WO2004062666A2 - Use of rho-kinase inhibitorrs for treatment of benign prostatic hyperplasia - Google Patents

Use of rho-kinase inhibitorrs for treatment of benign prostatic hyperplasia Download PDF

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Publication number
WO2004062666A2
WO2004062666A2 PCT/GB2004/000139 GB2004000139W WO2004062666A2 WO 2004062666 A2 WO2004062666 A2 WO 2004062666A2 GB 2004000139 W GB2004000139 W GB 2004000139W WO 2004062666 A2 WO2004062666 A2 WO 2004062666A2
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alkyl
hydrogen
aralkyl
phenyl
ring
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PCT/GB2004/000139
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French (fr)
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WO2004062666A3 (en
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Rowland Rees
Selim Cellek
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University College London
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4409—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 4, e.g. isoniazid, iproniazid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/08—Drugs for disorders of the urinary system of the prostate

Definitions

  • This invention relates to the treatment of benign prostatic hyperplasia (BPH).
  • BPH affects approximately 85% of men older than 50 years of age. BPH represents hyperplasia of both the glandular and stromal components of the prostate. Development of lower urinary tract symptoms (LUTS) due to BPH is caused by mechanical obstruction to urinary flow secondary to enlarged prostate (static component) and elevated smooth muscle tone in the f ⁇ bromuscular stroma, prostatic capsule and the bladder neck (dynamic prostate). Pharmacotherapy therefore aims to reduce the size of the prostate or to decrease the tone of prostatic smooth muscle.
  • the drugs which aim to decrease the size of the prostate are 5 ⁇ - reductase inhibitors; they block androgen stimulation of the organ without affecting androgen-dependent skeletal muscle strength, libido and potency.
  • ⁇ r Adrenoceptor antagonists aim to reduce the smooth muscle tone and urethral pressure.
  • Recently combination therapy using a 5 ⁇ -reductase inhibitor and a ⁇ r adrenoceptor antagonist has been found to be hopeful in the management of BPH.
  • Such a combination therapy has obvious theoretical appeal: ⁇ r adrenoceptor antagonist acts rapidly to reduce symptoms while the 5 ⁇ -reductase inhibitor reduces the size of the prostate over a longer period.
  • Rho-kinase inhibitor inhibits adrenergic contractions of the prostate tissue and proliferation of prostatic smooth muscle cells.
  • Rho-kinase inhibitors can therefore be used as a single agent treatment for BPH with dual effect; by relaxing the smooth muscle and by reducing the size of the prostate by inhibiting the smooth muscle cell proliferation.
  • the invention provides use of a Rho-kinase inhibitor for the manufacture of a medicament for use in the treatment of benign prostatic hyperplasia.
  • the invention also provides a method of treating benign prostatic hyperplasia in a man suffering therefrom, which method comprises the step of administering thereto a therapeutically effective amount of a Rho-kinase inhibitor.
  • FIG. 1 shows the expression of Rho-kinase in rat and human prostatic smooth muscle
  • FIG. 1 shows the expression of Rho-kinase in rat and human prostatic smooth muscle
  • FIG. 1 shows the expression of Rho-kinase in rat and human prostatic smooth muscle
  • FIG. 1 shows the expression of Rho-kinase in rat and human prostatic smooth muscle
  • FIG. 1 shows the expression of Rho-kinase in rat and human prostatic smooth muscle
  • Figure 2 shows that the Rho-kinase inhibitor Y-27632 inhibits proliferation but does not induce apoptosis in human prostatic smooth muscle cells
  • Cell viability after exposure to Y-27632 was measured using neutral red (a) and MTT (b) assays, c, Incorporation of BrdU to human prostatic smooth muscle cells was measured in the absence and presence of Y-27632, d, The effect of Y-27632 and staurosporine on the apoptotic index of human prostatic smooth muscle cells.
  • FIG. 3 shows that the Rho-kinase inhibitor Y-27632 inhibits adrenergic contractions of the rat prostate
  • a Contractions elicited with electrical field stimulation (EFS; denoted with dots) was reversibly inhibited by Y-27632. The contractions were inhibited by guanethidine confirming their noradrenergic nature
  • b Phenylephrine-induced contractions were reversibly inhibited by Y-27632.
  • c The effect of Y-27632 on contractions elicited by EFS ( ⁇ ) and exogenous phenylephrine
  • Rho-kinase inhibitor to treat benign prostatic hyperplasia (BPH).
  • Rho-kinase is activated by RhoA, a small monomeric G-protein believed to be coupled to excitatory receptors (i.e. ⁇ ,- adrenoceptors), Activated Rho-kinase inhibits smooth muscle myosin phosphatase (SMPP-1 ) by phosphorylating its regulatory subunit. This leads to sensitization of myofilaments to Ca 2+ in smooth muscle cells.
  • An inhibitor of Rho-kinase for use in the invention directly or indirectly causes a reduction in the activity and/or expression of Rho-kinase.
  • An inhibitor of Rho-kinase may thus inliibit the activity of Rho-kinase.
  • An inhibitor may act by binding to Rho-kinase or by binding to the substrate of Rho-kinase.
  • An inhibitor of the invention may act, at the level of Rho-kinase expression. Such an inhibitor may cause its effect, for example, at initiation, progression or termination of transcription, during transport of Rho-kinase mRNA from the nucleus to the cytoplasm, or during processing and/or translation of Rho-kinase mRNA. Alternatively or additionally, an inhibitor of the invention may reduce the stability of the Rho-kinase protein. For example, the inhibitor may increase the rate at which the protein is degraded or inactivated.
  • An inhibitor of expression may bind to a Rho-kinase gene 5' to the coding sequence and/or in the coding sequence and/or 3' to the coding sequence,
  • an inhibitory molecule may bind to the promoter, to reduce the rate of transcription
  • the inhibitor may bind and inhibit a protein factor required for transcription of the gene, preventing its activity
  • An inhibitory agent may bind to and stimulate a protein factor that causes down-regulation of Rho-kinase transcription.
  • the inhibitor may itself comprise such a factor or a nucleic acid encoding such a factor.
  • the present inhibitor is specific in its modulation of transcription from a Rho-kinase gene and has substantially no effect on transcription from other genes
  • An inhibitor of the invention may bind to the untranslated or translated regions of Rho-kinase mRNA, to inhibit initiation or progress of translation, to nuclear factors that bind to the mRNA and/or transport the mRNA to the cytoplasm, or to translation factors that contribute to translating the mRNA to protein.
  • An inhibitor of the invention may comprise a polynucleotide, for example DNA or RNA
  • the polynucleotide may encode a molecule with inhibitory activity such as any of those described above, for example a protein or mRNA molecule.
  • the inhibitory molecule thus becomes available upon expression of the polynucleotide, for example in a target cell.
  • the polynucleotide may itself have inhibitor ⁇ ' activity.
  • a polynucleotide may thus encode an inhibitory protein or mRNA or the polynucleotide may itself be an inhibitor, for example, an antisense molecule or iRNA,
  • the polynucleotide may include synthetic or modified nucleotides.
  • a number of different types of modification to polynucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule.
  • the polynucleotides described herein may be modified by any method available in the art, Such modifications may be carried out in order to enhance the in vivo activity or lifespan of polynucleotide.
  • an inhibitor for use in the invention may be identified by assaying a test substance for activity which reduces Rho-kinase activity and/or expression.
  • a typical assay comprises: (i) contacting a test substance with a source of Rho-kinase activity and/or expression, under conditions that would permit Rho-kinase activity and/or expression in the absence of the test substance; and (ii) determining whether the test substance causes a decrease in the activity and/or expression of the Rho-kinase.
  • Rho-kinase activity and/or expression may be a Rho-kinase such as ROC (Leung et al, J. Biol. Chem. 270, 29051-29054, 1995; sometimes also called ROCK-H) or pl ⁇ OROCK (Ishizaki et al, EMBO J. 15, 1885-1893, 1996; sometimes also called ROC ⁇ or ROCK-I).
  • ROC Rho-kinase
  • pl ⁇ OROCK Ishizaki et al, EMBO J. 15, 1885-1893, 1996; sometimes also called ROC ⁇ or ROCK-I
  • a control assay is generally also carried out to assess the activity and/or expression of the Rho-kinase, as the case may be, under the same reaction conditions but in the absence of the test substance.
  • the control assay is preferably carried out in parallel with the test assay, Preferably, multiple repetitions are made of both the test and control assays.
  • the samples may be assayed for any other enzyme to exclude the possibility that a test substance is a general modulator of enzyme activity,
  • An inhibitor of Rho-kinase transcription can be identified using a reporter gene assay.
  • a reporter gene assay comprises: (i) providing a test construct comprising a Rho-kinase promoter operably linked to a reporter polynucleotide to be expressed in the form of mRNA;" (ii) contacting a test substance with the test construct under conditions, which in the absence of the test substance, would permit expression of the reporter polynucleotide in the form of mRNA; (iii) carrying out a control assay under the same reaction conditions but in the absence of the test substance; and (iv) determining whether the test substance inhibits expression from the construct.
  • Rho-kinase promoter for use in the assay may be isolated via methods Icnown in the art. Any eukaryotic Rho-kinase promoter may be used but preferably a mammalian promoter in particular, a Rho-kinase promoter, is used.
  • the promoter sequence may be fused directly to a reporter sequence or via a linker.
  • the linker sequence may comprise a sequence having enhancer characteristics, to boost expression levels.
  • the reporter assay should be carried out under conditions that allow expression of the reporter polynucleotide as mRNA in the absence of the test substance.
  • the assay should include a suitable RNA polymerase and nucleotides to allow transcription of the reporter sequence, Levels of reporter mRNA in the presence and absence of the test substance may be compared to assess any modulatory effect of the test substance on transcription from the Rho-kinase promoter.
  • the assay is carried out under conditions which mimic those to which the Rho-kinase promoter is exposed in vivo.
  • one or more control assays is carried out using any other promoter in place of the Rho-kinase promoter, to exclude the possibility that a test substance is a general inhibitor of transcription,
  • Rho-kinase mRNA or Rho-kinase protein may be identified in a stability assay, hi a suitable assay, Rho-kinase mRNA or Rho-kinase protein is incubated in the presence and absence of a test substance, under conditions which mimic those to which the Rho-kinase mRNA or protein is exposed in vivo.
  • assay temperatures are those found in vivo, for example 25 to 37°C, in particular 37°C.
  • Samples are removed at regular time intervals and assayed for the presence of Rho-kinase mRNA or protein. This can be done by any suitable means, for example, by gel analysis or spectrophotometrically. In this- way, any alteration in the rate of degradation of Rho-kinase mRNA or protein, associated with the test substance, can be detected.
  • Rho-kinase An inhibitor which acts at the level of translation of Rho-kinase may be identified using an in vitro translation system. Suitable translation systems are available in the art: for example, the rabbit reticulocyte system. Rho-kinase mRNA is used in the system in the presence and absence of a test substance, under conditions which permit translation of the mRNA in the absence of the test substance, Again conditions will preferably mimic those to which Rho-kinase mRNA is exposed in vivo. The effect of a test substance is assessed by monitoring the production of Rho-kinase protein using any suitable means, for example, by gel analysis or by spectrophotometer.
  • Rho-kinase activity and/or expression is one which produces a measurable or detectable decrease in Rho-kinase activity and/or expression, for example in at least one of the assays described above, Generally such inhibitors are those which inhibit Rho-kinase activity and/or expression by at least 10%.
  • the percentage inhibition represents the percentage decrease in expression/activity in a comparison of assays carried out in the presence and absence of the test substance, Any combination of the above mentioned degrees of percentage inhibition and concentration of inhibitor may be used to define an inhibitor of the invention, with greater inhibition at lower concentrations being preferred,
  • suitable test substances include antibodies, combinatorial libraries, defined chemical identities, chemical compounds, peptide and peptide mimetics, oligonucleotides and natural product libraries, such as display libraries (e.g. phage display libraries).
  • organic molecules will be screened, preferably small organic molecules which have a molecular weight of from 50 to 2500 daltons.
  • Candidate products can be biomolecules including, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds.
  • Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
  • Test substances may include polynucleotides.
  • a potential modulator is a polynucleotide encoding an inhibitory molecule, for example, a protein or mRNA
  • the encoded molecule may be used as a "test substance" in an assay.
  • a modulator may include a single substance or a combination of two, three or more substances.
  • a modulator may refer to a single peptide, a mixture of two or more peptides or a mixture of a peptide and a defined chemical entity,
  • Test substances may be used in an initial screen of, for example, 10 substances per reaction, and the substances of these batches which show inhibition or stimulation tested individually.
  • Rho-kinase inhibitors for use in the invention are described in EP-A- 0956865. Specifically, such Rho-kinase inhibitors have of the formula (I):
  • Ra is a group of the formula:
  • R is hydrogen, alkyl or cycloalkyl, cycloalkylalkyl, phenyl or aralkyl, which optionally have a substituent on the ring, or a group of the formula:
  • R ⁇ is hydrogen, alkyl or formula : -NR S NR 9 wherein R 8 and R 9 are the same or different and each is hydrogen, alkyl, aralkyl or phenyl, R 7 is hydrogen, alkyl, aralkyl, phenyl, nitro or cyano, or R 6 and R 7 in combination form a heterocyclic group optionally having, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom,
  • R 1 is hydrogen, alkyl or cycloalkyl, cycloalkylalkyl, phenyl or aralkyl, which optionally have a substituent on the ring, or
  • R and R 1 in combination form, together with the adjacent nitrogen atom, a heterocyclic group optionally having, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom,
  • R 2 is hydrogen or alkyl
  • R 3 and R 4 are the same or different and each is hydrogen, alkyl, aralkyl, halogen, nitro, amino, alkylamino, acylamino, hydroxy, alkoxy, aralkyloxy, cyano, acyl, mercapto, alkylthio, aralkylthio, carboxy, alkoxycarbonyl, carbamoyl, alkylcarbamoyl or azide, and A is a group of the formula:
  • R 10 and R 11 are the same or different and each is hydrogen, alkyl, haloalkyl, aralkyl, hydroxyalkyl, carboxy or alkoxycarbonyl, or R 10 and R 11 in combination form a cycloalkyl group and 1, m and n are each 0 or an integer of l-3, and in which, in the formula (c): a broken line is a single bond or a double bond,
  • R 5 is hydrogen, hydroxy, alkoxy, alkoxycarbonyloxy, alkanoyloxy or aralkyloxycarbonyloxy, and
  • L is hydrogen, alkyl, aminoalkyl, mono or dialkylaminoalkyl,tetrahydrofurfuryl, carbamoylalkyl, phthalimidoalkyl, amidino or a group of the formula:
  • B is hydrogen, alkyl, alkoxy, aralkyl, aralkyloxy, aminoalkyl, hydroxyalkyl, hydroxyalkyl, alkanoyloxyalkyl, alkoxycarbonylalkyl, alpha-aminobenzyl, furyl, pyridyl, phenyl, phenylamino, styryl or imidazopyridyl,
  • Q 1 is hydrogen, halogen, hydroxy, aralkyloxy or thienylmethyl
  • W is alkylene
  • Q 2 is hydrogen, halogen, hydroxy or aralkyloxy
  • X is alkylene
  • Q 3 is hydrogen, halogen, hydroxy, alkoxy, nitro, amino, 2,3-dihydrofuryl or 5- methyl-3-oxo-2,3,4,5-tetrahydropyridazin-6-yl; and Y is a single bond, alkylene or alkenylene; and Rb is a hydrogen, an alkyl, an aralkyl, an aminoalkyl or a mono- or dialkylaminoalkyl; and
  • Re is an optionally substituted heterocycle containing nitrogen; an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.
  • Rho-kinase inhibitors disclosed in EP-A-0956865 have the formula
  • R 12 is hydrogen, chlorine or hydroxy, and when R !2 is a hydrogen,
  • Alk is an alkylene group having 2 to 6 carbon atoms, which optionally has alkyl having 1 to 10 carbon atoms, aryl or aralkyl as a substituent;
  • R 13 is hydrogen
  • R M is hydrogen, or linear or branched alkyl having 1 to 6 carbon atoms, aryl or aralkyl;
  • R 15 is hydrogen, linear or branched alkyl having 1 to 6 carbon atoms, aryl or aralkyl, or benzoyl, cinnamyl, cinnamoyl, furoyl or a group of the following formula
  • R 16 is linear or branched alkyl having 1 to 6 carbon atoms or a group of the following formula
  • R' 7 and R 18 are hydrogen or directly bonded to form alkylene having 2 to 4 carbon atoms; or R 13 and Rl4 are directly bonded to form alkylene having 4 or less carbon atoms, which is optionally substituted by alkyl having 1 to 10 carbon atoms, phenyl or benzyl, or
  • R 14 and R 15 directly or in combination via an oxygen atom form a heterocyclic group together with the adjacent nitrogen atom, and when R 12 is chlorine or hydroxy, Alk is an alkylene having 2 to 6 carbon atoms which is optionally substituted by alkyl having 1 to 6 carbon atoms,
  • R !3 and R 14 are each hydrogen, linear or branched alkyl having 1 to 6 carbon atoms or directly bonded to each other to form ethylene or trimethylene, wherein said alkyl, ethylene or trimethylene group is optionally substituted by alkyl having 1 to 6 carbon atoms; or
  • R 15 is hydrogen, linear or branched alkyl having 1 to 6 carbon atoms or an amidino group; an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.
  • - Alkyl at R and R l is linear or branched alkyl having 1 to 10 carbon atoms, which is exemplified by methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl and the like, with preference given to alkyl having 1 to 4 carbon atoms,
  • Cycloalkyl at R and R 1 has 3 to 7 carbon atoms and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like.
  • Cycloalkylalkyl at R and R ! has a cycloalkyl moiety having 3 to 7 carbon atoms as defined immediately above and the alkyl moiety is linear or branched alkyl having 1 to 6 carbon atoms (e.g. methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl and the like).
