof, wherein A is -C(=O)-, B is -NH-, D is -C(=NH)-NH2, and each of Ra, Rb, Rc and Rd is hydrogen.
29. A compound according to claim 1, or a salt form thereof, wherein A is -C(=O)-, B is -O-, D is -C(=NH)-NH2, each of Ra, Rb, Rc and Rd is hydrogen, and Ar1 is 4-substituted phenyl.
30. A compound according to claim 29, or a salt form thereof, wherein Ar1 is 4-monosubstituted phenyl.
31. A compound according to claim 29, or a salt form thereof, wherein the compound is selected from the group consisting of:
4-(2-guanidinothiazol-4-yl)phenyl 4-t-butylbenzoate; 4-(2-guanidinothiazol-4-yl)phenyl 4-n-butylbenzoate; 4-(2-guanidinothiazol-4-yl)phenyl 4-cyclopropylbenzoate; and salt forms of any thereof.
32. A compound according to claim 1, or a salt form thereof, wherein A is -C(=O)-, B is -O-, D is -C(=NH)-NH2, each of Ra, Rb, Rc and Rd is hydrogen, and Ar1 is biphenyl-4-yl.
33. A compound according to claim 32, or a salt form thereof, wherein the compound is selected from the group consisting of:
4-(2-guanidinothiazol-4-yl)phenyl biphenyl-4-carboxylate; 4-(2-guanidinothiazol-4-yl)phenyl 3'-methoxybiphenyl-4-carboxylate; 4-(2-guanidinothiazol-4-yl)phenyl 4'-methoxybiphenyl-4-carboxylate; and salt forms of any thereof.
34. A compound according to claim 1, or a salt form thereof, wherein A is -C(=O)-, B is -O-, D is -C(=NH)-NH2, each of Ra, Rb, Rc and Rd is hydrogen, and Ar1 is naphthyl.
35. A compound according to claim 34, or a salt form thereof, wherein the compound is selected from the group consisting of:
4-(2-guanidinothiazol-4-yl)phenyl 1 -naphthoate; 4-(2-guanidinothiazol-4-yl)phenyl 2-naphthoate; and salt forms of any thereof.
36. A compound according to claim 1, or a salt form thereof, wherein A is -C(=O)-, B is -NH-, D is -C(=NH)-NH2, each of Ra, Rb, Rc and Rd is hydrogen, and Ar1 is 4-substituted phenyl.
37. A compound according to claim 36, or a salt form thereof, wherein Ar1 is 4-monosubstituted phenyl.
38. A compound according to claim 36, or a salt form thereof, wherein the compound is selected from the group consisting of:
4-n-butyl-Λ/-(4-(2-guanidinothiazol-4-yl)phenyl)benzamide; 4-£-butyl-Λ/-(4-(2-guanidinothiazol-4-yl)phenyl)benzamide; and salt forms of any thereof.
39. A compound according to claim 1, or a salt form thereof, wherein A is -C(=O)-, B is -NH-, D is -C(=NH)-NH2, each of Ra, Rb, Rc and Rd is hydrogen, and Ar1 is biphenyl-4-yl.
40. A compound according to claim 34, or a salt form thereof, wherein the compound is selected from the group consisting of:
Λ/-(4-(2-guanidinothiazol-4-yl)phenyl)biphenyl-4-carboxamide; and salt forms thereof.
41. A compound according to claim 1, or a salt form thereof, wherein A is -C(=O)-, B is -NH-, D is -C(=NH)-NH2, each of Ra, Rb, Rc and Rd is hydrogen, and Ar1 is naphthyl.
42. A compound according to claim 41, or a salt form thereof, wherein the compound is selected from the group consisting of:
Λ/-(4-(2-guanidinothiazol-4-yl)phenyl)naphthalene-l-carboxamide; Λ/-(4-(2-guanidinothiazol-4-yl)phenyl)naphthalene-2-carboxamide; and salt forms of any thereof.
43. A compound according to claim 1, or a salt form thereof, wherein A is -SO2-, B is -O-, D is -C(=NH)-NH2, each of Ra, Rb, Rc and Rd is hydrogen, and Ar1 is 4-substituted phenyl.
44. A compound according to claim 43, or a salt form thereof, wherein Ar1 is 4-monosubstituted phenyl.
45. A compound according to claim 43, or a salt form thereof, wherein the compound is selected from the group consisting of:
4-(2-guanidinothiazol-4-yl)phenyl 4-(trifluoromethyl)benzenesulfonate; 4-(2-guanidinothiazol-4-yl)phenyl 4-methylbenzenesulfonate; 4-(2-guanidinothiazol-4-yl)phenyl 4-t-butylbenzenesulfonate; and salt forms of any thereof.
