+

WO2009146013A1 - Inhibiteurs de phosphatase de chaîne légère de myosine - Google Patents

Inhibiteurs de phosphatase de chaîne légère de myosine Download PDF

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
phenyl
salt form
alkyl
light chain
compound according
Prior art date
Application number
PCT/US2009/038783
Other languages
English (en)
Inventor
Milton L. Brown
Scott Grindrod
Original Assignee
Georgetown University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Georgetown University filed Critical Georgetown University
Publication of WO2009146013A1 publication Critical patent/WO2009146013A1/fr

Links

Classifications

    • 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.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne de nouveaux inhibiteurs de phosphatase de chaîne légère de myosine, des compositions comprenant ceux-ci, des procédés de préparation et d’utilisation de ceux-ci, et des procédés d’utilisation d’inhibiteurs de phosphatase de chaîne légère de myosine.
PCT/US2009/038783 2008-03-31 2009-03-30 Inhibiteurs de phosphatase de chaîne légère de myosine WO2009146013A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US4102608P 2008-03-31 2008-03-31
US61/041,026 2008-03-31

Publications (1)

Publication Number Publication Date
WO2009146013A1 true WO2009146013A1 (fr) 2009-12-03

Family

ID=41377469

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2009/038783 WO2009146013A1 (fr) 2008-03-31 2009-03-30 Inhibiteurs de phosphatase de chaîne légère de myosine

Country Status (1)

Country Link
WO (1) WO2009146013A1 (fr)

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110053847A1 (en) * 2007-09-21 2011-03-03 The Washington University Methods of treating vasodilatory shock
WO2011085128A1 (fr) * 2010-01-07 2011-07-14 Selexagen Therapeutics, Inc. Inhibiteurs de la voie hedgehog
KR20150081365A (ko) * 2012-11-09 2015-07-13 인쎄름 (엥스띠뛰 나씨오날 드 라 쌍떼 에 드 라 흐쉐르슈 메디깔) 신규한 벤젠 설폰아미드 티아졸 화합물
WO2017017004A1 (fr) * 2015-07-24 2017-02-02 INSERM (Institut National de la Santé et de la Recherche Médicale) Composés de thiazole sulfonamide de benzène hydrophobe substitué utilisables dans le traitement du cancer
US9676747B2 (en) 2011-12-21 2017-06-13 Novira Therapeutics, Inc. Hepatitis B antiviral agents
US9873671B2 (en) 2014-01-16 2018-01-23 Novira Therapeutics, Inc. Azepane derivatives and methods of treating hepatitis B infections
US9884818B2 (en) 2013-05-17 2018-02-06 Janssen Sciences Ireland Uc Sulphamoylpyrrolamide derivatives and the use thereof as medicaments for the treatment of hepatitis B
CN107674011A (zh) * 2013-09-10 2018-02-09 奥布赛瓦股份公司 作为催产素/加压素V1a受体拮抗剂的吡咯烷衍生物
US9895349B2 (en) 2013-04-03 2018-02-20 Janssen Sciences Ireland Us N-phenyl-carboxamide derivatives and the use thereof as medicaments for the treatment of hepatitis B
US10071961B2 (en) 2013-10-23 2018-09-11 Janssen Sciences Ireland Uc Carboxamide derivatives and the use thereof as medicaments for the treatment of hepatitis B
US10077239B2 (en) 2015-09-29 2018-09-18 Novira Therapeutics, Inc. Crystalline forms of a hepatitis B antiviral agent
US10125094B2 (en) 2013-02-28 2018-11-13 Janssen Sciences Ireland Uc Sulfamoyl-arylamides and the use thereof as medicaments for the treatment of hepatitis B
US10213420B2 (en) 2014-02-05 2019-02-26 Novira Therapeutics, Inc. Combination therapy for treatment of HBV infections
US10392349B2 (en) 2014-01-16 2019-08-27 Novira Therapeutics, Inc. Azepane derivatives and methods of treating hepatitis B infections
US10441589B2 (en) 2016-04-15 2019-10-15 Novira Therapeutics, Inc. Combinations and methods comprising a capsid assembly inhibitor
US10450270B2 (en) 2013-07-25 2019-10-22 Janssen Sciences Ireland Uc Glyoxamide substituted pyrrolamide derivatives and the use thereof as medicaments for the treatment of hepatitis B
US10537580B2 (en) 2015-03-19 2020-01-21 Novira Therapeutics, Inc. Azocane and azonane derivatives and methods of treating hepatitis B infections
US10676429B2 (en) 2012-08-28 2020-06-09 Janssen Sciences Ireland Uc Sulfamoyl-arylamides and the use thereof as medicaments for the treatment of hepatitis B
US10875876B2 (en) 2015-07-02 2020-12-29 Janssen Sciences Ireland Uc Cyclized sulfamoylarylamide derivatives and the use thereof as medicaments for the treatment of hepatitis B
US10973801B2 (en) 2018-03-14 2021-04-13 Janssen Sciences Ireland Unlimited Company Capsid assembly modulator dosing regimen
US11078193B2 (en) 2014-02-06 2021-08-03 Janssen Sciences Ireland Uc Sulphamoylpyrrolamide derivatives and the use thereof as medicaments for the treatment of hepatitis B
US11096931B2 (en) 2019-02-22 2021-08-24 Janssen Sciences Ireland Unlimited Company Amide derivatives useful in the treatment of HBV infection or HBV-induced diseases
US11491148B2 (en) 2019-05-06 2022-11-08 Janssen Sciences Ireland Unlimited Company Amide derivatives useful in the treatment of HBV infection or HBV-induced diseases