  • Such cycloalkylalkyl groups are exemplified by cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cycloheptylmethyl, cyclopropylethyl, cyclopentylethyl, cyclohexylethyl, cycloheptylethyl, cyclopropylpropyl, cyclopentylpropyl, cyclohexylpropyl, cycloheptylpropyl, cyclopropylbutyl, cyclopentylbutyl, cyclohexylbutyl, cycloheptylbutyl, cyclopropylhexyl, cyclopentylhexyl, cyclohexylhexyl, cycloheptylhexyl and the like.
  • Aralkyl at R and R 1 has an alkyl moiety of 1 to 4 carbon atoms and is exemplified by phenylalkyl such as benzyl, 1 -phenylethyl, 2-phenylethyl, 3- phenylpropyl, 4-phenylbutyl and the like.
  • the substituent of optionally substituted cycloalkyl, cycloalkylalkyl, phenyl and aralkyl on the ring at R and R 1 is halogen (e.g., chlorine, bromine, fluorine and iodine), alkyl (same as alkyl at R and R 1 ), alkoxy (linear or branched alkoxy having 1 to 6 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy and the like), aralkyl (same as aralkyl at R and R 1 ) or haloalkyl (alkyl at R and R 1 which is substituted by from 1 to 5 halogen atoms, and exemplified by fluoromethyl, difluoromefhyl, trifluoromethyl, 2,2,2- trifluoroeth
  • the group formed by R and R 1 in combination together with the adjacent nitrogen atom, which forms a heterocycle optionally having, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom is preferably a 5 or 6- membered ring and to which, optionally, a ring may be fused.
  • Examples thereof include 1-pyrrolidinyl, piperidino, 1-piperazinyl, morpholino, thiomorpholino, 1- imidazolyl, 2,3-dihydrothiazol-3-yl and the like,
  • the substituent of the optionally substituted nitrogen atom is exemplified by alkyl, aralkyl, haloalkyl and the like.
  • alkyl, aralkyl and haloalkyl are as defined for R and R 1
  • - Alkyl at R 2 is as defined for R and R 1 .
  • Halogen, alkyl, alkoxy and aralkyl at R 3 and R 4 are as defined for R and R ! .
  • Acyl at R 3 and R 4 is alkanoyl having 2 to 6 carbon atoms (e.g., acetyl, propionyl, butyryl, valeryl, pivaloyl and the like), benzoyl or phenylalkanoyl wherein the alkanoyl moiety has 2 to 4 carbon atoms (e.g., phenylacetyl, phenylpropionyl, phenylbutyryl and the like).
  • Alkylamino at R 3 and R 4 has a linear or branched alkyl moiety having 1 to 6 carbon atoms. Examples thereof include methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, sec-butylamino, tert-butylamino, pentylamino, hexylamino and the like.
  • - Acylamino at R 3 and R 4 has an acyl moiety which may be alkanoyl having 2 to 6 carbon atoms, benzyl or the alkanoyl moiety is phenylalkanoyl having 2 to 4 carbon atoms and the like, and is exemplified by acetylamino, propionylamino, butyrylamino, valeryl amino, pivaloylamino, benzoylamino, phenylacetylamino, phenylpropionylamino, phenylbutyrylamino and the like.
  • Alkylthio at R 3 and R 4 has a linear or branched alkyl moiety having 1 to 6 carbon atoms and is exemplified by methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, sec-butylthio, tert-butylthio, pentylthio, hexylthio and the like,
  • Aralkyloxy at R 3 and R 4 has an alkyl moiety having 1 to 4 carbon atoms and is exemplified by benzyloxy, 1-phenylethyloxy, 2-phenylethyloxy, 3- phenylpropyloxy, 4-phenylbutyloxy and the like.
  • Aralkylthio at R 3 and R 4 has an alkyl moiety having 1 to 4 carbon atoms and is exemplified by benzylthio,l-phenylethylthio,2-phenylethylthio,3- phenylpropylthio,4-phenylbutylthio and the like,
  • Alkoxycarbonyl at R 3 and R 4 has a linear or branched alkoxy moiety having 1 to 6 carbon atoms, which is exemplified by methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, sec- butoxycarbonyl, tert-butoxycarbonyl, pentyloxycarbonyl, hexyloxycarbonyl and the like.
  • Alkylcarbamoyl at R 3 and R 4 is carbamoyl mono- or di-substituted by alkyl having 1 to 4 carbon atoms and is exemplified by methylcarbamoyl, dimethylcarbamoyl, ethylcarbamoyl, diethylcarbamoyl, propylcarbamoyl, dipropylcarbamoyl, butylcarbamoyl, dibutylcarbamoyl and the like.
  • Alkoxy at R 5 is as defined for R and R 1 .
  • Alkoxycarbonyloxy at R 2 has a linear or branched alkoxy moiety having 1 to 6 carbon atoms and is exemplified by methoxycarbonyloxy, ethoxycarbonyloxy, propoxycarbonyloxy, isopropoxycarbonyloxy, butoxycarbonyloxy, isobutoxycarbonyloxy, sec-butoxycarbonyloxy, tert-butoxycarbonyloxy, pentyloxycarbonyloxy, hexyloxycarbonyloxy and the like.
  • Alkanoyloxy at R 5 has an alkanoyl moiety having 2 to 6 carbon atoms and is exemplified by acetyloxy, propionyloxy, butyryloxy, valeryloxy, pivaloyloxy and the like.
  • Aralkyloxycarbonyloxy at R 5 has an aralkyl moiety having C1-C4 alkyl and is exemplified by benzyloxycarbonyloxy, 1 -phenylethyloxycarbonyloxy, 2- phenylethyloxycarbonyloxy, 3-phenylpropyloxycarbonyloxy, 4- phenylbutyloxycarbonyloxy and the like.
  • Alkyl at R ⁇ is as defined for R and R 1 ; alkyl at R 8 and R 9 is as defined for R and R 1 ; and aralkyl at R 8 and R 9 is as defined for R and R 1 ,
  • Alkyl at R 7 is as defined for R and R 1 and aralkyl at R 7 is as defined for R and R 1 .
  • the group formed by R ⁇ and R 7 in combination, which forms a heterocycle optionally having, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom, is imidazol-2-yl, thiazol-2-yl, oxazol-2-yl, imidazolin-2-yl, 3,4,5,6- tetrahydropyridin-2-yl, 3,4,5,6-tetrahydropyrimidin-2-yl, l,3-oxazolin-2-yl, 1 ,3- thiazolin-2-yl or optionally substituted benzoimidazol-2-yl, benzothiazol-2-yl, benzoxazol-2-yl and the like having a substituent such as halogen, alkyl, alkoxy, haloalkyl, nitro, amino,
  • alkyl, aralkyl, haloalkyl and the like are as defined for R and R 1 .
  • Hydroxyalkyl at R 10 and R : is linear or branched alkyl having 1 to 6 carbon atoms which is substituted by 1 to 3 hydroxy, which is exemplified by hydroxymethyl, 2-hydroxyethyl, 1-hydroxyethyl, 3-hydroxypropyl, 4-hydroxybutyl and the like, Alkyl at R 10 and R 11 is as defined for R and R 1 ; haloalkyl and alkoxycarbonyl at R :0 and R 11 are as defined for R and R 1 ; aralkyl at R !0 and R 11 is as defined for R and R ! ; and cycloalkyl formed by R 10 and R ! 1 in combination is the same as cycloalkyl at R and R 1 .
  • Alkyl at L is as defined for R and R 1 .
  • Aminoalkyl at L is a linear or branched alkyl having 1 to 6 carbon atoms, which is substituted by amino, which is exemplified by aminomethyl, 2-aminoethyl, 1-aminoethyl, 3-aminopropyl, 4-aminobutyl, 5-aminopentyl, 6-aminohexyl and the like.
  • Mono- or dialkyiaminoalkyl at L is mono- or di-substituted aminoalkyl with alkyl having 1 to 4 carbon atoms, which is exemplified by methylaminomethyl, dimethylammomethyl, ethylaminomethyl, diethylammomethyl, propylaminomethyl, dipropylaminomethyl, butylaminomethyl, dibutylaminomethyl, 2- dimethylaminoethyl, 2-diethylaminoethyl and the like.
  • Carbamoylalkyl at L is linear or branched alkyl having 1 to 6 carbon atoms substituted by carbamoyl, which is exemplified by carbamoylmethyl, 2- carbamoylethyl, 1-carbamoylethyl, 3-carbamoylpropyl, 4-carbamoylbutyl, 5- carbamoylpentyl, 6-carbamoylhexyl and the like.
  • Phthalimido alkyl at L is linear or branched alkyl having 1 to 6 carbon atoms, which is substituted by phthalimide. Examples include phthalimidomethyl, 2- phthalimidoethyl, 1-phthalimidoethyl, 3-phthalimidopropyl, 4-phthalimidobutyl, 5- phthalimidopentyl, 6-phthalimidohexyl and the like, Alkyl at B is as defined for R and R 1 .
  • Alkoxy at B is as defined for R and R 1 .
  • Aralkyl at B is as defined for R and R 1 .
  • Aralkyloxy at B is as defined for R 3 and R 4 .
  • - Aminoalkyl at B is as defined for L.
  • Hydroxyalkyl at B is as defined for R 10 and R 11 .
  • Alkanoyloxyalkyl at B is linear or branched alkyl having 1 to 6 carbon atoms substituted by alkanoyloxy having an alkanoyl moiety having 2 to 6 carbon atoms, which is exemplified by acetyloxymethyl, propionyloxymethyl, butyryloxymethyl, valeryloxymethyl, pivaloyloxymethyl, acetyloxyethyl, propionyloxyethyl, butyryloxyethyl, valeryloxyethyl, pivaloyloxyethyl and the like.
  • Alkoxycarbonylalkyl at B is linear or branched alkyl having 1 to 6 carbon atoms substituted by alkoxycarbonyl having an alkoxy moiety having 1 to 6 carbon atoms, which is exemplified by methoxycarbonylmethyl, ethoxycarbonylmethyl, propoxycarbonylmethyl, isopropoxycarbonylmethyl, butoxycarbonylmethyl, isobutoxycarbonylmethyl, sec-butoxycarbonylmethyl, tert-butoxycarbonylmethyl, pentyloxycarbonylmethyl, hexyloxycarbonylmethyl, methoxycarbonylethyl, ethoxycarbonylethyl, propoxycarbonylethyl, isopropoxycarbonylethyl, butoxycarbonylethyl, isobutoxycarbonylethyl, sec-butoxycarbonylethyl, tert- butoxycarbonylethyl, pentyloxycarbonylethyl, hexyloxy
  • Flalogen at Q : , Q 2 and Q 3 is as defined for R and R 1 .
  • Aralkyloxy at Q 1 and Q 2 is as defined for R 3 and R 4 ,
  • Alkoxy at Q 3 is as defined for R and R 1 .
  • Alkylene at W, X and Y is linear or branched alkylene having 1 to 6 carbon atoms, which is exemplified by methylene, ethylene, trimethylene, propylene, tetramethylene, pentamethylene, hexamethylene and the like.
  • Alkenylene at Y is linear or branched alkenylene having 2 to 6 carbon atoms, which is exemplified by vinylene, propenylene, butenylene, pentenylene and the like.
  • Alkyl at Rb is as defined for R and R 1 .
  • - Aralkyl at Rb is as defined for R and R 1 ,
  • a ino alkyl at Rb is as defined for L.
  • Mono- or dialkylamino alkyl at Rb is as defined for L.
  • the heterocycle at Re is, when a single ring containing nitrogen, pyridine, pyrimidine, pyridazine, triazine, pyrazole, triazole and the like; and, when it is a condensed ring, pyrrolopyridine (e.g., lH-pyrrolo[2,3-b]pyridine, lH-pyrrolo[3,2- bjpyridine, lH-pyrrolo[3,4-b]pyridine and the like), pyrazolopyridine (e.g., 1H- pyrazolo[3,4-b]pyridine, lH-pyrazolo[4,3-b]pyridine and the like), imidazopyridine (e.g., lH-imidazo[4,5-b]pyridine and the like), pyrrolopyrimidine (e.g., 1H- pyrrolo[2,3-d]pyrimidine, lH-pyrrolo[3,2-d]pyrimidine, l
  • thienopyridine e.g., thieno[2,3- b]pyridine and the like
  • thienopyrimidine e.g., thieno[2,3-d]pyrimidine and the like
  • thiazolopyridine e.g., thiazolo[4,5-b]pyridine, thiazolo[5,4-b]pyridine and the like
  • thiazolopyrimidine e.g., thiazolo[4,5-d]pyrimidine, thiazolo[5,4-d]pyrimidine and the like
  • oxazolopyridine e.g., oxazolo[4,5 b]pyridine, oxazolo[5,4-b]pyridine and the like
  • oxazolopyrimidine e.g., oxazolo[4,5-d]pyrimidine, oxazolo[5,4-b]pyrimidine and the like
  • the carbon atom in the ring may be carbonyl and includes, for example, 2,3-dihydro-2-oxopyrrolopyridine, 2,3-dihydro-2,3-dioxopyrrolopyridine, 7,8-dihydro-7-oxo-l,8-naphthyridine, 5,6,7,8- tetrahydro-7-oxo-l,8-naphthyridine and the like.
  • These rings may be substituted by a substituent such as halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, amino, alkylammo, cyano, fomiyl, acyl, aminoalkyl, mono- or dialkylaminoalkyl, azide, carboxy, alkoxycarbonyl, carbamoyl, alkylcarbamoyl, alkoxyalkyl (e.g., methoxymethyl, methoxyethyl, methoxypropyl, ethoxymethyl, ethoxyethyl, ethoxypropyl and the like), optionally substituted hydrazino and the like.
  • a substituent such as halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, amino, alkylammo, cyano, fomiyl, acyl, aminoalkyl, mono- or dialkylaminoalkyl
  • the substituent of the optionally substituted hydrazino includes alkyl, aralkyl, nitro, cyano and the like, wherein alkyl and aralkyl are as defined for R and R and exemplified by methyl hydrazino, ethyl hydrazino, benzyl hydrazino and the like.
  • alkyl and aralkyl are as defined for R and R and exemplified by methyl hydrazino, ethyl hydrazino, benzyl hydrazino and the like.
  • the linear or branched alkyl having 1 to 6 carbon atoms at R 13 , R 14 , R 15 and R ' ' 6 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl and the like.
  • Aryl at R 14 and R 15 is phenyl, naphthyl and the like.
  • - Aralkyl at R 14 and R 15 is as defined for R and R 1 .
  • Alkylene having 4 or less carbon atoms which is formed by R 13 and R 14 directly bonded to each other, is methylene, ethylene, trimethylene, propylene, tetramethylene and the like.
  • Alkyl having 1 to 10 carbon atoms which substitutes alkylene having 4 or less carbon atoms formed by R 13 and R 14 directly bonded to each other, is linear or branched alkyl having 1 to 10 carbon atoms. Examples thereof include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl and the like.
  • Alkyl having 1 to 6 carbon atoms which substitutes ethylene and trimethylene formed by R' 3 and R 14 directly bonded to each other is linear or branched alkyl having 1 to 6 carbon atoms, which is the same as those for R.
  • the heterocycle formed by R 14 and R 15 directly or via oxygen atom bonded together with the adjacent nitrogen atom is pyrrolidino, piperidino, morpholino, homopiperidino, homomorpholino and the like.
  • - Alkylene having 2 to 4 carbon atoms formed by R 17 and R 18 directly bonded to each other is ethylene, trimethylene, propylene, tetramethylene and the like.
  • Alkylene having 2 to 6 carbon atoms at Alk is ethylene, trimethylene. propylene, tetramethylene, pentamethylene, hexamethylene and the like.
  • Alkyl having 1 to 6 carbon atoms and alkyl having 1 to 10 carbon atoms, which are the substituents of alkylene having 2 to 6 carbon atoms at Alk, are as defined for R 13 .
  • Aryl and aralkyl which are the substituents of alkylene having 2 to 6 carbon atoms at Alk, are as defined for R 14 .
  • Rho kinase inhibitors of the formula (I) include: (1) 4-(2-pyridylcarbamoyl)piperidine
  • N-(4-pyridyl)-4-aminomethylbenzamide (1 1) N-(4-pyridyl)-4-aminomethyl-2-hydroxybenzamide (172) N-(4-pyridyl)-4-(2-aminoethyl)benzamide (173) N-(4-pyridyl)-4-aminomethyl-3-nitrobenzamide
  • Rho kinase inhibitors of the formula (II) include:
  • N-(4-aminobutyl)-l-hydroxy-5-isoquinolinesulfonamide N-(2-amino-l -methylethyl)- l-hydroxy-5-isoquinolinesulfonamide
  • N-(2-amino-l-methylheptyl)-l-hydroxy-5-isoquinolinesulfonamide N-(3-amino-2-methylbutyl)-l-hydroxy-5-isoquinolinesulfonamide
  • N-[3-( N, N-dibutylamino)propyl]-l-hydroxy-5-isoquinolinesulfonamide N-[2-( N-cyclohexyl-N-methylamino)ethyl]-l-hydroxy-5- isoquinolinesulfonamide
  • Preferred compounds are (204) and (308).
  • the compounds may be provided as a pharmaceutically acceptable acid addition salt
  • the acid may be an inorganic acid such as hydrochloric acid, hydrobromic acid or sulfuric acid or an organic acid such as n ethanesulfonic acid, fumaric acid, maleic acid, mandelic acid, citric acid, tartaric acid or salicylic acid.
  • Compounds having a carboxyl group can be converted to a salt with a metal such as sodium, potassium, calcium, magnesium or aluminum or an amino acid such as lysine and the like.