46. A compound according to claim 1, or a salt form thereof, wherein A is -SO2-, B is -O-, D is -C(=NH)-NH2, each of Ra, Rb, Rc and Rd is hydrogen, and Ar1 is biphenyl-4-yl.
47. A compound according to claim 46, or a salt form thereof, wherein the compound is selected from the group consisting of:
4-(2-guanidinothiazol-4-yl)phenyl biphenyl-4-sulfonate; and salt forms thereof.
48. A compound according to claim 1, or a salt form thereof, wherein A is -SO2-, B is -O-, D is -C(=NH)-NH2, each of Ra, Rb, Rc and Rd is hydrogen, and Ar1 is naphthyl.
49. A compound according to claim 48, or a salt form thereof, wherein the compound is selected from the group consisting of:
4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene- 1 -sulfonate; and salt forms thereof.
50. A compound according to claim 6, wherein each of Ra, Rb, Rc and Rd is hydrogen.
51. A compound according to claim 50, or a salt form thereof, wherein the compound is selected from the group consisting of:
4-(2-aminothiazol-4-yl)phenyl biphenyl-4-carboxylate; 4-(2-aminothiazol-4-yl)phenyl 4-t-butylbenzoate; 4-£-butyl-Λ/-(4-(2-aminothiazol-4-yl)phenyl)benzamide; 4-(2-aminothiazol-4-yl)phenyl 4-n-butylbenzoate; 4-/?-butyl-Λ/-(4-(2-aminothiazol-4-yl)phenyl)benzamide; 4-(2-aminothiazol-4-yl)phenyl 4-cyclopropylbenzoate; (4-(2-aminothiazol-4-yl)phenyl)(phenyl)methanone; Λ/-(4-(2-aminothiazol-4-yl)phenyl)- 1 -naphthamide; Λ/-(4-(2-aminothiazol-4-yl)phenyl)-2-naphthamide; 4-(2-aminothiazol-4-yl)phenyl 2-naphthoate; and salt forms thereof.
52. A compound according to any one of claims 1 to 51, wherein the compound is fluorescent.
53. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound according to any one of claims 1 to 52, or a pharmaceutically acceptable salt form thereof.
54. A pharmaceutical composition according to claim 53, wherein the compound is selected from the group consisting of:
JV-(4-(2-guanidinothiazol-4-yl)phenyl)- 1 -naphthamide; Λ/-(4-(2-guanidinothiazol-4-yl)phenyl)-2-naphthamide; 4-(2-guanidinothiazol-4-yl)phenyl 4-t-butylbenzoate; 4-(2-guanidinothiazol-4-yl)phenyl 4-n-butylbenzoate; 4-(2-guanidinothiazol-4-yl)phenyl 4-cyclopropylbenzoate; N-[4-(4-benzoylphenyl)-thiazol-2-yl]-guanidine; 4-(2-guanidinothiazol-4-yl)phenyl biphenyl-4-carboxylate; 4-(2-guanidinothiazol-4-yl)phenyl 3'-methoxybiphenyl-4-carboxylate; 4-(2-guanidinothiazol-4-yl)phenyl 4'-methoxybiphenyl-4-carboxylate; 4-(2-guanidinothiazol-4-yl)phenyl 1 -naphthoate; 4-(2-guanidinothiazol-4-yl)phenyl 2-naphthoate; 4-n-butyl-Λ/-(4-(2-guanidinothiazol-4-yl)phenyl)benzamide; 4-£-butyl-Λ/-(4-(2-guanidinothiazol-4-yl)phenyl)benzamide; Λ/-(4-(2-guanidinothiazol-4-yl)phenyl)biphenyl-4-carboxamide; 4-(2-guanidinothiazol-4-yl)phenyl 4-(trifluoromethyl)benzenesulfonate; 4-(2-guanidinothiazol-4-yl)phenyl 4-methylbenzenesulfonate; 4-(2-guanidinothiazol-4-yl)phenyl 4-t-butylbenzenesulfonate; 4-(2-guanidinothiazol-4-yl)phenyl biphenyl-4-sulfonate; 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene- 1 -sulfonate; 4-(2-aminothiazol-4-yl)phenyl biphenyl-4-carboxylate; 4-(2-aminothiazol-4-yl)phenyl 4-t-butylbenzoate; 4-t-butyl-Λ/-(4-(2-aminothiazol-4-yl)phenyl)benzamide; 4-(2-aminothiazol-4-yl)phenyl 4-n-butylbenzoate; 4-/?-butyl-Λ/-(4-(2-aminothiazol-4-yl)phenyl)benzamide; 4-(2-aminothiazol-4-yl)phenyl 4-cyclopropylbenzoate; (4-(2-aminothiazol-4-yl)phenyl)(phenyl)methanone; N-(4-(2-aminothiazol-4-yl)phenyl)- 1 -naphthamide; Λ/-(4-(2-aminothiazol-4-yl)phenyl)-2-naphthamide; 4-(2-aminothiazol-4-yl)phenyl 2-naphthoate;
Λ/-(4-(2-guanidinothiazol-4-yl)phenyl)-4-methyl-Λ/-tosylbenzenesulfonamide; 3 -(2-guanidinothiazol-4-yl)phenyl 4-t-butylbenzenesulfonate; and pharmaceutically acceptable salt forms of any thereof.