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007056496A1 (fr) * 2005-11-07 2007-05-18 Irm Llc Modulateurs des ppar contenant de l'oxazole et du thiazole
US20080045579A1 (en) * 2006-08-15 2008-02-21 Wyeth Oxazolidine derivatives as PR modulators

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007056496A1 (fr) * 2005-11-07 2007-05-18 Irm Llc Modulateurs des ppar contenant de l'oxazole et du thiazole
US20080045579A1 (en) * 2006-08-15 2008-02-21 Wyeth Oxazolidine derivatives as PR modulators

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"Abstracts of Papers. 230th ACS National Meeting", 28 August 2005, WASHINGTON, DC, UNITED STATES, article GRINDROD, S. C. ET AL.: "Novel thiazole compounds that inhibit myosin phosphatase activity and hyman cancer cell proliferation." *
JOHNSTON, P. A. ET AL.: "Development and implementation of a 384-well homogeneous fluorescence intensity high-throughput screening assay to identify mitogen-activated protein kinase phosphatase-1 dual-specificity protein phosphatase inhibitors.", ASSAY AND DRUG DEVELOPMENT TECHNOLOGIES., vol. 5, no. 3, 2007, pages 319 - 332 *

Cited By (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110053847A1 (en) * 2007-09-21 2011-03-03 The Washington University Methods of treating vasodilatory shock
US9629827B2 (en) * 2007-09-21 2017-04-25 Washington University Methods of treating vasodilatory shock
US9174949B2 (en) 2010-01-07 2015-11-03 Selexagen Therapeutics, Inc. Hedgehog inhibitors
WO2011085128A1 (fr) * 2010-01-07 2011-07-14 Selexagen Therapeutics, Inc. Inhibiteurs de la voie hedgehog
CN102802419A (zh) * 2010-01-07 2012-11-28 西莱瑟根治疗公司 刺猬(hedgehog)抑制剂
US10196376B2 (en) 2011-12-21 2019-02-05 Novira Therapeutics, Inc. Hepatitis B antiviral agents
US9676747B2 (en) 2011-12-21 2017-06-13 Novira Therapeutics, Inc. Hepatitis B antiviral agents
US9751857B2 (en) 2011-12-21 2017-09-05 Novira Therapeutics, Inc. Hepatitis B antiviral agents
US10676429B2 (en) 2012-08-28 2020-06-09 Janssen Sciences Ireland Uc Sulfamoyl-arylamides and the use thereof as medicaments for the treatment of hepatitis B
US10995064B2 (en) 2012-08-28 2021-05-04 Janssen Sciences Ireland Uc Sulfamoyl-arylamides and the use thereof as medicaments for the treatment of hepatitis B
CN104797565A (zh) * 2012-11-09 2015-07-22 法国国家健康医学研究院 新的苯磺酰胺噻唑化合物
US9567310B2 (en) * 2012-11-09 2017-02-14 Inserm (Institut National De La Sante Et De La Recharche Medicale) Benzene sulfonamide thiazole compounds
US20150284347A1 (en) * 2012-11-09 2015-10-08 INSERM (Institut National de la Santé et de la Recherche Médicale) New benzene sulfonamide thiazole compounds
KR20150081365A (ko) * 2012-11-09 2015-07-13 인쎄름 (엥스띠뛰 나씨오날 드 라 쌍떼 에 드 라 흐쉐르슈 메디깔) 신규한 벤젠 설폰아미드 티아졸 화합물
KR102271794B1 (ko) * 2012-11-09 2021-07-01 인쎄름 (엥스띠뛰 나씨오날 드 라 쌍떼 에 드 라 흐쉐르슈 메디깔) 항암 활성을 나타내는 벤젠 설폰아미드 티아졸 화합물
US10125094B2 (en) 2013-02-28 2018-11-13 Janssen Sciences Ireland Uc Sulfamoyl-arylamides and the use thereof as medicaments for the treatment of hepatitis B
US10941113B2 (en) 2013-02-28 2021-03-09 Janssen Sciences Ireland Uc Sulfamoyl-arylamides and the use thereof as medicaments for the treatment of hepatitis B
US10398677B2 (en) 2013-04-03 2019-09-03 Janssen Sciences Ireland Uc N-phenyl-carboxamide derivatives and the use thereof as medicaments for the treatment of hepatitis B
US9895349B2 (en) 2013-04-03 2018-02-20 Janssen Sciences Ireland Us N-phenyl-carboxamide derivatives and the use thereof as medicaments for the treatment of hepatitis B