  • Rho-kinase inhibitors are disclosed in US-A-5478838. These Rho- kinase inhibitors are: (1) 4-amino(alkyl)cyclohexane-l-carboxamide compounds of the formula (III):
  • R 1 and R 2 are the same or different and each is hydrogen, alkyl, or cycloalkyl, cycloalkylalkyl, phenyl, aralkyl, piperidyl or pyrrolidinyl, any of which may have a substituent on the ring, or a group of the formula:
  • R is hydrogen, alkyl, -NR'R" (where R' and R" are the same or different and each is hydrogen, alkyl, aralkyl or phenyl) and R° is hydrogen, alkyl, aralkyl, phenyl, nitro or cyano, or R and R° may in combination form a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom, or
  • R 1 and R 2 in combination represent alkylidene or phenylalkylidene, or R 1 and R 2 form, together with the nitrogen atom, a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom;
  • R 3 and R 4 are each hydrogen or alkyl;
  • A is a single bond or alkylene;
  • Ra and Rb are the same or different and each is hydrogen, halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, -NRcRd
  • Re and Rd are the same or different and each is hydrogen, alkyl, -COR 9 , -COOR 9 or -S02R 9' (where R 9 is hydrogen, alkyl, phenyl or aralkyl and R 9' is alkyl, phenyl or aralkyl) or Re and Rd fo ⁇ n, together with the nitrogen atom to which they are attached, a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom], cyano, azide, optionally substituted hydrazino, -COOR 10 , or -CONR u R 12 (wherein R 10"12 are each hydrogen, alkyl,
  • R 1 and R 2 in combination represent alkylidene or phenylalkylidene, or R 1 and R 2 form, together with the nitrogen atom to which they are attached, a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom;
  • R 3 and R 4 are each hydrogen or alkyl
  • A is a single bond or alkylene
  • Ra and Rb are the same or different and each is hydrogen, halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, -NRcRd
  • Re and Rd are the same or different and each is hydrogen, alkyl, -COR 9 , -COOR 9 ' or -S02R 9 ' (where R 9 is hydrogen, alkyl, phenyl or aralkyl and R Sl is alkyl, phenyl or aralkyl) or Re and Rd form, together with the nitrogen atom to which they are attached, a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom], cyano, azido, optionally substituted hydrazino, -COOR 10 or -CONR n R 12 (wherein R 10"12 are each hydrogen, alkyl, phenyl or aralkyl), or
  • R is hydrogen, alkyl or -NR'R" (wherein R 1 and R" are the same or different and each is hydrogen, alkyl, aralkyl or phenyl) and R° is hydrogen, alkyl, aralkyl, phenyl, nitro or cyano, or R and R° in combination form a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom;
  • Ra and Rb are the same or different and each is hydrogen, halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, -NRcRd
  • Re and Rd are the same or different and each is hydrogen, alkyl, -COR 9 , -COOR 9 ' or -S02R 9 ' (where R 9 is hydrogen, alkyl, phenyl or aralkyl and R 9 ' is alkyl, phenyl or aralkyl) or Re and Rd form, together with the nitrogen atom to which they are attached, a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom], cyano, azido, optionally substituted hydrazino, -COOR 10 or -CONR u R 12 (wherein R 10"12 are each hydrogen, alkyl,
  • Ra and Rb in combination form an optionally hydrogenated 5- or 6-membered aromatic ring which may have, in the ring, at least one of a nitrogen atom, sulfur atom and oxygen atom provided that, when R 5 and R b are of the formula (b) or (d), Ra and Rb together form an optionally hydrogenated 5- or 6-membered aromatic ring which may have at least one of a nitrogen atom, sulfur atom and oxygen atom;
  • R 7 and R 8 are the same or different and each is hydrogen, halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, -NReRf [wherein Re and Rf are the same or different are each is hydrogen, alkyl, -COR 9 , -COOR 9 ' or -S02R 9 ' (where R 9 is hydrogen, alkyl, phenyl or aralkyl and R 9 ' is alkyl, phenyl or aralkyl) or Re and Rf form, together
  • Preferred such compounds are: trans-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-aminomethylcyclohexanecarboxamide, trans-N-(l H-pyrazolo [3, 4-b] ⁇ yridin-4-yl)-4-aminomethylcyclohexanecarboxamide,
  • halogen means chlorine, bromine, fluorine or iodine
  • alkyl means straight- or branched chain alkyl having 1 to 10, preferably 1 to 6 ' carbon atoms, which is exemplified by methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, 2- ethylhexyl, octyl, nonyl or decyl; haloalkyl means the aforementioned alkyl substituted by 1 to 5 halogens, which is exemplified by trifluoromethyl, 2,2,2- trifluoroethyl or 2,2,3,3,3-pentafluoropropyl; alkoxy means straight- or branched chain alkoxy having 1 to 6 carbon
  • substituents for cycloalkyl having 3 to 7 carbon atoms include halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, -NRcRd (wherein Re and Rd may be the same or different and each is hydrogen, alkyl, -COR 9 , -COOR 9 ' or -S02R 9 ' or Re and Rd, together with the nitrogen atom to which they are attached, form a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom), cyano, azido, formyl, acyl, -COOR 10 , -CONR ⁇ R 12 or optionally substituted hydrazino, As used herein, halogen, alkyl, alkoxy
  • substituents for the optionally substituted hydrazino include alkyl, aralkyl, nitro and cyano, wherein alkyl and aralkyl are as defined above.
  • the heterocyclic ring optionally having, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom, which is formed by R 1 and R 2 , Re and Rd or Re and Rf, together with the nitrogen atom to which they are attached, is preferably a 5- or 6-membered ring or a fused ring system and is exemplified by pyrrolidinyl, piperidyl, piperazinyl, morpholino and thiomorpholmo.
  • substituent for the optionally substituted nitrogen atom include alkyl, aralkyl and haloalkyl, wherein alkyl, aralkyl and haloalkyl are as defined above,
  • the heterocyclic ring optionally having, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom, which is formed by R° and R 1 in combination, is exemplified by imidazol-2-yl, thiazol-2-yl, oxazol-2-yl, imidazolin-2- yl, 3,4,5,6-tetrahydropyridin-2-yl, 3,4,5,6-tetrahydropyrimidin-2-yl, l,3-oxazolin-2- yl, l,3-thiazolin-2-yl, and benzimidazol-2-yl, benzothiazol-2-yl, benzoxazol-2-yl and indol-2-yl which may have substituent such as halogen, alkyl, alkoxy, haloalkyl, nitro, amino, phenyl or aralkyl, wherein halogen, alkyl, alkoxy, hal
  • substituent for the optionally substituted nitrogen atom examples include alkyl, aralkyl and haloalkyl, wherein alkyl, aralkyl and haloalkyl are as defined above.
  • R 5 and R ⁇ are of the formula (a), (c), (e) or (f) and form a single ring
  • the ring is pyridine, pyrimidine, pyridazine, triazine, pyrazole or triazole.
  • R 5 and R ⁇ are of the formula (a), (b) or (d) and form a condensed ring
  • the ring is pyrrolopyridine (e.g. lH-pyrrolo[2,3-b]pyridine, lH-pyrrolo[3,2-b]pyridine, 1H- pyrrolo[3,4-b]pyridine), pyrazolopyridine (e.g.
  • lH-pyrazolo[3,4-b]pyridine 1H- pyrazolo[4,3-b]pyridine
  • imidazopyridine e.g. lH-imidazo[4,5-b]pyridine
  • pyrrolopyrimidme e.g. lH-pyrrolo[2,3-d]pyrimidine, lH-pyrrolo[3,2-d]py ⁇ imidine, lH-pyrrolo[3,4-d]pyrimidine
  • pyrazolopyrimidine e.g.
  • lH-pyrazolo[3,4- d]pyrimidine pyrazolo[l,5-a]pyrimidine, lH-pyrazolo[4,3-d]pyrimidine
  • imidazopyrimidine e.g. imidazo[l,2-a]pyrimidine, lN-imidazo[4,5-d]pyrimidine
  • pyrrolotriazine e.g. pyrrolo[l,2-a]-l,3,5-triazine, pyrrolo[2,l-f]-l,2,4-triazine
  • pyrazolotriazine e.g.
  • pyrazolo[l,5-a]-l,3,5-triazine triazolopyridme (e.g. 1H-1,2,3- triazolo[4,5-b]pyridine), triazolopyrimidine (e.g. l ,2,4-triazolo[l,5-a]pyrimidine, l,2,4-triazolo[4,3-a]pyrimidine, lH-l,2.3-triazolo[4,5-d]pyrimidine), cinnoline, quinazoline, quinoline, pyridopyridazine (e.g. pyrido[2,3-c]pyridazine), pyridopyrazine (e.g.
  • pyrido[2,3-b]pyrazine pyrido[2,3-b]pyrazine
  • pyridopyrimidine e.g. pyrido[2,3- d]pyrimidine, pyrido[3,2-d]pyrimidine
  • pyrimidopyrimidine e.g. pyrimido[4,5- djpyrimidine, pyrimido[5,4-d]pyrimidine
  • pyrazinopyrimidine e.g. pyrazino[2,3- djpyrimidine
  • naphthyridine e.g. 1,8-naphthyridine
  • tetrazolopyrimidine e.g.
  • thienopyridine e.g. thieno[2,3-b]py ⁇ idine
  • thienopyrimidine e.g. [2,3-d]pyrimidine
  • thiazolopyridine e.g. thiazolo[4,5- bjpyridine, thiazolo[5,4-b]pyridine
  • thiazolopyrimidine e.g, thiazolo[4,5- d]pyrimidine, thiazolo[5,4-d]pyrimidine
  • oxazolopyridine e.g.
  • 2,3-dihydropyrrolopyridine e.g, 2,3-dihydro-lH-py ⁇ olo[
  • the carbon atom in the ring may be carbonyl and, for example, 2,3- dihydro-2-oxopyrrolopyridine, 2,3-dihydro-2,3-dioxopyrrolopyridine, 7,8-dihydro-7- oxonaphthyridine and 5,6,7,8-tetrahydro-7-oxonaphthyridine are included,
  • These rings may be substituted by substituent such as halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, -NRcRd, cyano, formyl, acyl, aminoalkyl, mono- or dialkylammoalkyl, azido, -COOR 10 , -CONR n R 12 or optionally substituted hydrazino,
  • Pharmaceutically acceptable acid addition salts of a compound of formula (III) or (V) include salts with an inorganic acid or organic acid.
  • the compound may talce the fo ⁇ n of a hydrate or solvate.
  • the salt may be a metal salt such as a sodium salt, potassium salt, calcium salt or aluminum salt or a salt with an amino acid such as lysine and ornithine.
  • the compound may take the form of a cis- or trans-geometrical isomer or a mixture thereof,
  • the compound of formula (III) or (V) has an asymmetric carbon, it may be an optical isomer or a racemate.
  • Rho-kinase inhibitors for use in the invention are disclosed in WO 02/076977, These inhibitors are compounds of formula (VI) to (XI):
  • Suitable pharmaceutically acceptable salts include basic salts of inorganic and organic acids, such as hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, sulphonic acid, acetic acid, trifiuoroacetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicyclic acid, phenyl acetic acid and mandelic acid, ln addition, pharmaceutically acceptable salts include acid salts of inorganic bases, such as salts containing alkaline cations (e.g, Li-, Na + or K ⁇ ), alkaline earth cations (e.g, Mg + , Ca + or Ba ⁇ ), the ammonium cation, as well as acid salts of organic bases, including aliphatic and aromatic substituted ammonium, and quaternary ammoni
  • Rho-kinase inhibitors are disclosed in EP-A-0187371.
  • a particularly prefe ⁇ ed Rho-kinase inhibitor is (-)-(R)-/ra «-s-4-(l-aminoethyl)-N-(4- pyridyl)cyclohexanecarboxamide or a pharmaceutically acceptable acid addition salt thereof, especially the dihydrochloride which has the formula:
  • This compound is typically provided as a monohydrate.
  • Rho-kinase inhibitors are used to treat benign prostatic hyperplasia (BPH).
  • BPH benign prostatic hyperplasia
  • An effective amount of a Rho-kinase inhibitor is administered to a man suffering from BPH, The condition of the patient can thus be improved.
  • the symptoms of BPFI can be alleviated.
  • an inhibitor is provided in a pharmaceutical pack together with instructions for use in treating BPH.
  • Administration of an inhibitor may be in a variety of dosage forms, Thus, an inhibitor may be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules.
  • An inhibitor may also be administered parenterally, either subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques.
  • An inhibitor may be administered locally using a catheter (intraurethrally), An inhibitor may be administered as a suppository, The dosage form and route of administration will depend largely on the nature of
  • An inhibitor modulator is typically formulated for administration in the present invention with a pharmaceutically acceptable carrier or diluent.
  • the pha ⁇ naceutical carrier or diluent may be, for example, an isotonic solution.
  • solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g, silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g.
  • starches gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations.
  • Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film-coating processes.
  • Liquid dispersions for oral administration may be syrups, emulsions or suspensions.
  • the syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
  • Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate,- pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol.
  • the suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g, sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
  • Solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.
  • a therapeutically effective amount of an inhibitor is administered to man having BPH.
  • the dose of inhibitor may be determined according to various parameters, especially according to the particular inhibitor used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen.
  • a physician will be able to determine the required route of administration and dosage for any particular patient.
  • a typical daily dose is from about 0.1 to 50 mg per kg of body weight, according to the activity of the specific inhibitor, the age, weight and conditions of the subject to be treated, the severity of the BPH and the frequency and route of administration.
  • daily dosage levels are from 5 mg to 2 g.
  • a daily dose may be given in one administration or more than one administration, for example 2, 3 or 4 administrations.
  • an inhibitor may be administered as a polynucleotide.
  • the polynucleotide may comprise coding sequence for a molecule with inhibitory activity, for example a protein or mRNA.
  • the polynucleotide is generally constructed so as to permit expression of the inhibitory molecule in a target cell.
  • the coding sequence may be operably linked to a suitable promoter sequence, which will direct expression in a target cell.
  • the polynucleotide may itself have inhibitory activity.
  • an inhibitor ⁇ ' polynucleotide may encode an inhibitory protein or mRNA, or the polynucleotide may itself be an inhibitor, for example, an antisense molecule or iRNA,
  • the polynucleotide may be administered as a naked nucleic acid construct,
  • a nucleic acid may also be administered using a viral vector.
  • a polynucleotide composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, transdermal or intraurethral administration, Typically, a polynucleotide inhibitor is mixed with a transfection agent to produce a composition.
  • the naked polynucleotide construct, viral vector comprising the polynucleotide or polynucleotide composition is combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition
  • a pharmaceutically acceptable carrier or diluent include isotonic saline solutions, for example phosphate-buffered saline,
  • a polynucleotide is administered in such a way that the polynucleotide, for example viral vector can be incorporated into cells of the prostate.
  • the amount of virus administered is in the range of from 10 6 to 10 :o pfu, preferably from 10 7 to 10 9 pfu, more preferably about 10 8 pfu for adenoviral vectors.
  • typically 1-2 ml of virus in a pharmaceutically acceptable suitable carrier or diluent is administered.
  • the amount of nucleic acid administered is typically in the range of from l ⁇ g to 10 mg,
  • Human prostate tissue was obtained from four patients subjected to transurethral resection of the prostate (TURP) due to symptomatic BPH, Specimens were resected from normal appearing, non-adenomatous parts of the prostate close to tl ⁇ e bladder neck.
  • PBS phosphate buffered saline solution
  • Tissue specimens were transferred into sterile culture medium (MCDB-131 with 10% fetal calf serum, 1% antibiotics, lOmM HEPES, 1% penicillin- streptomycin solution, 1% non-essential aminoacids, 5 ⁇ g/ml insulin, lO ⁇ g/ml transfe ⁇ in, 5ng/ml sodium-selenite, 0.1 ⁇ M dexamethasone and 0.1 ⁇ M ⁇ -estradiol).
  • This medium has been previously shown to stimulate the growth of smooth muscle cells and to suppress the growth of fibroblasts from prostatic explants (Corvin et al, Prostate 37, 209-214, 1998).
  • the specimens were then cut into 1 mm 3 pieces using a Mcllwain tissue chopper (MLE, UK). 5-10 small pieces were seeded into each well of 6- well culture plates (NUNC, Gibco, UK) each containing 0.5 ml of medium. After incubating the pieces at 37'C in 5% C0 2 in a humidified incubator for
  • 0.5-1.0x10 5 cells on each 22x22mm coverslip were fixed in methanol at -20°C and washed in PBS containing 1% bovine serum albumin.
  • the coverslips were incubated with Rho-kinase (1 : 100, AutogenBioclear, UK) and smooth muscle specific ⁇ -actin (1 :200, Sigma, UK) antibodies overnight at 4°C. They were then incubated with biotinylated anti-goat IgG (1 :200, Vector Laboratories, USA) for 2 hours followed by FITC avidin-D (1 :200) and anti-mouse IgG-Texas Red (1 :200, Vector Laboratories, USA) for 1 hour at room temperature.
  • the cover slips were mounted on glass-slides with a drop of Vectashield mounting medium (Vector
  • the cell membranes were disrupted using a sonicator at 20 ⁇ m for 5 s at 4°C.
  • the lysate was centrifuged at 13,000 g for 15 min at 4°C.
  • Tissue specimens human and rat prostate
  • the homogenate was resuspended in 1 ml homogenising buffer (composition as above), vortexed vigorously for 20 s and centrifuged at 13,000 g for 15 min at 4°C.
  • Rho-kinase inhibitor Y-27632 ((+)-(R)-trans-4-(l-aminoethyl)-N-(4- pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate; Calbiochem, UK; 0.01-100 ⁇ M) in the culture medium as above but containing only 1% FCS for 24, 48 and 72 hours.
  • 5-bromo-2'-deoxyuridine (BrdL ) labelling (Roche Diagnostics, UK) was performed according to the protocol provided by the manufacturer.
  • the pyrimidine analogue of BrdU is incorporated in place of thymidine into the DNA of proliferating cells. After removing the culture medium the cells are fixed and the DNA is denatured, Incorporated BrdU is then detected by immunoassay.