55. A pharmaceutical composition according to claim 53, wherein the compound is selected from the group consisting of:
4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene- 1 -sulfonate; and pharmaceutically acceptable salt forms thereof.
56. A therapeutic method comprising administering a compound according to any one of claims 1 to 52, or a pharmaceutically acceptable salt form thereof, to an individual.
57. A method of inhibiting myosin light chain phosphatase comprising contacting an effective amount of a compound according to any one of claims 1 to 52, or a salt form thereof, with myosin light chain phosphatase.
58. A method according to claim 57, wherein the contacting is achieved by causing an effective amount of the compound, or a salt form thereof, to be present in an individual, thereby inhibiting myosin light chain phosphatase in vivo.
59. A method according to claim 58, wherein the contacting is achieved by administering the compound, or a pharmaceutically acceptable salt form thereof, to an individual.
60. A method according to claim 57, wherein the contacting is performed in vitro.
61. A method of inhibiting protein phosphatase 1C, comprising contacting an effective amount of a compound according to any one of claims 1 to 52, or a salt form thereof, with protein phosphatase 1C.
62. A method according to claim 61, wherein the contacting is achieved by causing an effective amount of the compound, or a salt form thereof, to be present in an individual, thereby inhibiting protein phosphatase 1C in vivo.
63. A method according to claim 62, wherein the contacting is achieved by administering the compound, or a pharmaceutically acceptable salt form thereof, to an individual.
64. A method according to claim 61, wherein the contacting is performed in vitro.
65. A method of increasing the level of phosphorylation of myosin light chain in a cell comprising contacting an effective amount of a compound according to any one of claims 1 to 52, or a salt form thereof, with the cell.
66. A method according to claim 65, wherein the contacting is achieved by causing an effective amount of the compound, or a salt form thereof, to be present in an individual, thereby increasing the level of phosphorylation of myosin light chain phosphatase in vivo.
67. A method according to claim 66, wherein the contacting is achieved by administering an effective amount of the compound, or a pharmaceutically acceptable salt form thereof, to an individual.
68. A method according to claim 65, wherein the contacting is performed in vitro.
69. A method of increasing the level of phosphorylation of Ras-1 in a cell comprising contacting an effective amount of a compound according to any one of claims 1 to 52, or a salt form thereof, with the cell.
70. A method according to claim 69, wherein the contacting is achieved by causing an effective amount of the compound, or a salt form thereof, to be present in an individual, thereby increasing the level of Ras-1 in vivo.
71. A method according to claim 70, wherein the contacting is achieved by administering an effective amount of the compound, or a pharmaceutically acceptable salt form thereof, to an individual.
72. A method according to claim 69, wherein the contacting is performed in vitro.
73. A method of inhibiting actin polymerization in a cell comprising contacting an effective amount of a compound according to any one of claims 1 to 52, or a salt form thereof, with the cell.
74. A method according to claim 73, wherein the contacting is achieved by causing an effective amount of the compound, or a salt form thereof, to be present in an individual, inhibiting actin polymerization in vivo.
75. A method according to claim 72, wherein the contacting is achieved by administering an effective amount of the compound, or a pharmaceutically acceptable salt form thereof, to an individual.
76. A method according to claim 74, wherein the contacting is performed in vitro.
11. A method of inhibiting tubulin polymerization in a cell comprising contacting an effective amount of a compound according to any one of claims 1 to 52, or a salt form thereof, with the cell.