US10457638B2 (en) 2013-05-17 2019-10-29 Janssen Sciences Ireland Uc Sulphamoylpyrrolamide derivatives and the use thereof as medicaments for the treatment of hepatitis B
US9884818B2 (en) 2013-05-17 2018-02-06 Janssen Sciences Ireland Uc Sulphamoylpyrrolamide derivatives and the use thereof as medicaments for the treatment of hepatitis B
US10450270B2 (en) 2013-07-25 2019-10-22 Janssen Sciences Ireland Uc Glyoxamide substituted pyrrolamide derivatives and the use thereof as medicaments for the treatment of hepatitis B
CN109928905A (zh) * 2013-09-10 2019-06-25 奥布赛瓦股份公司 作为催产素/加压素V1a受体拮抗剂的吡咯烷衍生物
CN107674011A (zh) * 2013-09-10 2018-02-09 奥布赛瓦股份公司 作为催产素/加压素V1a受体拮抗剂的吡咯烷衍生物
US10071961B2 (en) 2013-10-23 2018-09-11 Janssen Sciences Ireland Uc Carboxamide derivatives and the use thereof as medicaments for the treatment of hepatitis B
US10377709B2 (en) 2013-10-23 2019-08-13 Janssen Sciences Ireland Uc Carboxamide derivatives and the use thereof as medicaments for the treatment of hepatitis B
US10392349B2 (en) 2014-01-16 2019-08-27 Novira Therapeutics, Inc. Azepane derivatives and methods of treating hepatitis B infections
US9873671B2 (en) 2014-01-16 2018-01-23 Novira Therapeutics, Inc. Azepane derivatives and methods of treating hepatitis B infections
US10632112B2 (en) 2014-02-05 2020-04-28 Novira Therapeutics, Inc. Combination therapy for treatment of HBV infections
US10213420B2 (en) 2014-02-05 2019-02-26 Novira Therapeutics, Inc. Combination therapy for treatment of HBV infections
US11078193B2 (en) 2014-02-06 2021-08-03 Janssen Sciences Ireland Uc Sulphamoylpyrrolamide derivatives and the use thereof as medicaments for the treatment of hepatitis B
US10537580B2 (en) 2015-03-19 2020-01-21 Novira Therapeutics, Inc. Azocane and azonane derivatives and methods of treating hepatitis B infections
US10875876B2 (en) 2015-07-02 2020-12-29 Janssen Sciences Ireland Uc Cyclized sulfamoylarylamide derivatives and the use thereof as medicaments for the treatment of hepatitis B
US10815207B2 (en) 2015-07-24 2020-10-27 Inserm (Institut National De La Sante Et De La Recherche Medicale) Substituted hydrophobic benzene sulfonamide thiazole compounds for use in treating cancer
JP2018527324A (ja) * 2015-07-24 2018-09-20 アンスティチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル ガンを処置するのに使用するための置換疎水性ベンゼンスルホンアミドチアゾール化合物
WO2017017004A1 (fr) * 2015-07-24 2017-02-02 INSERM (Institut National de la Santé et de la Recherche Médicale) Composés de thiazole sulfonamide de benzène hydrophobe substitué utilisables dans le traitement du cancer
US10077239B2 (en) 2015-09-29 2018-09-18 Novira Therapeutics, Inc. Crystalline forms of a hepatitis B antiviral agent
US10441589B2 (en) 2016-04-15 2019-10-15 Novira Therapeutics, Inc. Combinations and methods comprising a capsid assembly inhibitor
US11129834B2 (en) 2016-04-15 2021-09-28 Novira Therapeutics, Inc. Combinations and methods comprising a capsid assembly inhibitor
US10973801B2 (en) 2018-03-14 2021-04-13 Janssen Sciences Ireland Unlimited Company Capsid assembly modulator dosing regimen
US11096931B2 (en) 2019-02-22 2021-08-24 Janssen Sciences Ireland Unlimited Company Amide derivatives useful in the treatment of HBV infection or HBV-induced diseases
US11491148B2 (en) 2019-05-06 2022-11-08 Janssen Sciences Ireland Unlimited Company Amide derivatives useful in the treatment of HBV infection or HBV-induced diseases