  • the viability of human prostatic smooth muscle cells was assessed using the neutral red and MTT assays as described previously (Morgan, J. Immunol, Methods 145. 259-262, 1991 ; Mosmann, Immunol. Methods 65, 55-63, 19S3).
  • the cells were cultured in 6-well plates. After serum starvation for 24 hours, the cells were incubated in the presence or absence of Y-27632 (0.01-100 ⁇ M) in the culture medium as above but containing only 1% FCS for 24, 48 and 72 hours.
  • the cells in 10cm dishes were restimulated with 1% serum in the presence or absence of Y-27632 (0.01-100 ⁇ M) for 48 hours.
  • the cells were collected and incubated in the presence of TRIS (10 mM), NaCl (ImM), RNase (100 ⁇ g ml), Tween 20 (0, 1%) and propidium iodide (0.004%) at 37°C for 25 min.
  • Apoptotic cells were distinguished from non-apoptotic cells by their decreased DNA content, as determined by their lower propidium iodide staining intensity in the presence of RNase.
  • the cells were counted using a flow cytometer (Becton Dickinson, FACSCalibur, UK), Human prostatic smooth muscle cells treated with 0.1 ⁇ M staurosporine for 4 hours were used as positive controls,
  • Rat prostate was obtained from male Wistar rats (250-275g), The ventral part of the prostate was dissected, cleaned from adherent tissue and divided vertically into three equally sized strips, The prostatic strips were placed horizontally between two ring electrodes in tissue chambers at 37°C.
  • the tissues were superfused at a constant flow of 1 ml/min by means of peristaltic pumps (Miniplus 2, Gilson, UK) with a medium of the following composition (mM): NaCl 136.9, KC1 2.7, CaCl 2 1 ,8, MgSO, 0.6, NaHC0 3 11.9, KH 2 PO 4 0.5, glucose 11.5, indomethacin 0.01, dexamethasone 0.01 and gassed with 5% CO 2 in 0 2 (pH 7.4-7.6).
  • mM composition
  • One end of the preparation was tied to a Grass FT 03 C force-displacement transducer connected to a Linearcorder WR 3101 (Graphtec, USA) for registration of isometric changes in tension.
  • the preparations were stretched (0.2 to 0.5g) until they reached approximately the in situ length and allowed to equilibrate for 90 min,
  • the preparations were stimulated electrically (electrical field stimulation; EFS) with 5 s trains of rectangular pulses of 50 V, 0,3 ms pulse duration and frequencies ranging from 1 to 50 Hz, delivered by Grass S88 stimulators every 120 s throughout the experiment. Drugs were applied into the medium reservoir,

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Abstract

Rho-kinase inhibitors are used to treat benign prostatic hyperplasia in man. Such inhibitors have a dual effect: they can relax the smooth muscle of the prostate and reduce the size of the prostate by inhibiting smooth muscle cell proliferation. A suitable Rho-kinase inhibitor is (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride.

Description

TREATMENT OFBENIGN PROSTATICHYPERPLASIA
Field of the invention
This invention relates to the treatment of benign prostatic hyperplasia (BPH).
Background of the invention
BPH affects approximately 85% of men older than 50 years of age. BPH represents hyperplasia of both the glandular and stromal components of the prostate. Development of lower urinary tract symptoms (LUTS) due to BPH is caused by mechanical obstruction to urinary flow secondary to enlarged prostate (static component) and elevated smooth muscle tone in the fϊbromuscular stroma, prostatic capsule and the bladder neck (dynamic prostate). Pharmacotherapy therefore aims to reduce the size of the prostate or to decrease the tone of prostatic smooth muscle. Among the drugs which aim to decrease the size of the prostate are 5α- reductase inhibitors; they block androgen stimulation of the organ without affecting androgen-dependent skeletal muscle strength, libido and potency. αrAdrenoceptor antagonists on the other hand aim to reduce the smooth muscle tone and urethral pressure. Recently combination therapy using a 5α-reductase inhibitor and a αr adrenoceptor antagonist has been found to be hopeful in the management of BPH. Such a combination therapy has obvious theoretical appeal: αradrenoceptor antagonist acts rapidly to reduce symptoms while the 5α-reductase inhibitor reduces the size of the prostate over a longer period.
Summary of the invention We have found that a Rho-kinase inhibitor inhibits adrenergic contractions of the prostate tissue and proliferation of prostatic smooth muscle cells. Rho-kinase inhibitors can therefore be used as a single agent treatment for BPH with dual effect; by relaxing the smooth muscle and by reducing the size of the prostate by inhibiting the smooth muscle cell proliferation. Accordingly, the invention provides use of a Rho-kinase inhibitor for the manufacture of a medicament for use in the treatment of benign prostatic hyperplasia. The invention also provides a method of treating benign prostatic hyperplasia in a man suffering therefrom, which method comprises the step of administering thereto a therapeutically effective amount of a Rho-kinase inhibitor.
Brief Description of the Figures
Figure 1 shows the expression of Rho-kinase in rat and human prostatic smooth muscle, Confocal microscopy images of double immunofluorescence in smooth muscle cells from human (a,b) and rat prostate (c,d). Note perinuclear localisation of Rho-kinase in a and c. Scale bar 25 μm. e, Western immunoblotting of Rho-kinase in rat brain (lane 1), human prostatic smooth muscle cells (lane 2), rat prostatic smooth muscle cells (lane 3), human prostate (lane 4), rat prostate (lane 5), The band corresponding to Rho-kinase (- 180 kD) is indicated with an arrow.
Figure 2 shows that the Rho-kinase inhibitor Y-27632 inhibits proliferation but does not induce apoptosis in human prostatic smooth muscle cells, Cell viability after exposure to Y-27632 was measured using neutral red (a) and MTT (b) assays, c, Incorporation of BrdU to human prostatic smooth muscle cells was measured in the absence and presence of Y-27632, d, The effect of Y-27632 and staurosporine on the apoptotic index of human prostatic smooth muscle cells. Figure 3 shows that the Rho-kinase inhibitor Y-27632 inhibits adrenergic contractions of the rat prostate, a, Contractions elicited with electrical field stimulation (EFS; denoted with dots) was reversibly inhibited by Y-27632. The contractions were inhibited by guanethidine confirming their noradrenergic nature, b, Phenylephrine-induced contractions were reversibly inhibited by Y-27632. c, The effect of Y-27632 on contractions elicited by EFS (■) and exogenous phenylephrine
Detailed description of the invention
The present invention is concerned with the use of a Rho-kinase inhibitor to treat benign prostatic hyperplasia (BPH). Rho-kinase is activated by RhoA, a small monomeric G-protein believed to be coupled to excitatory receptors (i.e. α,- adrenoceptors), Activated Rho-kinase inhibits smooth muscle myosin phosphatase (SMPP-1 ) by phosphorylating its regulatory subunit. This leads to sensitization of myofilaments to Ca2+ in smooth muscle cells. An inhibitor of Rho-kinase for use in the invention directly or indirectly causes a reduction in the activity and/or expression of Rho-kinase. An inhibitor of Rho-kinase may thus inliibit the activity of Rho-kinase. An inhibitor may act by binding to Rho-kinase or by binding to the substrate of Rho-kinase.
An inhibitor of the invention may act, at the level of Rho-kinase expression. Such an inhibitor may cause its effect, for example, at initiation, progression or termination of transcription, during transport of Rho-kinase mRNA from the nucleus to the cytoplasm, or during processing and/or translation of Rho-kinase mRNA. Alternatively or additionally, an inhibitor of the invention may reduce the stability of the Rho-kinase protein. For example, the inhibitor may increase the rate at which the protein is degraded or inactivated.
An inhibitor of expression may bind to a Rho-kinase gene 5' to the coding sequence and/or in the coding sequence and/or 3' to the coding sequence, Thus an inhibitory molecule may bind to the promoter, to reduce the rate of transcription, The inhibitor may bind and inhibit a protein factor required for transcription of the gene, preventing its activity, An inhibitory agent may bind to and stimulate a protein factor that causes down-regulation of Rho-kinase transcription. The inhibitor may itself comprise such a factor or a nucleic acid encoding such a factor. Preferably, the present inhibitor is specific in its modulation of transcription from a Rho-kinase gene and has substantially no effect on transcription from other genes, An inhibitor of the invention may bind to the untranslated or translated regions of Rho-kinase mRNA, to inhibit initiation or progress of translation, to nuclear factors that bind to the mRNA and/or transport the mRNA to the cytoplasm, or to translation factors that contribute to translating the mRNA to protein.
An inhibitor of the invention may comprise a polynucleotide, for example DNA or RNA, The polynucleotide may encode a molecule with inhibitory activity such as any of those described above, for example a protein or mRNA molecule. The inhibitory molecule thus becomes available upon expression of the polynucleotide, for example in a target cell. Alternatively the polynucleotide may itself have inhibitor}' activity. A polynucleotide may thus encode an inhibitory protein or mRNA or the polynucleotide may itself be an inhibitor, for example, an antisense molecule or iRNA,
The polynucleotide may include synthetic or modified nucleotides. A number of different types of modification to polynucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. The polynucleotides described herein may be modified by any method available in the art, Such modifications may be carried out in order to enhance the in vivo activity or lifespan of polynucleotide.
In general, an inhibitor for use in the invention may be identified by assaying a test substance for activity which reduces Rho-kinase activity and/or expression. A typical assay comprises: (i) contacting a test substance with a source of Rho-kinase activity and/or expression, under conditions that would permit Rho-kinase activity and/or expression in the absence of the test substance; and (ii) determining whether the test substance causes a decrease in the activity and/or expression of the Rho-kinase.
Any suitable assay form may be adopted, The source of Rho-kinase activity and/or expression may be a Rho-kinase such as ROC (Leung et al, J. Biol. Chem. 270, 29051-29054, 1995; sometimes also called ROCK-H) or plβOROCK (Ishizaki et al, EMBO J. 15, 1885-1893, 1996; sometimes also called ROCβ or ROCK-I).
A control assay is generally also carried out to assess the activity and/or expression of the Rho-kinase, as the case may be, under the same reaction conditions but in the absence of the test substance. The control assay is preferably carried out in parallel with the test assay, Preferably, multiple repetitions are made of both the test and control assays. Also, as a control, the samples may be assayed for any other enzyme to exclude the possibility that a test substance is a general modulator of enzyme activity,
An inhibitor of Rho-kinase transcription can be identified using a reporter gene assay. Typically such an assay comprises: (i) providing a test construct comprising a Rho-kinase promoter operably linked to a reporter polynucleotide to be expressed in the form of mRNA;" (ii) contacting a test substance with the test construct under conditions, which in the absence of the test substance, would permit expression of the reporter polynucleotide in the form of mRNA; (iii) carrying out a control assay under the same reaction conditions but in the absence of the test substance; and (iv) determining whether the test substance inhibits expression from the construct.
A Rho-kinase promoter for use in the assay may be isolated via methods Icnown in the art. Any eukaryotic Rho-kinase promoter may be used but preferably a mammalian promoter in particular, a Rho-kinase promoter, is used. The promoter sequence may be fused directly to a reporter sequence or via a linker. The linker sequence may comprise a sequence having enhancer characteristics, to boost expression levels.
The reporter assay should be carried out under conditions that allow expression of the reporter polynucleotide as mRNA in the absence of the test substance. For example, the assay should include a suitable RNA polymerase and nucleotides to allow transcription of the reporter sequence, Levels of reporter mRNA in the presence and absence of the test substance may be compared to assess any modulatory effect of the test substance on transcription from the Rho-kinase promoter. Preferably the assay is carried out under conditions which mimic those to which the Rho-kinase promoter is exposed in vivo. Preferably one or more control assays is carried out using any other promoter in place of the Rho-kinase promoter, to exclude the possibility that a test substance is a general inhibitor of transcription,
An inhibitor which affects the stability of Rho-kinase mRNA or Rho-kinase protein may be identified in a stability assay, hi a suitable assay, Rho-kinase mRNA or Rho-kinase protein is incubated in the presence and absence of a test substance, under conditions which mimic those to which the Rho-kinase mRNA or protein is exposed in vivo. Typically, assay temperatures are those found in vivo, for example 25 to 37°C, in particular 37°C. Samples are removed at regular time intervals and assayed for the presence of Rho-kinase mRNA or protein. This can be done by any suitable means, for example, by gel analysis or spectrophotometrically. In this- way, any alteration in the rate of degradation of Rho-kinase mRNA or protein, associated with the test substance, can be detected.
An inhibitor which acts at the level of translation of Rho-kinase may be identified using an in vitro translation system. Suitable translation systems are available in the art: for example, the rabbit reticulocyte system. Rho-kinase mRNA is used in the system in the presence and absence of a test substance, under conditions which permit translation of the mRNA in the absence of the test substance, Again conditions will preferably mimic those to which Rho-kinase mRNA is exposed in vivo. The effect of a test substance is assessed by monitoring the production of Rho-kinase protein using any suitable means, for example, by gel analysis or by spectrophotometer.
An inhibitor of Rho-kinase activity and/or expression is one which produces a measurable or detectable decrease in Rho-kinase activity and/or expression, for example in at least one of the assays described above, Generally such inhibitors are those which inhibit Rho-kinase activity and/or expression by at least 10%. at least 20%, at least 30%, at least 40% at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% at a concentration of the inhibitor of lμg ml"1, lOμg ml'1, lOOμg ml"1, SOOμg mr1, lmg rnl"1' lOmg mϊ' or lOOmg ml"1. The percentage inhibition represents the percentage decrease in expression/activity in a comparison of assays carried out in the presence and absence of the test substance, Any combination of the above mentioned degrees of percentage inhibition and concentration of inhibitor may be used to define an inhibitor of the invention, with greater inhibition at lower concentrations being preferred, In the above assays, suitable test substances include antibodies, combinatorial libraries, defined chemical identities, chemical compounds, peptide and peptide mimetics, oligonucleotides and natural product libraries, such as display libraries (e.g. phage display libraries). Typically, organic molecules will be screened, preferably small organic molecules which have a molecular weight of from 50 to 2500 daltons. Candidate products can be biomolecules including, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds, Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. Test substances may include polynucleotides. Where a potential modulator is a polynucleotide encoding an inhibitory molecule, for example, a protein or mRNA, the encoded molecule may be used as a "test substance" in an assay. A modulator may include a single substance or a combination of two, three or more substances. For example, a modulator may refer to a single peptide, a mixture of two or more peptides or a mixture of a peptide and a defined chemical entity,
Test substances may be used in an initial screen of, for example, 10 substances per reaction, and the substances of these batches which show inhibition or stimulation tested individually.
Suitable Rho-kinase inhibitors for use in the invention are described in EP-A- 0956865. Specifically, such Rho-kinase inhibitors have of the formula (I):
0 Rb Ra— C-N-Rc ( I )
wherein Ra is a group of the formula:
Figure imgf000008_0001
Figure imgf000009_0001
h, in the formulae (a) and (b):
R is hydrogen, alkyl or cycloalkyl, cycloalkylalkyl, phenyl or aralkyl, which optionally have a substituent on the ring, or a group of the formula:
NR7
( d )
RΛ
wherein Rδ is hydrogen, alkyl or formula : -NRSNR9 wherein R8 and R9 are the same or different and each is hydrogen, alkyl, aralkyl or phenyl, R7 is hydrogen, alkyl, aralkyl, phenyl, nitro or cyano, or R6 and R7 in combination form a heterocyclic group optionally having, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom,
R1 is hydrogen, alkyl or cycloalkyl, cycloalkylalkyl, phenyl or aralkyl, which optionally have a substituent on the ring, or
R and R1 in combination form, together with the adjacent nitrogen atom, a heterocyclic group optionally having, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom,
R2 is hydrogen or alkyl,
R3 and R4 are the same or different and each is hydrogen, alkyl, aralkyl, halogen, nitro, amino, alkylamino, acylamino, hydroxy, alkoxy, aralkyloxy, cyano, acyl, mercapto, alkylthio, aralkylthio, carboxy, alkoxycarbonyl, carbamoyl, alkylcarbamoyl or azide, and A is a group of the formula:
( e )
Figure imgf000010_0001
wherein R10 and R11 are the same or different and each is hydrogen, alkyl, haloalkyl, aralkyl, hydroxyalkyl, carboxy or alkoxycarbonyl, or R10 and R11 in combination form a cycloalkyl group and 1, m and n are each 0 or an integer of l-3, and in which, in the formula (c): a broken line is a single bond or a double bond,
R5 is hydrogen, hydroxy, alkoxy, alkoxycarbonyloxy, alkanoyloxy or aralkyloxycarbonyloxy, and
L is hydrogen, alkyl, aminoalkyl, mono or dialkylaminoalkyl,tetrahydrofurfuryl, carbamoylalkyl, phthalimidoalkyl, amidino or a group of the formula:
O
II B— C- ( f)
Figure imgf000010_0002
wherein B is hydrogen, alkyl, alkoxy, aralkyl, aralkyloxy, aminoalkyl, hydroxyalkyl, hydroxyalkyl, alkanoyloxyalkyl, alkoxycarbonylalkyl, alpha-aminobenzyl, furyl, pyridyl, phenyl, phenylamino, styryl or imidazopyridyl,
Q1 is hydrogen, halogen, hydroxy, aralkyloxy or thienylmethyl,
W is alkylene,
Q2 is hydrogen, halogen, hydroxy or aralkyloxy,
X is alkylene, and
Q3 is hydrogen, halogen, hydroxy, alkoxy, nitro, amino, 2,3-dihydrofuryl or 5- methyl-3-oxo-2,3,4,5-tetrahydropyridazin-6-yl; and Y is a single bond, alkylene or alkenylene; and Rb is a hydrogen, an alkyl, an aralkyl, an aminoalkyl or a mono- or dialkylaminoalkyl; and
Re is an optionally substituted heterocycle containing nitrogen; an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof.