78. A method according to claim 77, wherein the contacting is achieved by causing an effective amount of the compound, or a salt form thereof, to be present in an individual, thereby inhibiting tubulin polymerization in vivo.
79. A method according to claim 78, wherein the contacting is achieved by administering an effective amount of the compound, or a pharmaceutically acceptable salt form thereof, to an individual.
80. A method according to claim 77, wherein the contacting is performed in vitro.
81. A method of treating a disease or condition associated with myosin light chain phosphatase activity, comprising causing an effective amount of a compound according to any one of claims 1 to 52, or a salt form thereof, to be present in an individual in need of such treatment.
82. A method according to 81, wherein the causing is achieved by administering an effective amount of the compound, or a pharmaceutically acceptable salt form thereof, to the individual.
83. A method according to claim 81 or 82, wherein the disease or condition is a cancer.
84. A method of inducing cell-cycle arrest and/or apoptosis of a cell comprising contacting the cell with a myosin light chain phosphatase inhibitor.
85. A method according to claim 84, wherein the myosin light chain phosphatase inhibitor is a compound according to any one of claims 1 to 52, or a salt thereof.
86. A method according to claim 85, wherein the contacting is achieved by causing an effective amount of the compound, or a salt form thereof, to be present in an individual, thereby inducing cell-cycle arrest and/or apoptosis in vivo.
87. A method according to claim 86, wherein the contacting is achieved by administering an effective amount of the compound, or a pharmaceutically acceptable salt form thereof, to an individual, thereby inducing cell-cycle arrest and/or apoptosis in vivo.
88. A method according to claim 84 or 85, wherein the contacting is performed in vivo.
89. A method according to claim 84 or 85, wherein the contacting is performed in vitro.
90. A method according to any one of claims 84 to 89, wherein the cell is a cancer cell.
91. A method for treating cancer comprising administering an effective amount of a myosin light chain phosphatase inhibitor to an individual in need of such treatment.
92. A method according to claim 91 wherein the myosin light chain phosphatase inhibitor is a compound according to any one of claims 1 to 52, or a salt form thereof.
93. A method according to claim 91 or 92, wherein the cancer is selected from the group consisting of bladder cancer, brain cancer, breast cancer, colorectal cancer, cervical cancer, gastrointestinal cancer, genitourinary cancer, head and neck cancer, lung cancer, ovarian cancer, prostate cancer, renal cancer, skin cancer, and testicular cancer.
94. A method according to claim 93, wherein the cancer is prostate cancer.
95. A method according to claim 94, wherein the cancer is androgen-independent.
96. A method of killing a tumor cell comprising: contacting the tumor cell with an effective amount of a myosin light chain phosphatase inhibitor; and irradiating the tumor cell with an effective amount of ionizing radiation.
97. A method according to claim 96 wherein the myosin light chain phosphatase inhibitor is a compound according to any one of claims 1 to 52, or a salt form thereof.
98. A method of killing a tumor cell comprising: contacting the tumor cell with an effective amount of a myosin light chain phosphatase inhibitor; and contacting the tumor cell with an effective amount of at least one further chemotherapeutic agent.
99. A method according to claim 98 wherein the myosin light chain phosphatase inhibitor is a compound according to any one of claims 1 to 54, or a salt form thereof.
100. A method according to any one of claims 96 to 99, wherein the tumor cell is a prostate cancer cell.
101. A method according to claim 100, wherein the cancer is androgen independent.
102. A method of treating a tumor in an individual comprising: causing an effective amount of a myosin light chain phosphatase inhibitor, to be present in the individual; and irradiating the tumor with an effective amount of ionizing radiation.
103. A method according to claim 102, wherein the myosin light chain phosphatase inhibitor is a compound according to any one of claims 1 to 52, or a salt form thereof.
104. A method according to claim 102 or 103, wherein the tumor is a prostate cancer.
105. A method according to claim 104, wherein the cancer is androgen independent.
106. A method according to any one of claims 102 to 105, wherein the causing is achieved by administering an effective amount of the myosin light chain phosphatase inhibitor to the individual.
107. A method according to claim 106 wherein the myosin light chain phosphatase inhibitor is a compound according to any one of claims 1 to 52, and the causing is achieved by administering an effective amount of the compound, or a pharmaceutically acceptable salt form thereof, to the individual.