Similar Documents

Publication Publication Date Title
WO2009146013A1 (fr) Inhibiteurs de phosphatase de chaîne légère de myosine
AU2020241258A1 (en) Novel small molecule inhibitors of tead transcription factors
CN105492444B (zh) 作为rock抑制剂的三环吡啶-甲酰胺衍生物
CN103038231B (zh) Hec1活性调节剂及其方法
CA2671426C (fr) Analogues de sulfure, sulfoxyde et sulfone de chalcone utiles dans le traitement du cancer et autres troubles proliferatifs
JP5551613B2 (ja) ６−アミノイソキノリン化合物および７−アミノイソキノリン化合物、ならびにそれらの製造方法および使用方法
AU2007248341C1 (en) Benzimidazole modulators of VR1
CA2667644A1 (fr) Composes tricycliques servant d'inhibiteurs des metalloproteases matricielles
EP2231596A1 (fr) Inhibiteurs de l'histone déacétylase
JP4667384B2 (ja) イオンチャネルリガンドとしてのアミド誘導体および薬学的組成物、ならびにこれらを使用する方法
JP2011507848A (ja) Ｒｈｏキナーゼインヒビターとしてのベンゾピランおよびアナログ
BRPI0807626B1 (pt) Processo para a síntese de compostos de 2-aminotiazol como inibidores de quinase
JP2008056660A (ja) インドールスルフォンアミド化合物
JP2011507850A (ja) Ｒｈｏキナーゼインヒビターとしてのアニリドおよびアナログ
BRPI0721169A2 (pt) Inibidores de benzimidazol trpv1
TWI844004B (zh) 二唑硫羰基化合物及包含該等化合物之醫藥組合物
EP2046322A2 (fr) Aryl vinyl sulfures, sulfones, sulfoxides et sulfonamides, dérivés de ceux-ci et utilisations thérapeutiques de ceux-ci
KR102493005B1 (ko) 신경통, 통증, 만성 폐색성 폐질환 (copd) 및 천식 치료시 trpm8-억제제로서 유용한 2-아릴-4-하이드록시-1,3-티아졸 유도체
CA3135921C (fr) Compose contenant du quinolyle, composition pharmaceutique et utilisation associee
CA2784749A1 (fr) Pyrido[2,3-d]pyrimidin-7(8h)-ones substitues et leurs utilisations therapeuthiques
EP2299996A1 (fr) Inhibiteurs de la 5-lipoxygénase
CN104211682B (zh) 吡啶类化合物及其用途
ES2552100T3 (es) Nuevos derivados de 1,2-bis-sulfonamida como moduladores de receptores de quimiocinas
US20080114041A1 (en) Benzothiazolesulfonamides
CN109134451A (zh) 1,3-二取代脲类与硫脲类衍生物及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09755376

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 09755376

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载