Other Rho-kinase inhibitors disclosed in EP-A-0956865 have the formula
(H) :
Figure imgf000011_0001
wherein
R12 is hydrogen, chlorine or hydroxy, and when R!2 is a hydrogen,
Alk is an alkylene group having 2 to 6 carbon atoms, which optionally has alkyl having 1 to 10 carbon atoms, aryl or aralkyl as a substituent;
R13 is hydrogen;
RM is hydrogen, or linear or branched alkyl having 1 to 6 carbon atoms, aryl or aralkyl;
R15 is hydrogen, linear or branched alkyl having 1 to 6 carbon atoms, aryl or aralkyl, or benzoyl, cinnamyl, cinnamoyl, furoyl or a group of the following formula
Figure imgf000012_0001
wherein R16 is linear or branched alkyl having 1 to 6 carbon atoms or a group of the following formula
Figure imgf000012_0002
wherein R'7 and R18 are hydrogen or directly bonded to form alkylene having 2 to 4 carbon atoms; or R13 and Rl4 are directly bonded to form alkylene having 4 or less carbon atoms, which is optionally substituted by alkyl having 1 to 10 carbon atoms, phenyl or benzyl, or
R14 and R15 directly or in combination via an oxygen atom form a heterocyclic group together with the adjacent nitrogen atom, and when R12 is chlorine or hydroxy, Alk is an alkylene having 2 to 6 carbon atoms which is optionally substituted by alkyl having 1 to 6 carbon atoms,
R!3 and R14 are each hydrogen, linear or branched alkyl having 1 to 6 carbon atoms or directly bonded to each other to form ethylene or trimethylene, wherein said alkyl, ethylene or trimethylene group is optionally substituted by alkyl having 1 to 6 carbon atoms; or
R15 is hydrogen, linear or branched alkyl having 1 to 6 carbon atoms or an amidino group; an isomer thereof and/or a pharmaceutically acceptable acid addition salt thereof. Typically in formula (I): - Alkyl at R and Rl is linear or branched alkyl having 1 to 10 carbon atoms, which is exemplified by methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl and the like, with preference given to alkyl having 1 to 4 carbon atoms,
Cycloalkyl at R and R1 has 3 to 7 carbon atoms and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and the like.
Cycloalkylalkyl at R and R! has a cycloalkyl moiety having 3 to 7 carbon atoms as defined immediately above and the alkyl moiety is linear or branched alkyl having 1 to 6 carbon atoms (e.g. methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl and the like). Such cycloalkylalkyl groups are exemplified by cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cycloheptylmethyl, cyclopropylethyl, cyclopentylethyl, cyclohexylethyl, cycloheptylethyl, cyclopropylpropyl, cyclopentylpropyl, cyclohexylpropyl, cycloheptylpropyl, cyclopropylbutyl, cyclopentylbutyl, cyclohexylbutyl, cycloheptylbutyl, cyclopropylhexyl, cyclopentylhexyl, cyclohexylhexyl, cycloheptylhexyl and the like. - Aralkyl at R and R1 has an alkyl moiety of 1 to 4 carbon atoms and is exemplified by phenylalkyl such as benzyl, 1 -phenylethyl, 2-phenylethyl, 3- phenylpropyl, 4-phenylbutyl and the like.
The substituent of optionally substituted cycloalkyl, cycloalkylalkyl, phenyl and aralkyl on the ring at R and R1 is halogen (e.g., chlorine, bromine, fluorine and iodine), alkyl (same as alkyl at R and R1), alkoxy (linear or branched alkoxy having 1 to 6 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, hexyloxy and the like), aralkyl (same as aralkyl at R and R1) or haloalkyl (alkyl at R and R1 which is substituted by from 1 to 5 halogen atoms, and exemplified by fluoromethyl, difluoromefhyl, trifluoromethyl, 2,2,2- trifluoroethyl, 2,2,3,3,3-pentafluoropropyl and the like), nitro, amino, cyano, azide and the like.
The group formed by R and R1 in combination together with the adjacent nitrogen atom, which forms a heterocycle optionally having, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom is preferably a 5 or 6- membered ring and to which, optionally, a ring may be fused. Examples thereof include 1-pyrrolidinyl, piperidino, 1-piperazinyl, morpholino, thiomorpholino, 1- imidazolyl, 2,3-dihydrothiazol-3-yl and the like, The substituent of the optionally substituted nitrogen atom is exemplified by alkyl, aralkyl, haloalkyl and the like. As used herein, alkyl, aralkyl and haloalkyl are as defined for R and R1, - Alkyl at R2 is as defined for R and R1.
Halogen, alkyl, alkoxy and aralkyl at R3 and R4 are as defined for R and R!. Acyl at R3 and R4 is alkanoyl having 2 to 6 carbon atoms (e.g., acetyl, propionyl, butyryl, valeryl, pivaloyl and the like), benzoyl or phenylalkanoyl wherein the alkanoyl moiety has 2 to 4 carbon atoms (e.g., phenylacetyl, phenylpropionyl, phenylbutyryl and the like).
Alkylamino at R3 and R4 has a linear or branched alkyl moiety having 1 to 6 carbon atoms. Examples thereof include methylamino, ethylamino, propylamino, isopropylamino, butylamino, isobutylamino, sec-butylamino, tert-butylamino, pentylamino, hexylamino and the like. - Acylamino at R3 and R4 has an acyl moiety which may be alkanoyl having 2 to 6 carbon atoms, benzyl or the alkanoyl moiety is phenylalkanoyl having 2 to 4 carbon atoms and the like, and is exemplified by acetylamino, propionylamino, butyrylamino, valeryl amino, pivaloylamino, benzoylamino, phenylacetylamino, phenylpropionylamino, phenylbutyrylamino and the like. - Alkylthio at R3 and R4 has a linear or branched alkyl moiety having 1 to 6 carbon atoms and is exemplified by methylthio, ethylthio, propylthio, isopropylthio, butylthio, isobutylthio, sec-butylthio, tert-butylthio, pentylthio, hexylthio and the like,
Aralkyloxy at R3 and R4 has an alkyl moiety having 1 to 4 carbon atoms and is exemplified by benzyloxy, 1-phenylethyloxy, 2-phenylethyloxy, 3- phenylpropyloxy, 4-phenylbutyloxy and the like.
Aralkylthio at R3 and R4 has an alkyl moiety having 1 to 4 carbon atoms and is exemplified by benzylthio,l-phenylethylthio,2-phenylethylthio,3- phenylpropylthio,4-phenylbutylthio and the like,
Alkoxycarbonyl at R3 and R4 has a linear or branched alkoxy moiety having 1 to 6 carbon atoms, which is exemplified by methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, sec- butoxycarbonyl, tert-butoxycarbonyl, pentyloxycarbonyl, hexyloxycarbonyl and the like.
Alkylcarbamoyl at R3 and R4 is carbamoyl mono- or di-substituted by alkyl having 1 to 4 carbon atoms and is exemplified by methylcarbamoyl, dimethylcarbamoyl, ethylcarbamoyl, diethylcarbamoyl, propylcarbamoyl, dipropylcarbamoyl, butylcarbamoyl, dibutylcarbamoyl and the like.
Alkoxy at R5 is as defined for R and R1.
Alkoxycarbonyloxy at R2 has a linear or branched alkoxy moiety having 1 to 6 carbon atoms and is exemplified by methoxycarbonyloxy, ethoxycarbonyloxy, propoxycarbonyloxy, isopropoxycarbonyloxy, butoxycarbonyloxy, isobutoxycarbonyloxy, sec-butoxycarbonyloxy, tert-butoxycarbonyloxy, pentyloxycarbonyloxy, hexyloxycarbonyloxy and the like.
Alkanoyloxy at R5 has an alkanoyl moiety having 2 to 6 carbon atoms and is exemplified by acetyloxy, propionyloxy, butyryloxy, valeryloxy, pivaloyloxy and the like.
Aralkyloxycarbonyloxy at R5 has an aralkyl moiety having C1-C4 alkyl and is exemplified by benzyloxycarbonyloxy, 1 -phenylethyloxycarbonyloxy, 2- phenylethyloxycarbonyloxy, 3-phenylpropyloxycarbonyloxy, 4- phenylbutyloxycarbonyloxy and the like.
Alkyl at Rδ is as defined for R and R1; alkyl at R8 and R9 is as defined for R and R1; and aralkyl at R8 and R9 is as defined for R and R1,
Alkyl at R7 is as defined for R and R1 and aralkyl at R7 is as defined for R and R1. - The group formed by Rδ and R7 in combination, which forms a heterocycle optionally having, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom, is imidazol-2-yl, thiazol-2-yl, oxazol-2-yl, imidazolin-2-yl, 3,4,5,6- tetrahydropyridin-2-yl, 3,4,5,6-tetrahydropyrimidin-2-yl, l,3-oxazolin-2-yl, 1 ,3- thiazolin-2-yl or optionally substituted benzoimidazol-2-yl, benzothiazol-2-yl, benzoxazol-2-yl and the like having a substituent such as halogen, alkyl, alkoxy, haloalkyl, nitro, amino, phenyl, aralkyl and the like. As used herein, halogen, alkyl, alkoxy, haloalkyl and aralkyl are as defined for R and R1,
The substituent of the above-mentioned optionally substituted nitrogen atom is exemplified by alkyl, aralkyl, haloalkyl and the like, As used herein, alkyl, aralkyl and haloalkyl are as defined for R and R1.
Hydroxyalkyl at R10 and R: : is linear or branched alkyl having 1 to 6 carbon atoms which is substituted by 1 to 3 hydroxy, which is exemplified by hydroxymethyl, 2-hydroxyethyl, 1-hydroxyethyl, 3-hydroxypropyl, 4-hydroxybutyl and the like, Alkyl at R10 and R11 is as defined for R and R1; haloalkyl and alkoxycarbonyl at R:0 and R11 are as defined for R and R1; aralkyl at R!0 and R11 is as defined for R and R!; and cycloalkyl formed by R10 and R! 1 in combination is the same as cycloalkyl at R and R1.
Alkyl at L is as defined for R and R1.
Aminoalkyl at L is a linear or branched alkyl having 1 to 6 carbon atoms, which is substituted by amino, which is exemplified by aminomethyl, 2-aminoethyl, 1-aminoethyl, 3-aminopropyl, 4-aminobutyl, 5-aminopentyl, 6-aminohexyl and the like.
Mono- or dialkyiaminoalkyl at L is mono- or di-substituted aminoalkyl with alkyl having 1 to 4 carbon atoms, which is exemplified by methylaminomethyl, dimethylammomethyl, ethylaminomethyl, diethylammomethyl, propylaminomethyl, dipropylaminomethyl, butylaminomethyl, dibutylaminomethyl, 2- dimethylaminoethyl, 2-diethylaminoethyl and the like.
Carbamoylalkyl at L is linear or branched alkyl having 1 to 6 carbon atoms substituted by carbamoyl, which is exemplified by carbamoylmethyl, 2- carbamoylethyl, 1-carbamoylethyl, 3-carbamoylpropyl, 4-carbamoylbutyl, 5- carbamoylpentyl, 6-carbamoylhexyl and the like.
Phthalimido alkyl at L is linear or branched alkyl having 1 to 6 carbon atoms, which is substituted by phthalimide. Examples include phthalimidomethyl, 2- phthalimidoethyl, 1-phthalimidoethyl, 3-phthalimidopropyl, 4-phthalimidobutyl, 5- phthalimidopentyl, 6-phthalimidohexyl and the like, Alkyl at B is as defined for R and R1.
Alkoxy at B is as defined for R and R1.
Aralkyl at B is as defined for R and R1.
Aralkyloxy at B is as defined for R3 and R4. - Aminoalkyl at B is as defined for L.
Hydroxyalkyl at B is as defined for R10 and R11.
Alkanoyloxyalkyl at B is linear or branched alkyl having 1 to 6 carbon atoms substituted by alkanoyloxy having an alkanoyl moiety having 2 to 6 carbon atoms, which is exemplified by acetyloxymethyl, propionyloxymethyl, butyryloxymethyl, valeryloxymethyl, pivaloyloxymethyl, acetyloxyethyl, propionyloxyethyl, butyryloxyethyl, valeryloxyethyl, pivaloyloxyethyl and the like.
Alkoxycarbonylalkyl at B is linear or branched alkyl having 1 to 6 carbon atoms substituted by alkoxycarbonyl having an alkoxy moiety having 1 to 6 carbon atoms, which is exemplified by methoxycarbonylmethyl, ethoxycarbonylmethyl, propoxycarbonylmethyl, isopropoxycarbonylmethyl, butoxycarbonylmethyl, isobutoxycarbonylmethyl, sec-butoxycarbonylmethyl, tert-butoxycarbonylmethyl, pentyloxycarbonylmethyl, hexyloxycarbonylmethyl, methoxycarbonylethyl, ethoxycarbonylethyl, propoxycarbonylethyl, isopropoxycarbonylethyl, butoxycarbonylethyl, isobutoxycarbonylethyl, sec-butoxycarbonylethyl, tert- butoxycarbonylethyl, pentyloxycarbonylethyl, hexyloxycarbonylethyl and the like.
Flalogen at Q:, Q2 and Q3 is as defined for R and R1.
Aralkyloxy at Q1 and Q2 is as defined for R3 and R4,
Alkoxy at Q3 is as defined for R and R1.
Alkylene at W, X and Y is linear or branched alkylene having 1 to 6 carbon atoms, which is exemplified by methylene, ethylene, trimethylene, propylene, tetramethylene, pentamethylene, hexamethylene and the like.
Alkenylene at Y is linear or branched alkenylene having 2 to 6 carbon atoms, which is exemplified by vinylene, propenylene, butenylene, pentenylene and the like.
Alkyl at Rb is as defined for R and R1. - Aralkyl at Rb is as defined for R and R1, A ino alkyl at Rb is as defined for L. Mono- or dialkylamino alkyl at Rb is as defined for L. The heterocycle at Re is, when a single ring containing nitrogen, pyridine, pyrimidine, pyridazine, triazine, pyrazole, triazole and the like; and, when it is a condensed ring, pyrrolopyridine (e.g., lH-pyrrolo[2,3-b]pyridine, lH-pyrrolo[3,2- bjpyridine, lH-pyrrolo[3,4-b]pyridine and the like), pyrazolopyridine (e.g., 1H- pyrazolo[3,4-b]pyridine, lH-pyrazolo[4,3-b]pyridine and the like), imidazopyridine (e.g., lH-imidazo[4,5-b]pyridine and the like), pyrrolopyrimidine (e.g., 1H- pyrrolo[2,3-d]pyrimidine, lH-pyrrolo[3,2-d]pyrimidine, lH-pyrrolo[3,4-d]pyrimidine and the like), pyrazolopyrimidine (e.g., lH-pyrazolo[3,4-d]pyrimidine, pyrazolo[l,5- ajpyrimidine, lH-pyrazolo[4,3-d]pyrimidine and the like), imidazopyrimidine (e.g., imidazo[l,2-a]pyrimidine, lH-imidazo[4,5-d]pyrimidine and the like), pyrrolotriazine (e.g., pyrrolo[l,2-a]-l,3,5-triazine, pyrrolo[2,l-f]-l,2,4-triazine), pyrazolotriazine (e.g., pyrazolo[l,5-a]-l,3,5-triazine and the like), triazolopyridine (e.g., lH-l,2,3-triazolo[4,5-b]pyridine and the like), triazolopyrimidine (e.g., 1,2,4- triazolo[l,5-a]pyrimidine, l,2,4-triazolo[4,3-a]pyrimidine, lH-l,2,3-triazolo[4,5- d]pyrimidine and the like), cinnoline, quinazoline, quinoline, pyridopyridazine (e.g., pyrido[2,3-c]pyridazine and the like), pyridopyrazine (e.g., pyrido[2,3-b]pyrazine and the like), pyridopyrimidine (e.g., pyrido[2,3-d]pyrimidine, pyrido[3,2-d]pyrimidine and the like), pyrimidopyrimidine (e.g., pyrimido[4,5-d]pyrimidine, pyrimido[5,4- djpyrimidine and the like), pyrazinopyrimidine (e.g., pyrazino[2,3-d]pyrimidine and the like), naphthyridine (e.g., 1,8-naphthyridine and the like), tetrazolopyrimidine (e.g.. tetrazolo[l,5-a]pyrimidine and the like), thienopyridine (e.g., thieno[2,3- b]pyridine and the like), thienopyrimidine (e.g., thieno[2,3-d]pyrimidine and the like), thiazolopyridine (e.g., thiazolo[4,5-b]pyridine, thiazolo[5,4-b]pyridine and the like), thiazolopyrimidine (e.g., thiazolo[4,5-d]pyrimidine, thiazolo[5,4-d]pyrimidine and the like), oxazolopyridine (e.g., oxazolo[4,5 b]pyridine, oxazolo[5,4-b]pyridine and the like), oxazolopyrimidine (e.g., oxazolo[4,5-d]pyrimidine, oxazolo[5,4- djpyrimidine and the like), furopyridine (e.g., furo[2,3-b]pyridine, furo[3,2- bjpyridine and the like), furopyrimidine (e.g., furo[2,3-d]pyrimidine, furo[3,2- djpyrimidine and the like), 2,3-dihydropyrrolopyridine (e.g., 2,3-dihydro-lH- pyrrolo[2,3-b]pyridine, 2,3-dihydro-lH-pyrrolo[3,2-b]pyridine and the like), 2,3- dihydropyrrolopyrimidine (e.g., 2,3-dihydro-lH-pyrrolo[2,3-d]pyrimidine, 2,3- dihydro-lH-pyrrolo[3,2-d]pyrimidine and the like), 5,6,7,8-tetrahydropyrido[2,3- djpyrimidine, 5,6,7,8-tetrahydro-l,8-naphthyridine, 5,6,7,8-tetrahydroquinoline and the like. When these rings form a hydrogenated aromatic ring, the carbon atom in the ring may be carbonyl and includes, for example, 2,3-dihydro-2-oxopyrrolopyridine, 2,3-dihydro-2,3-dioxopyrrolopyridine, 7,8-dihydro-7-oxo-l,8-naphthyridine, 5,6,7,8- tetrahydro-7-oxo-l,8-naphthyridine and the like. These rings may be substituted by a substituent such as halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, amino, alkylammo, cyano, fomiyl, acyl, aminoalkyl, mono- or dialkylaminoalkyl, azide, carboxy, alkoxycarbonyl, carbamoyl, alkylcarbamoyl, alkoxyalkyl (e.g., methoxymethyl, methoxyethyl, methoxypropyl, ethoxymethyl, ethoxyethyl, ethoxypropyl and the like), optionally substituted hydrazino and the like.