108. A method of detecting the presence of an elevated amount of myosin light chain phosphatase in a subject cell comprising: providing a fluorescent myosin light chain phosphatase inhibitor; contacting the myosin light chain phosphatase inhibitor with the subject cell and with a control cell; observing fluorescence of the cells after the contacting; wherein an elevated level of fluorescence of the subject cells relative to the level of fluorescence of the control cells is indicative of an elevated amount of myosin light chain phosphatase in the subject cell as compared to the control cell.
109. A method according to claim 108, wherein the fluorescent myosin light chain phosphatase inhibitor is a fluorescent compound to any one of claims 1 to 52, or a salt form thereof.
110. A method according to claim 109, wherein the fluorescent myosin light chain phosphatase inhibitor is 4-(2-guanidinothiazol-4-yl)phenyl 5- (dimethylamino)naphthalene-l -sulfonate, or a salt form thereof.
111. A method of detecting diseased cells, wherein the diseased cells comprise elevated amounts of myosin light chain phosphatase comprising: providing a fluorescent myosin light chain phosphatase inhibitor; contacting the fluorescent myosin light chain phosphatase inhibitor with tissue; observing for fluorescence of the cells of the tissue after the contacting; wherein an elevated level of fluorescence of the some of the cells relative to others in the tissue or relative to control non-diseased cells that have been contacted with the fluorescent myosin light chain phosphatase inhibitor is indicative that the fluorescent cells may be diseased cells comprising elevated amounts of myosin light chain phosphatase.
112. A method according to claim 111, wherein the contacting is performed in vitro.
Ill
113. A method according to claim 111, wherein the contacting is performed in vivo by administering an effective amount of a fluorescent myosin light chain phosphatase inhibitor to an individual.
114. A method according to any one of claims 111 to 113, wherein the fluorescent myosin light chain phoasphatase inhibitor is a fluorescent compound according to any one of claims 1 to 54, or a salt form thereof.
115. A method according to claim 114, wherein the fluorescent myosin light chain phosphatase inhibitor is 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate, or a salt form thereof.
116. A method of detecting diseased cells in a tissue of an individual comprising: providing a fluorescent myosin light chain phosphatase inhibitor; contacting the fluorescent myosin light chain phosphatase inhibitor with the tissue; observing for fluorescence of at least some cells of the tissue after the contacting; wherein an elevated level of fluorescence of the some of the cells relative to others in the tissue or relative to control non-diseased cells that have been contacted with the fluorescent myosin light chain phosphatase inhibitor is indicative that the fluorescent cells may be diseased cells comprising elevated amounts of myosin light chain phosphatase.
117. A method according to claim 116, wherein the contacting is performed in vitro.
118. A method according to claim 116, wherein the contacting is performed in vivo by administering an effective amount of a fluorescent myosin light chain phosphatase inhibitor to an individual.
119. A method according to any one of claims 116 to 118, wherein the fluorescent myosin light chain phoasphatase inhibitor is a fluorescent compound according to any one of claims 1 to 54, or a salt form thereof.
120. A method according to claim 119, wherein the fluorescent myosin light chain phosphatase inhibitor is 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate, or a salt form thereof.
121. A method of radiotherapy of tumors, wherein the tumors comprise cells comprise an elevated amount of myosin light chain phosphatase relative to non-tumor cells, comprising: providing a fluorescent myosin light chain phosphatase inhibitor; causing the fluorescent myosin light chain phosphatase inhibitor to be present in the tumor cells in an effective amount to inhibit myosin light chain phosphatase and for fluorescence to be observable; observing the fluorescence; and directing an effective amount of ionizing radiation to the fluorescent tumor cells.
122. A method of surgery to remove tumor tissue from an individual comprising: providing a fluorescent myosin light chain phosphatase inhibitor; causing the fluorescent myosin light chain phosphatase inhibitor to be present in at least some cells of the tumor tissue in an effective amount for fluorescence of at least some of the tumor tissue to be observable; observing the fluorescence; and surgically removing at least some of the fluorescent tumor tissue, whereby at least a portion of the tumor that comprises fluorescent tumor cells is removed.
123. A method according to claim 121 or 122, wherein the fluorescent myosin light chain phosphatase inhibitor is a fluorescent compound according to any one of claims 1 to 52, or a salt form thereof.
124. A method according to claim 123, wherein the fluorescent myosin light chain phosphatase inhibitor is 4-(2-guanidinothiazol-4-yl)phenyl 5-(dimethylamino)naphthalene-l -sulfonate, or a salt form thereof.
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