As used herein, the substituent of the optionally substituted hydrazino includes alkyl, aralkyl, nitro, cyano and the like, wherein alkyl and aralkyl are as defined for R and R and exemplified by methyl hydrazino, ethyl hydrazino, benzyl hydrazino and the like. Typically in formula (II):
The linear or branched alkyl having 1 to 6 carbon atoms at R13, R14, R15 and R''6 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl and the like.
Aryl at R14 and R15 is phenyl, naphthyl and the like. - Aralkyl at R14 and R15 is as defined for R and R1.
Alkylene having 4 or less carbon atoms, which is formed by R13 and R14 directly bonded to each other, is methylene, ethylene, trimethylene, propylene, tetramethylene and the like.
Alkyl having 1 to 10 carbon atoms, which substitutes alkylene having 4 or less carbon atoms formed by R13 and R14 directly bonded to each other, is linear or branched alkyl having 1 to 10 carbon atoms. Examples thereof include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl and the like.
Alkyl having 1 to 6 carbon atoms which substitutes ethylene and trimethylene formed by R'3 and R14 directly bonded to each other is linear or branched alkyl having 1 to 6 carbon atoms, which is the same as those for R.
The heterocycle formed by R14 and R15 directly or via oxygen atom bonded together with the adjacent nitrogen atom is pyrrolidino, piperidino, morpholino, homopiperidino, homomorpholino and the like. - Alkylene having 2 to 4 carbon atoms formed by R17 and R18 directly bonded to each other is ethylene, trimethylene, propylene, tetramethylene and the like.
Alkylene having 2 to 6 carbon atoms at Alk is ethylene, trimethylene. propylene, tetramethylene, pentamethylene, hexamethylene and the like.
Alkyl having 1 to 6 carbon atoms and alkyl having 1 to 10 carbon atoms, which are the substituents of alkylene having 2 to 6 carbon atoms at Alk, are as defined for R13.
Aryl and aralkyl, which are the substituents of alkylene having 2 to 6 carbon atoms at Alk, are as defined for R14.
Rho kinase inhibitors of the formula (I) include: (1) 4-(2-pyridylcarbamoyl)piperidine
(2) 1 -benzyloxycarbonyl-4-(4-pyridylcarbamoyl)piperidine
(3) 1 -benzoyl-4-(4-pyridylcarbamoyl)piperidine
(4) 1 -propyl-4-(4-pyridylcarbamoyl)piperidine
(5) [3-(2-(2-thienylmethyl)phenoxy)-2-hydroxypropyl]-4-(4- pyridylcarbamoyl)piperidine
(6) 4-(4-pyridylcarbamoyl)piperidine
(7) l-benzyl-4-(4-pyridylcarbamoyl)-l,2,5,6-tetrahydropyridine
(8) 3-(4-pyridylcarbamoyl)piperidine
(9) l-benzyl-3-(4-pyridylcarbamoyl)piperidine (10) l-(2-(4-benzyloxyphenoxy)ethyl)-4-(N-(2-pyridyl)-N-benzylcarbamoyl)pyridine (11) l-formyl-4-(4-pyridylcarbamoyl)piperidine
(12) 4-(3-pyridylcarbamoyl)piperidine
(13) l-isopropyl-4-(4-pyridylcarbamoyl)piperidine
(14) l-methyl-4-(4-pyridylcarbamoyl)piperidine (15) l-hexyl-4-(4-pyridylcarbamoyl)piperidine
(16) l-benzyl-4-(4-pyridylcarbamoyl)piperidine
(17) l-(2-phenylethyl)-4-(4-pyridylcarbamoyl)piperidine
(18) l-(2-(4-methoxyphenyl)ethyl)-4-(4-pyridylcarbamoyl)piperidine
(19) l-(2-(4-methoxyphenyl)ethyl)-4-(2-pyridylcarbamoyl)piperidine (20) 1 -(2-(4-chlorophenyl)ethyl)-4-(4-pyridy lcarbamoyl)piperidine
(21) l-diphenylmethyl-4-(2-pyridylcarbamoyl)piperidine
(22) l-[2-(4-(5-methyl-3-oxo-2,3,4,5-tetrahydropyridazin-6-yl)phenyl)ethyl]-4-(2- pyridylcarbamoyl)piperidine
(23) l-(4-(4,5-dihydro-2-fuιyl)phenyl)-4-(4-pyridylcarbamoyl)piperidine (24) l-(2-nitrophenyl)-4-(4-pyridylcarbamoyl)piperidine
(25) l-(2-aminophenyl)-4-(4-pyridylcarbamoyl)piperidine
(26) l-nicotinoyl-4-(4-pyridylcarbamoyl)piperidine
(27) 1 -isonicotinoyl-4-(4-pyridylcarbamoyl)piperidine
(28) l-(3,4,5-trimethoxybenzoyl)-4-(4-pyridylcarbamoyl)piperidine (29) l-acetyl-4-(4-pyridylcarbamoyl)piperidine
(30) l-(3-(4-fluorobenzoyl)propyl)-4-(4-pyridylcarbamoyl)piperidine
(31) l-(3-(4-fluorobenzoyl)propyl)-4-(2-pyridylcarbamoyl)piperidine
(32) l-(l-(4-hydroxybenzoyl)ethyl)-4-(2-pyridylcarbamoyl)piperidine
(33) l-(l-(4-benzyloxybenzoyl)ethyl)-4-(2-pyridylcarbamoyl)piperidine (34) l-(2-(4-hydroxphenoxy)ethyl)-4-(2-pyridylcarbamoyl)piperidine
(35) l-(4-(4-fluorophenyl)-4-hydroxybutyl)-4-(4-pyridylcarbamoyl)piperidine (36) 1 -(1 -methyl-2-(4-hydroxyphenyl)-2-hydroxyethyl)-4-(2- pyridylcarbamoyl)piperidine (37) l-cinnamyl-4-(2-pyridylcarbamoyl)piperidine (3S) l-(2-hydroxy-3-phenoxypropyl)-4-(4-pyridylcarbamoyl)piperidine (39) l-(2-hydroxy-3-phenoxypropyl)-4-(3-pyridylcarbamoyl)piperidine
(40) l-(2-hydroxy-3-phenoxypropyl)-4-(2-pyridylcarbamoyl)piperidine
(41) l-(2-phenylethyl)-4-[ N-(2-pyridyl)-N-(2-( N, N- dimethylamino)ethyl)carbamoyl]piperidine (42) l-benzyloxycarbonyl-4-(2-pyridylcarbamoyl)piperidine
(43) l-(3-chlorophenyl)carbamoyl-4-(4-pyridylcarbamoyl)piperidine
(44) l-[N-(2-pyridyl)-N-(2-( N, N-dimethylamino)ethyl)carbamoyl]piperidine
(45) l-methyl-4-(4-pyridylcarbamoyl)-l,2,5,6-tetrahydropyridine
(46) l-nicotinoyl-3-(4-pyridylcarbamoyl)piperidine (47) l-[2-(4-fluorobenzoyl)ethyl]-4-(4-pyridylcarbamoyl)piperidine
(48) l-(6-chloro-2-methylimidazo[l ,2-a]pyridine-3-carbonyl)-4-(4- pyridylcarbamoyl)piperidine
(49) l-(4-nitrobenzyl)-4-(4-pyridylcarbamoyl)piperidine
(50) l-hexyl-4-(4-pyridylcarbamoyl)piperidine (51) l-benzyloxycarbonyl-4-(2-chloro-4-pyridylcarbamoyl)piperidine
(52) 4-(2-chloro-4-pyridylcarbamoyl)piperidine
(53) l-(2-chloronicotinoyl)-4-(4-pyridylcarbamoyl)piperidine
(54) 3-(2-chloiO-4-pyridylcarbamoyl)piperidine
(55) l-(4-phthalimidobutyl)-4-(4-pyridylcarbamoyl)piperidine (56) l-(3,5-di-tert-butyl-4-hydroxycinnamoyl)-4-(4-pyridylcarbamoyl)piperidine
(57) 1 -carbamoylmethyl-4-(4-pyridylcarbamoyl)piperidine
(58) l-benzyloxycarbonyl-4-(5-nitro-2-pyridylcarbamoyl)piperidine
(59) 4-(5-nitro-2-pyridylcarbamoyl)piperidine
(60) trans-4-benzyloxycarboxamidomethyl-l-(4-pyridylcarbamoyl)cyclohexane (61) trans-4-aminomethyl-l-(4-pyridylcarbamoyl)cyclohexane
(62) trans-4-foπnamidomethyl- 1 -(4-pyridylcarbamoyl)cyclohexane
(63) trans-4-dimethylaminomethyl- 1 -(4-pyridylcarbamoyl)cyclohexane
(64) N-benzylidene-trans-(4-pyridylcarbamoyl)cyclohexylmethylamine
(65) trans-4-benzylaminomethyl-l-(4-pyridylcarbamoyl)cyclohexane (66) trans-4-isopropylaminomethyl- 1 -(4-pyridylcarbamoyl)cyclohexane (67) trans-4-nicotinoylaminomethyl-l-(4-pyridylcarbamoyl)cyclohexane
(68) trans-4-cyclohexylaminomethyl-l-(4-pyridylcarbamoyl)cyclohexane
(69) trans-4-benzyloxycarboxamide-l-(4-pyridylcarbamoyl)cyclohexane
(70) trans-4-amino-l-(4-pyridylcarbamoyl)cyclohexane (71) trans-4-(l-aminoethyl)-l-(4-pyridylcarbamoyl)cyclohexane
(72) trans-4-aminomethyl-cis-2-methyl-l-(4-pyridylcarbamoyl)cyclohexane
(73) (~)-trans-4-(l-benzyloxycarboxamidopropyl)-l-cyclohexanecarboxylic acid
(74) (+)-trans-4-(l -benzyloxycarboxamidopropyl)- 1 -(4- pyridylcarbamoyl)cyclohexane (75) (-)-trans-4-(l-benzyloxycarboxamidpropyl)-l-(4-pyridylcarbamoyl)cyclohexane
(76) (-)-trans-4-(l-aminopropyl)-l-(4-pyridylcarbamoyl)cyclohexane
(77) (-)-trans-4-(l-aminopropyl)-l-(4-pyridylcarbamoyl)cyclohexane
(78) (-)-trans-4-(l-benzyloxycarboxamidoethyl)-l-(4-pyridylcarbamoyl)cyclohexane
(79) (-)-trans-4- l-benzyloxycarboxamidoethyl)-l-(4-pyridylcarbamoyl)cyclohexane (80) (-)-trans-4-(l-aminoethyl)-l-(4-pyridylcarbamoyl)cyclohexane
(81) (-)-trans-4-(l-aminoethyl)-l-(4-pyridylcarbamoyl)cyclohexane
(82) trans-4-(4-chlorobenzoyl)aminomethyl-l-(4-pyridylcarbamoyl)cyclohexane
(83) trans-4-aminomethyl-l-(2-pyridylcarbamoyl)cyclohexane
(84) trans-4-benzyloxycarboxamidomethyl-l-(2-pyridylcarbamoyl)cyclohexane (85) trans-4-methylaminomethyl-l-(4-pyridylcarbamoyl)cyclohexane
(86) trans-4-(N-benzyl-N-methylamino)methyl-l-(4-pyridylcarbamoyl)cyclohexane
(87) trans-4-aminomethyl-l-(3-pyridylcarbamoyl)cyclohexane
(88) trans-4-aminomethyl-l-[(3-hydroxy-2-pyridyl)carbamoyl]cyclohexane
(89) trans-4-benzyloxycarboxamidomethyl-l-(3-pyridylcarbamoyl)cyclohexane (90) trans-4-benzyloxycarboxamidomethyl-l-[(3-benzyloxy-2- pyridyl)carbamoyl]cyclohexane
(91) trans-4-phthalimidomethyl-l-(4-pyridylcarbamoyl)cyclohexane
(92) trans-4-benzyloxycarboxamidomethyl-l-(3-methyl-4- pyridylcarbamoyl)cyclohexane (93) trans-4-aminomethyl-l-(3-methyl-4-pyridylcarbamoyl)cyclohexane (94) 4-(trans-4-benzyloxycarboxamidomethylcyclohexylcarbonyl)amino-2,6- dimethylpyridine-N-oxide
(95) 4-(trans-4-aminomethylcyclohexylcarbonyl)amino-2,6-dimethylpyridine-N- oxide (96) trans-4-aminomethyl- 1 -(2-methyl-4-pyridylcarbamoyl)cyclohexane
(97) trans-4-(l-benzyloxycarboxamidoethyl)-l-(4-pyridylcarbamoyl)cyclohexane
(98) trans-4-( 1 -amino- 1 -methylethyl)- 1 -(4-pyridylcarbamoyl)cyclohexane
(99) trans-4-(2-aminoethyl)- 1 -(4-pyridylcarbamoyl)cyclohexane
(100) trans-4-(2-amino-l -methylethyl)- l-(4-pyridylcarbamoyl)cyclohexane (101) trans-4-( 1 -aminopropyl)- 1 -(4-pridylcarbamoyl)cyclohexane
( 102) trans-4-aminomethyl-trans- 1 -methyl- 1 -(4-pyridylcarbamoyl)cyclohexane
(103) trans-4-benzylaminomethyl-cis-2-methyl-l-(4-pyridylcarbamoyl)cyclohexane
(104) trans-4-(l-benzyloxycarboxamide-l-methylethyl)-l-(4- pyridylcarbamoyl)cyclohexane (105) trans-4-benzyloxycarboxamidomethyl-l-( N-methyl-4- pyridylcarbamoyl)cyclohexane
(106) trans-4-( 1 -acetamide- 1 -methylethyl)- 1 -(4-pyridylcarbamoyl)cyclohexane
(107) trans-N-(6-amino-4-pyrimidyl)-4-aminomethylcyclohexanecarboxamide (108) trans-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4- aminomethylcyclohexanecarboxamide
(109) (-L)-trans-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(l- aminoethyl)cyclohexanecarboxamide
(1 10) trans-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(l-amino-l- methylethyl)cyclohexanecarboxamide (11 1) trans-N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-4- aminomethylcyclohexanecarboxamide
(1 12) (+)-trans-N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-4-(l- aminoethyl)cyclohexanecarboxamide
(113) trans-N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-4-(l-amino-l- methylethyl)cyclohexanecarboxamide (114) (-)-trans-NT-(2-amino-4-pyridyl)-4-(l-aminoethyl)cyclohexanecarboxamide
(115) trans-N-(lH-pyrazolo[3,4-d]pyrimidin-4-yl)-4- aminomethylcyclohexanecarboxamide
(116) (+)-trans-N-(lH-pyrazolo[3,4-d]ρyrimidin-4-yl)-4-(l- aminoethyl)cyclohexanecarboxamide
(117) trans-N-(lH-pyrazolo[3,4-d]pyrimidin-4-yl)-4-(l-amino-l- methylethyl)cyclohexanecarboxamide
(118) trans-N-(4-pyrimidinyl)-4-aminomethylcyclohexanecarboxamide
(119) trans-N-(3-amino-4-pyridyl)-4-aminomethylcyclohexanecarboxamide (120) trans-N-(7H-imidazo[4,5-d]pyrimidin-6-yl)-4- aminomethylcyclohexanecarboxamide
(121) trans-N-(3H-l,2,3-triazolo[4,5-d]pyrimidin-7-yl)-4- aminomethylcyclohexanecarboxamide
(122) trans-N-( 1 -benzyl- 1 H-pyrazolo [3 ,4-b]pyridin-4-yl)-4- aminomethylcyclohexanecarboxamide
(123) trans-N-(lH-5-pyrazolyl)-4-aminomethylcyclohexanecarboxamide
(124) trans-N-( 1 Fl-pyrazolo [3 ,4-b]pyridin-4-yl)-4- aminomethylcyclohexanecarboxamide
(125) trans-N-(4-pyridazinyl)-4-aminomethylcyclohexanecarboxamide (126) trans-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-4- aminomethylcyclohexanecarboxamide
(127) trans-N-(2-amino-4-pyridyl)-4-aminomethylcyclohexanecarboxamide
(128) trans-N-(thieno[2,3-d]pyrimidin-4-yl)-4-aminomethylcyclohexanecarboxamide
(129) trans-N-(5-methyl-l,2,4-triazolo[l ,5-a]pyrimidin-7-yl)-4- aminomethylcyclohexanecarboxamide
(130) trans-N-(3-cyano-5-methylpyrazolo[l,5-a]pyrimidin-7-yl)-4- aminomethylcyclohexanecarboxamide
(131) trans-N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-4-(l -amino- 1- methylethyl)cyclohexanecarboxamide (132) trans-N-(2-(l-pyrrolidinyl)-4-pyridyl)-4-aminomethylcyclohexanecarboxamide (133) trans-N-(2,6-diamino-4-pyrimidyl)-4-aminomethylcyclohexanecarboxamide
(134) (-)-trans-N-(7-methyl-l,8-naρhthyridin-4-yl)-4-(l- aminoethyl)cyclohexanecarboxamide
(135) trans-N-(l-benzyloxymethylpyrrolo[2,3-b]pyridin-4-yl)-4- aminomethylcyclohexanecarboxamide
(136) (+)-trans-N-(l-methylρyτrolo[2,3-b]pyridin-4-yl)-4-(l- aminoethyl)cyclohexanecarboxamide
(137) trans-N-benzyl-N-(2-benzylamino-4-pyridyl)-4-(l-amino-l- methylethyl)cyclohexanecarboxamide (138) trans-N-(2-azide-4-pyridyl)-4-aminomethylcyclohexanecarboxamide
(139) trans-N-(2,3-dihydro-lH-pyrrolo[2,3-b]pyridin-4-yl)-4- aminomethylcyclohexanecarboxamide
(140) trans-N-(2,3-dihydiO-lFI-pyrrolo[2,3-b]pyridin-4-yl)-4-(l-amino-l- methylethyl)cyclohexanecarboxamide (141-1) trans-N-(2-carboxy-4-pyridyl)-4-aminomethylcyclohexanecarboxamide
(141-2) (R)-(+)-trans-N-(3-bromo-lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(l- aminoethyl)cyclohexanecarboxamide
(142) trans-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4- guanidinomethylcyclohexanecarboxamide (143) trans-N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-4- guanidinomethylcyclohexanecarboxamide
(144) trans-N-(4-pyridyl)-4-guanidinomethylcyclohexanecarboxamide
(145) trans-N-(l-methylpyrrolo[2,3-b]pyridin-4-yl)-4- (guanidinomethyl)cyclohexanecarboxamide (146) trans-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(2-imidazolin-2- yl)aminomethylcyclohexanecarboxamide
(147) trans-N-( 1 -benzyloxymethylpyrrolo [2,3 -b]pyridin-4-yl)-4- guanidinomethylcyclohexanecarboxamide
(148) trans-N-(2-amino-4-pyridyl)-4-guanidinomethylcyclohexanecarboxamide (149) trans-N-(l-benzyloxymethyl-lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(2-imidazolin-2- yl)aminomethylcyclohexanecarboxamide
(150) trans-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(3- benzylguanidinomethyl)cyclohexanecarboxamide
(151) trans-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(3- phenylguanidinomethyl)cyclohexanecarboxamide
(152) trans-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(3- propylguanidinomethyl)cyclohexanecarboxamide
(153) trans-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(3- octylguanidinomethyl)cyclohexanecarboxamide (154) trans-N-(l-benzyloxymethylpyrrolo[2,3-b]pyridin-4-yl)-4-(2-benzyl-3- ethylguanidinomethyl)cyclohexanecarboxamide
(155) trans-N-(lH-pyrrolo[2,3-b]pyridin 4 yl)-4-(imidazol-2- yl)aminomethylcyclohexanecarboxamide
(156) trans-N-(lFI-pyrrolo[2,3-b]pyridin-4-yl)-4-(thiazol-2- yl)aminomethylcyclohexanecarboxamide
(157) (R)-(+)-N-(4-pyridyl)-4-(l-aminoethyl)benzamide
(158) N-(4-pyridyl)-4-(l -amino- l-methylethyl)benzamide
(159) N-(4-pyridyl)-4-aminomethyl-2-benzyloxybenzamide
(160) N-(4-pyridyl)-4-aminomethyl-2-ethoxybenzamide (161) (R)-(-)-N-(4-pyridyl)-4-(l-aminoethyl)-3-nitrobenzamide
(162) (R)-(-)-N-(4-pyridyl)-3-amino-4-(l-aminoethyl)benzamide
(163) (R)-(-)-N-(4-pyridyl)-4-(l-aminoethyl)-3-chlorobenzamide
(164) N-(4-pyridyl)-3-aminomethylbenzamide
(165) (R)-(+)-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(l-aminoethyl)benzamide (166) (R)-(+)-N-(lFI-pyrazolo[3,4-b]pyridin-4-yl)-4-(l-aminoethyl)benzamide
(167) N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-4-guanidinomethylbenzamide
(168) N-(4-pyridyl)-4-guanidinomethylbenzamide
(169) (R)-(÷)-N-(4-pyridyl)-4-(l-aminoethyl)-3-fluorobenzamide
(170) N-(4-pyridyl)-4-aminomethylbenzamide (1 1) N-(4-pyridyl)-4-aminomethyl-2-hydroxybenzamide (172) N-(4-pyridyl)-4-(2-aminoethyl)benzamide (173) N-(4-pyridyl)-4-aminomethyl-3-nitrobenzamide
(174) N-(4-pyridyl)-3-amino-4-aminomethylbenzamide
(175) (S)-(-)-N-(4-pyridyl)-4-(l-aminoethyl)benzamide (176) (S)-(-)-N-(4-pyridyl)-2-(l-aminoethyl)benzamide
(177) (R)-(-)-N-(4-pyridyl)-4-(l-aminoethyl)-2-chloroben-zamide (178) (R)-(+)-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(l-(3- propylguanidino)ethyl)benzamide
(179) (R)-(-)-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(T-aminoethyl)-3-azidebenzamide (180) (R)-(÷)-N-(4-pyridyl)-4-(l-aminoethyl)-2-nitrobenzamide
(181) (R)-(-)-N-(4-pyridyl)-4-(l-aminoethyl)-3-ethoxybenzamide
(182) (R)-(+)-N-(3-iodo-lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(l-aminoethyl)benz amide (183) (R)-(+)-N-(3-iodo-lH-pyπOlo[2,3-b]pyridin-4-yl)-4-(l-aminoethyl)-3- azidebenzamide (184) (R)-(-)-N-(4-pyridyl)-4-(l-aminoethyl)-3-hydroxybenzamide
(185) N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-4-guanidinomethyl-3-nitrobenzamide
(186) (R)-N-( 1 H-pyrazolo [3 ,4-b]pyridin-4-yl)-4-( 1 -guanidinoethyl)-3 - nitrobenzamide
(187) (R)-N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-4-(l-aminoethyl)-2-nitrobenzamide (188) N-(lFI-pyrazolo[3,4-b]pyridin-4-yl)-4-guanidinobenzamide
(189) (R)-N-( 1 H-pyrazolo [3 ,4-b]pyridin-4-yl)-4-( 1 -aminoethyl)-3-nitrobenzamide
(190) (R)-N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-4-(l-guanidinoethyl)benzamide
(191) N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-4-(l-amino-2-hydroxyethyl)benzamide
(192) N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-4-aminomethyl-3-nitrobenzamide (193) N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidinecarboxamide
(194) N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-4-piperidinecarboxamide
(195) N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-l-aminoacetyl-4-piperidinecarboxamide
(196) N-(l-methoxymethyl-l H-pyrazolo [3, 4-b]pyridin-4-yl)-4- piperidinecarboxamide (197) N-(2,3-dihydro-lH-pyrrolo[2,3-b]pyridin-4-yl)-4-piperidinecarboxamide (198) N-(lH-pyπolo[2,3-b]pyridin-4-yl)-l-(2-phenylethyl)-4-piperidinecarboxamide (199) N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-l-amidino-4-piperidinecarboxamide (200) N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-l-(3-phenylpropyl)-4- piperidinecarboxamide (201) N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-l-benzyl-4-piperidinecarboxamide (202) N-(l H-pyrazolo [3, 4-b]pyridin-4-yl)-l -(2 -phenylethyl)-4- piperidinecarboxamide
(203) N-(lH-ρyrazolo[3,4-b]pyridin-4-yl)-l-(3-phenylpropyl)-4- piperidinecarboxamide Preferred are compounds (80), (109), (110), (112), (115), (142), (143), (144),
(145), (153), (157), (163), (165), (166) and (179).
Rho kinase inhibitors of the formula (II) include:
(204) 1 -(5-isoquinolinesulfonyl)homopiperazine
(205) l-(5-isoquinolinesulfonyl)-2-methylhomopiperazine (206) l-(5-isoquinolinesulfonyl)-3-methylhomopiperazine
(207) l-(5-isoquinolinesulfonyl)-6-methylhomopiperazine
(208) l-(5-isoquinolinesulfonyl)-2,3-dimethylhomopiperazine
(209) l-(5-isoquinolinesulfonyl)-3,3-dimethylhomopiperazine
(210) l-(5-isoquinolinesulfonyl)-3-ethylhomopiperazine (211) l-(5-isoquinolinesulfonyl)-3-propylhomopiperazine
(212) l-(5-isoquinolinesulfonyl)-3-isobutylhomopiperazine
(213) l-(5-isoquinolinesulfonyl)-3-phenylhomopiperazine
(214) l-(5-isoquinolinesulfonyl)-3-benzylhomopiperazine
(215) l-(5-isoquinolinesulfonyl)-6-ethylhomopiperazine (216) l-(5-isoquinolinesulfonyl)-6-propylhomopiperazine
(217) l-(5-isoquinolinesulfonyl)-6-butylhomopiperazine
(218) l-(5-isoquinolinesulfonyl)-6-pentylhomopiperazine
(219) l-(5-isoquinolinesulfonyl)-6-hexylhomopiperazine
(220) 1 -(5-isoquinolinesulfonyl)-6-phenylhomopiperazine (221) l-(5-isoquinolinesulfonyl)-6-benzylhomopiperazine (222) l-(5-isoquinolinesulfonyl)-4-methylhomopiperazine
(223) l-(5-isoquinolinesulfonyl)-4-ethylhomopiperazine
(224) l-(5-isoquinolinesulfonyl)-4-propylhomopiperazine
(225) l-(5-isoquinolinesulfonyl)-4-butylhomopiperazine
(226) l-(5-isoquinolinesulfonyl)-4-hexylhomopiperazine
(227) N-(2-aminoethyl)-l-chloro-5-isoquinolinesulfonamide
(228) N-(4-aminoethyl)-l-chloro-5-isoquinolinesulfonamide
(229) N-(2-amino-l-methylethyl)-l-chloro-5-isoquinolinesulfonamide
(230) N-(2-amino-l-methylpentyl)-l-chloro-5-isoquinoline
(231) N-(3-amino-2-methylbutyl)-l-chloro-5-isoquinolinesulfonamide (232) N-(3-di-n-butylaminopropyl)-l-chloro-5-isoquinolinesulfonamide
(233) N-( N-cyclohexyl-N-methylaminoethyl)-l-chloro-5-isoquinolinesulfonamide
(234) N-(2-guanidinoethyl)-l-chloro-5-isoquinolinesulfonamide
(235) N-(2-guanidinobutyl)-l-chloro-5-isoquinolinesulfonamide
(236) N-(2-guanidino- 1 -methylethyl)- 1 -chloro-5-isoquinolinesulfonamide
(237) N-(2-guanidinomethylpentyl)-l-chloiO-5-isoquinolinesulfonamide (238) N-(2-guanidino-3-methylbutyl)-l-chloro-5-isoquinolinesulfonamide
(239) N-(3-guanidino-2-methylpropyl)-l-chloro-5-isoquinolinesulfonamide
(240) N-(4-guanidino-3-methylbutyl)-l-chloro-5-isoquinolinesulfonamide
(241) 2-methyl-4-(l-chloro-5-isoquinolinesulfonyl)piperazine
(242) 2-ethyl-4-(l-chloro-5-isoquinolinesulfonyl)piperazine
(243) 2-isobutyl-4-(l-chloro-5-isoquinolinesulfonyl)piperazine
(244) 2,5-dimethyl-4-(l-chloro-5-isoquinolinesulfonyl)piperazine
(245) l-methyl-4-(l-chloro-5-isoquinolinesulfonyl)piperazine
(246) l-amidino-4-(l-chloro-5-isoquinolinesulfonyl)piperazine
(247) l-amidino-4-(l-chloro-5-isoquinolinesulfonyl)homopiperazine
(248) l-amidino-3-methyl-4-(l-chloro-5-isoquinolinesulfonyl)piperazine
(249) l-amidino-2,5-dimethyl-4-(l-chloro-5-isoquinolinesulfonyl)piperazine
(250) N-(2-aminoethyl)-l-hydroxy-5-isoquinolinesulfonamide
(251) N-(4-aminobutyl)-l-hydroxy-5-isoquinolinesulfonamide (252) N-(2-amino-l -methylethyl)- l-hydroxy-5-isoquinolinesulfonamide (253) N-(2-amino-l-methylheptyl)-l-hydroxy-5-isoquinolinesulfonamide (254) N-(3-amino-2-methylbutyl)-l-hydroxy-5-isoquinolinesulfonamide (255) N-[3-( N, N-dibutylamino)propyl]-l-hydroxy-5-isoquinolinesulfonamide (256) N-[2-( N-cyclohexyl-N-methylamino)ethyl]-l-hydroxy-5- isoquinolinesulfonamide
(257) N-(2-guanidinoethyl)-l-hydroxy-5-isoquinolinesulfonamide (258) N-(4-guanidinobutyl)-l-hydroxy-5-isoquinolinesulfonamide (259) N-(2-guanidino-l -methylethyl)- l-hydroxy-5-isoquinolinesulfonamide (260) N-(l-guanidinomethylpentyl)-l-hydroxy-5-isoquinolinesulfonamide
(261) N-(2-guanidino-3-methylbutyl)-l-hydroxy-5-isoquinolinesulfonamide
(262) N-(3-guanidino-2-methylpropyl)-l-hydoxy-5-isoquinolinesulfonamide (263) N-(4-guanidino-3-methylbutyl)-l-hydroxy-5-isoquinolinesulfonamide (264) 2-methyl-4-(l-hydroxy-5-isoquinolinesulfonyl)piperazine (265) 2-ethyl-4-(l-hydroxy-5-isoquinolinesulfonyl)piperazine
(266) 2-isobutyl-4-(l-hydroxy-5-isoquinolinesulfonyl)piperazine
(267) 2,5-dimethyl-4-(l-hydroxy-5-isoquinolinesulfonyl)piperazine
(268) l-methyl-4-(l-hydroxy-5-isoquinolinesulfonyl)piperazine
(269) l-amidino-4-(l-hydroxy-5-isoquinolinesulfonyl)piperazine (270) l-amidino-4-(l-hydroxy-5-isoquinolinesulfonyl)homopiperazine
(271) l-amidino-3-methyl-4-(l-hydroxy-5-isoquinolinesulfonyl)piperazine
(272) l-amidino-2,5-dimethyl-4-(l-hydroxy-5-isoquinolinesulfonyl)piperazine
(273) N-(2-methylaminoethyl)- 1 -chloro-5-isoquinolinesulfonamide
(274) N-(2-ethylaminoethyl)-l-chloro-5-isoquinolinesulfonamide (275) N-(2-propylaminoethyl)-l-chloro-5-isoquinolinesulfonamide
(276) N-(2-butylaminoethyl)-l-chloro-5-isoquinolinesulfonamide
(277) N-(2-hexylaminoethyl)-l-chloro-5-isoquinolinesulfonamide ι
(278) l-(l-chloro-5-isoquinolinesulfonyl)piperazine
(279) l-(l-chloro-5-isoquinolinesulfonyl)homopiperazine (280) N-(2-methylaminoethyl)-l-hydroxy-5-isoquinolinesulfonamide 281 N-(2-ethylaminoethyl)-l-hydroxy-5-isoquinolinesulfonamide 282 N-(2-propylaminoethyl)-l-hydroxy-5-isoquinolinesulfonamide 283; N-(2-butylaminoethyl)-l-hydroxy-5-isoquinolinesulfonamide 284; N-(2-hexylaminoethyl)-l-hydroxy-5-isoquinolinesulfonamide 285; l-(l-hydroxy-5-isoquinolinesulfonyl)piperazine 286 l-(l-hydroxy-5-isoquinolinesulfonyl)homopiperazine 287 l-(5-isoquinolinesulfonyl)-4-methylpiperazine 288; l-(5-isoquinolinesulfonyl)-4-n-hexylpiperazine 289 l-(5-isoquinolinesulfonyl)-4-cinnamylpiperazine 290 l-(5-isoquinolinesulfonyl)piperazine 291 N-(2-aminoethyl)-5-isoquinolinesulfonamide 292 N-(4-aminobutyl)-5-isoquinolinesulfonamide 293 N-(3-di-n-butylaminopropyl)-5-isoquinolinesulfonamide 294 l-(5-isoquinolinesulfonyl)-3-methylpiperazine 295 l-(5-isoquinolinesulfonyl)-3-isobutylpiperazine 296 l-(5-isoquinolinesulfonyl)-2,5-dimethylpiperazine 297 N-(3-guanidino-2-phenylpropyl)-5-isoquinolinesulfonamide 298 N-(6-guanidino-l-methylheptyl)-5-isoquinolinesulfonamide 299 2-[2-(5-isoquinolinesulfonamide)ethylamino]-2-imidazoline 300 2-amidino-l-(5-isoquinolinesulfonyl)piperazine
30i; 4-amidino-2,5-dimethyl-l-(5-isoquinolinesulfonyl)piperazine
302 4-amidino-l-(5-isoquinolinesulfonyl)homopiperazine 303 4-(Nl, N2-dimethylamidino)-l-(5-isoquinolinesulfonyl)piperazine 304' 4-amidino-3-butyl-l-(5-isoquinolinesulfonyl)piperazine 305 4-hexyl-l-(5-isoquinolinesulibnyl)ethylenediamine 306 N-(4-guanidinobutyl)-5-isoquinolinesulfonamide 307 N-(2-guanidinoethyl)-5-isoquinolinesulfonamide 308 l-(5-isoquinolinesulfonyl)-2-methylpiperazine
Preferred compounds are (204) and (308).
The compounds may be provided as a pharmaceutically acceptable acid addition salt, The acid may be an inorganic acid such as hydrochloric acid, hydrobromic acid or sulfuric acid or an organic acid such as n ethanesulfonic acid, fumaric acid, maleic acid, mandelic acid, citric acid, tartaric acid or salicylic acid. Compounds having a carboxyl group can be converted to a salt with a metal such as sodium, potassium, calcium, magnesium or aluminum or an amino acid such as lysine and the like.
Other Rho-kinase inhibitors are disclosed in US-A-5478838. These Rho- kinase inhibitors are: (1) 4-amino(alkyl)cyclohexane-l-carboxamide compounds of the formula (III):
Figure imgf000033_0001
wherein R1 and R2 are the same or different and each is hydrogen, alkyl, or cycloalkyl, cycloalkylalkyl, phenyl, aralkyl, piperidyl or pyrrolidinyl, any of which may have a substituent on the ring, or a group of the formula:
NR° 'i
— Cχ ( IV )
R
wherein R is hydrogen, alkyl, -NR'R" (where R' and R" are the same or different and each is hydrogen, alkyl, aralkyl or phenyl) and R° is hydrogen, alkyl, aralkyl, phenyl, nitro or cyano, or R and R° may in combination form a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom, or
R1 and R2 in combination represent alkylidene or phenylalkylidene, or R1 and R2 form, together with the nitrogen atom, a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom; R3 and R4 are each hydrogen or alkyl; A is a single bond or alkylene; X is =C(R7)- or =N-;
R5 and R6 together form a group of the formula: (a) -CRa=CRb-,
(b) -NRa-C(=Rb)-,
(c) -N=CRb-,
(d) -C(=Ra)-NRb-,
(e) -CRa=N-, or (f) -NRa-, wherein Ra and Rb are the same or different and each is hydrogen, halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, -NRcRd [wherein Re and Rd are the same or different and each is hydrogen, alkyl, -COR9, -COOR9 or -S02R9' (where R9 is hydrogen, alkyl, phenyl or aralkyl and R9' is alkyl, phenyl or aralkyl) or Re and Rd foπn, together with the nitrogen atom to which they are attached, a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom], cyano, azide, optionally substituted hydrazino, -COOR10, or -CONRuR12 (wherein R10"12 are each hydrogen, alkyl, phenyl or aralkyl), or Ra and Rb in combination form an optionally hydrogenated 5- or 6-membered aromatic ring which may have at least one of a nitrogen atom, sulfur atom and oxygen atom provided that, when R3 and R6 are of the formula (b) or (d), Ra and Rb together form an optionally hydrogenated 5- or 6-membered aromatic ring which may have at least one of a nitrogen atom, sulfur atom and oxygen atom; R7 and R8 are the same or different and each is hydrogen, halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, -NReRf [wherein Re and Rf are the same or different and each is hydrogen, alkyl, -COR9, -COOR9' or -S02R9' (where R9 is hydrogen, alkyl, phenyl or aralkyl and R9' is alkyl, phenyl or aralkyl) or Re and Rf form, together with the nitrogen atom to which they are attached, a heterocyclic ring which may have, in the ring, oxygen atom, sulfur atom or optionally substituted nitrogen atom], cyano, azido, optionally substituted hydrazino, -COOR10 or -CONR1 'R12 (wherein R10"12 are each hydrogen, alkyl, phenyl or aralkyl); and n is 0 or 1; with the proviso that, when R5 and Rb are of the formula (a), X is =C(R7)- and either one of Ra, Rb, R7 and Rs is -NRcRd, -NReRf, azido, optionally substituted hydrazino, -COOR10 or -CONRnR12, or Ra and Rb together form an optionally hydrogenated 5- or 6-membered aromatic ring which may have, in the ring, at least one of nitrogen atom, sulfur atom and oxygen atom; isomers thereof and pharmaceutically acceptable acid addition salts thereof:
(2) 4-amino(alkyl)cyclohexane-l-carboxamide compounds of formula (HI) above wherein R1 and R2 are the same or different and each is hydrogen, alkyl having 1 to
10 carbon atoms, cycloalkyl having 3 to 7 carbon atoms, cycloalkylalkyl, phenyl, aralkyl, piperidyl or pyrrolidinyl which may have substituent on the ring, or R1 and R2 in combination represent alkylidene or phenylalkylidene, or R1 and R2 form, together with the nitrogen atom to which they are attached, a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom;
R3 and R4 are each hydrogen or alkyl;
A is a single bond or alkylene;
X is =C(R7)- or =N-; R3 and Rδ together form a group of the formula
(a) -CRa=CRb-,
(b) -NRa-C(=Rb)-,
(c) -N=CRb-,
(d) -C(=Ra)-NRb-, (e) -CRa=N- or
(f) -NRa-, wherein Ra and Rb are the same or different and each is hydrogen, halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, -NRcRd [wherein Re and Rd are the same or different and each is hydrogen, alkyl, -COR9, -COOR9' or -S02R9' (where R9 is hydrogen, alkyl, phenyl or aralkyl and RSl is alkyl, phenyl or aralkyl) or Re and Rd form, together with the nitrogen atom to which they are attached, a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom], cyano, azido, optionally substituted hydrazino, -COOR10 or -CONRnR12 (wherein R10"12 are each hydrogen, alkyl, phenyl or aralkyl), or Ra and Rb in combination form an optionally hydrogenated 5- or 6-membered aromatic ring which may have, in the ring, at least one of a nitrogen atom, sulfur atom and oxygen atom provided that, when R3 and Rδ are of the formula (b) or (d), Ra and Rb together fonn an optionally hydrogenated 5- or 6-membered aromatic ring which may have at least one of a nitrogen atom, sulfur atom and oxygen atom; R7 and R8 are the same or different and each is hydrogen, halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, -NReRf [wherein Re and Rf are the same or different are each is hydrogen, alkyl, -COR9, -COOR9' or -S02R9' (where R9 is hydrogen, alkyl, phenyl or aralkyl and R9' is alkyl, phenyl or aralkyl) or Re and Rf form, together with the nitrogen atom to which they are attached, a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom], cyano, azido, optionally substituted hydrazino, -COOR10 or -CONRπR12 (wherein R10"12 are each hydrogen, alkyl, phenyl or aralkyl); and n is 0 or 1 ; with the proviso that, when R5 and R6 are of the formula (a), X is =C(R7)- and either one of Ra, Rb, R7 and R8 is -NRcRd, -NReRf, azido, optionally substituted hydrazino, -COOR10 or -CONRnRi2, or Ra and Rb together form an optionally hydrogenated 5- or 6-membered aromatic ring which may have, in the ring, at least one of a nitrogen atom, sulfur atom and oxygen atom; isomers thereof and pharmaceutically acceptable acid addition salts thereof: (3) 4-amino(alkyl)cyclohexane-l-carboxamide compounds of the formula (V):
Figure imgf000036_0001
wherein R is hydrogen, alkyl or -NR'R" (wherein R1 and R" are the same or different and each is hydrogen, alkyl, aralkyl or phenyl) and R° is hydrogen, alkyl, aralkyl, phenyl, nitro or cyano, or R and R° in combination form a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom;
R2 is hydrogen, alkyl or aralkyl; R3 and R4 are each hydrogen or alkyl; A is a single bond or alkylene; X is =C(R7)- or =N-;
R5 and Rϋ together form a group of the formula
(a) -CRa=CRb-,
(b) -NRa-C(=Rb)-,
(c) -N=CRb-, (d) -C(=Ra)-NRb-,
(e) -CRa-N-, or (1) or -NRa-, wherein Ra and Rb are the same or different and each is hydrogen, halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, -NRcRd [wherein Re and Rd are the same or different and each is hydrogen, alkyl, -COR9, -COOR9' or -S02R9' (where R9 is hydrogen, alkyl, phenyl or aralkyl and R9' is alkyl, phenyl or aralkyl) or Re and Rd form, together with the nitrogen atom to which they are attached, a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom], cyano, azido, optionally substituted hydrazino, -COOR10 or -CONRuR12 (wherein R10"12 are each hydrogen, alkyl, phenyl or aralkyl), or
Ra and Rb in combination form an optionally hydrogenated 5- or 6-membered aromatic ring which may have, in the ring, at least one of a nitrogen atom, sulfur atom and oxygen atom provided that, when R5 and Rb are of the formula (b) or (d), Ra and Rb together form an optionally hydrogenated 5- or 6-membered aromatic ring which may have at least one of a nitrogen atom, sulfur atom and oxygen atom; R7 and R8 are the same or different and each is hydrogen, halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, -NReRf [wherein Re and Rf are the same or different are each is hydrogen, alkyl, -COR9, -COOR9' or -S02R9' (where R9 is hydrogen, alkyl, phenyl or aralkyl and R9' is alkyl, phenyl or aralkyl) or Re and Rf form, together with the nitrogen atom to which they are attached, a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom], cyano, azido, optionally substituted hydrazino, -COOR10 or -CONRnR12 (wherein R10'12 are each hydrogen, alkyl, phenyl or aralkyl); and n is 0 or 1 ; with the proviso that, when R5 and Rb are of the foπnula (a), X is =C(R7)- and either one of Ra, Rb, R7 and Rs is -NRcRd, -NReRf, azido, optionally substituted hydrazino, -COOR10 or -CONRπR:2, or Ra and Rb together form an optionally hydrogenated 5- or 6-membered aromatic ring which may have, in the ring, at least one of a nitrogen atom, sulfur atom and oxygen atom; isomers thereof and pharmaceutically acceptable acid addition salts thereof. Preferred such compounds are: trans-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-aminomethylcyclohexanecarboxamide, trans-N-(l H-pyrazolo [3, 4-b]ρyridin-4-yl)-4-aminomethylcyclohexanecarboxamide,
(-)-trans-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(l- aminomethyl)cyclohexanecarboxamide, trans-N-(lIT-pyrrolo[2,3-b]pyridin-4-yl)-4-(l -amino- 1- methylethyl)cyclohexanecarboxamide,
(-)-trans-N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-4-(l- aminomethyl)cyclohexanecarboxamide, trans-N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-4-(l-amino-l- methylethyl)cyclohexanecarboxamide. trans-N-(2-methanesulfonylamino-4-pyridyl)-4- aminomethylcyclohexanecarboxamide,
(-)-trans-N-(2,3-dihydro-lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(l- aminoethyl)cyclohexanecarboxamide, (-)-trans-N-(2,3-dihydro-2-oxo-lH-ρyrrolo[2,3-b]pyridin-4-yl)-4-(l- aminoethyl)cyclohexanecarboxamide,
(+)-trans-N-(2-methylcarbamoyl-4-pyridyl)-4-(l - aminoethyl)cyclohexanecarboxamide, (-)-trans-N-(2-(2,2-dimethylhydrazino)-4-pyridyl)-4-(l- aminoethyl)cyclohexanecarboxamide,
(-)-trans-N-(2-methylamino-4-pyridyl)-4-(l-aminoethyl)cyclohexanecarboxamide,
(-)-trans-N-(2-ethylamino-4-pyridyl)-4-(l-aminoethyl)cyclohexanecarboxamide, trans-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-guanidinomethylcyclohexanecarboxamide, trans-N-(lH-pyrazolo[3,4-b]pyridin-4-yl)-4- guanidinomethylcyclohexanecarboxamide, trans-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(2-imidazolin-2-yl)- aminomethylcyclohexanecarboxamide, trans-N-(lH-pyrrolo[2,3-b]pyridin-4-yl)-4-(l- methylguanidinomethyl)cyclohexanecarboxamide,
N'-[trans-(4-(lH-pyrrolo[2,3-b]pyridin-4- ' yl)carbamoyl)cyclohexylmethyl]formamidine, trans-N-(2,3-dihydro-2-oxo-lH-pyrrolo[2,3-b]pyridin-4-yl)-4- guanidinomethylcyclohexanecarboxamide, and trans-N-(2,3-dihydro-lH-pyπolo[2,3-b]pyridin-4-yl)-4- guanidinomethylcyclohexanecarboxamide; isomers thereof and pharmaceutically acceptable acid addition salts thereof.
In the formulae (HI) to (IV) halogen means chlorine, bromine, fluorine or iodine; alkyl means straight- or branched chain alkyl having 1 to 10, preferably 1 to 6' carbon atoms, which is exemplified by methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, 2- ethylhexyl, octyl, nonyl or decyl; haloalkyl means the aforementioned alkyl substituted by 1 to 5 halogens, which is exemplified by trifluoromethyl, 2,2,2- trifluoroethyl or 2,2,3,3,3-pentafluoropropyl; alkoxy means straight- or branched chain alkoxy having 1 to 6 carbon atoms, which is exemplified by methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, pentyloxy, isopentyloxy, neopentyloxy or hexyloxy; cycloalkyl means that having 3 to 7 carbon atoms, which is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl; cycloalkylalkyl means that wherein the cycloalkyl moiety is the aforementioned cycloalkyl having 3 to 7 carbon atoms and the alkyl moiety is straight- or branched chain alkyl having 1 to 6 carbon atoms (e.g. methyl, ethyl, propyl, isopropyl, butyl, pentyl, hexyl) and is exemplified by cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, cycloheptylmethyl, cyclopropylethyl, cyclopentylethyl, cyclohexylethyl, cycloheptylethyl, cyclopropylpropyl, cyclopentylpropyl, cyclohexylpropyl, cycloheptylpropyl, cyclopropylbutyl, cyclopentylbutyl, cyclohexylbutyl, cycloheptylbutyl, cyclopropylhexyl, cyclopentylhexyl, cyclohexylhexyl or cyloheptylhexyl; alkylene means straight- or branched chain alkylene having 1 to 6 carbon atoms, which is exemplified by methylene, ethylene, trimethylene, tetramethylene, pentamethylene, hexamethylene, methylmethylene, dimethylmethylene, ethylmethylene, diethylmethylene, propylmethylene, propylene, methyltrimethylene, dimethylethylene, dimethyltrimethylene or dimethyltetramethylene; aralkyl means that wherein the alkyl moiety is alkyl having 1 to 4 carbon atoms and is exemplified by phenylalkyl such as benzyl, 1 -phenylethyl, 2-phenylethyl, 3-phenylpropyl and 4- phenylbutyl; alkylidene means straight- or branched chain alkylidene having 1 to 6 carbon atoms, which is exemplified by methylidene, ethylidene, propylidene, isopropylidene, butylidene, pentylidene or hexylidene; phenylalkylidene means that wherein the alkylidene moiety is alkylidene having 1 to 6 carbon atoms, and is exemplified by benzylidene, phenylethylidene, phenylpropylidene, phenylbutylidene, phenylpentylidene or phenylhexylidene.
Examples of the substituents for cycloalkyl having 3 to 7 carbon atoms, and for cycloalkylalkyl, phenyl, aralkyl, piperidyl and pyrrolidinyl which may have a substituent, include halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, -NRcRd (wherein Re and Rd may be the same or different and each is hydrogen, alkyl, -COR9, -COOR9' or -S02R9' or Re and Rd, together with the nitrogen atom to which they are attached, form a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom), cyano, azido, formyl, acyl, -COOR10, -CONRπR12 or optionally substituted hydrazino, As used herein, halogen, alkyl, alkoxy, aralkyl and haloalkyl are as defined above and acyl is exemplified by acetyl, propionyl, butyryl, valeryl, pivaloyl, benzoyl, phenylacetyl, phenylpropionyl or phenylbutyryl,
Examples of the substituent for the optionally substituted hydrazino include alkyl, aralkyl, nitro and cyano, wherein alkyl and aralkyl are as defined above.
The heterocyclic ring optionally having, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom, which is formed by R1 and R2, Re and Rd or Re and Rf, together with the nitrogen atom to which they are attached, is preferably a 5- or 6-membered ring or a fused ring system and is exemplified by pyrrolidinyl, piperidyl, piperazinyl, morpholino and thiomorpholmo. Examples of the substituent for the optionally substituted nitrogen atom include alkyl, aralkyl and haloalkyl, wherein alkyl, aralkyl and haloalkyl are as defined above,
The heterocyclic ring optionally having, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom, which is formed by R° and R1 in combination, is exemplified by imidazol-2-yl, thiazol-2-yl, oxazol-2-yl, imidazolin-2- yl, 3,4,5,6-tetrahydropyridin-2-yl, 3,4,5,6-tetrahydropyrimidin-2-yl, l,3-oxazolin-2- yl, l,3-thiazolin-2-yl, and benzimidazol-2-yl, benzothiazol-2-yl, benzoxazol-2-yl and indol-2-yl which may have substituent such as halogen, alkyl, alkoxy, haloalkyl, nitro, amino, phenyl or aralkyl, wherein halogen, alkyl, alkoxy, haloalkyl and aralkyl are as defined above,
Examples of the substituent for the optionally substituted nitrogen atom include alkyl, aralkyl and haloalkyl, wherein alkyl, aralkyl and haloalkyl are as defined above.
When R5 and Rύ are of the formula (a), (c), (e) or (f) and form a single ring, the ring is pyridine, pyrimidine, pyridazine, triazine, pyrazole or triazole. When R5 and Rδ are of the formula (a), (b) or (d) and form a condensed ring, the ring is pyrrolopyridine (e.g. lH-pyrrolo[2,3-b]pyridine, lH-pyrrolo[3,2-b]pyridine, 1H- pyrrolo[3,4-b]pyridine), pyrazolopyridine (e.g. lH-pyrazolo[3,4-b]pyridine, 1H- pyrazolo[4,3-b]pyridine), imidazopyridine (e.g. lH-imidazo[4,5-b]pyridine), pyrrolopyrimidme (e.g. lH-pyrrolo[2,3-d]pyrimidine, lH-pyrrolo[3,2-d]pyτimidine, lH-pyrrolo[3,4-d]pyrimidine), pyrazolopyrimidine (e.g. lH-pyrazolo[3,4- d]pyrimidine, pyrazolo[l,5-a]pyrimidine, lH-pyrazolo[4,3-d]pyrimidine), imidazopyrimidine (e.g. imidazo[l,2-a]pyrimidine, lN-imidazo[4,5-d]pyrimidine), pyrrolotriazine (e.g. pyrrolo[l,2-a]-l,3,5-triazine, pyrrolo[2,l-f]-l,2,4-triazine), pyrazolotriazine (e.g. pyrazolo[l,5-a]-l,3,5-triazine), triazolopyridme (e.g. 1H-1,2,3- triazolo[4,5-b]pyridine), triazolopyrimidine (e.g. l ,2,4-triazolo[l,5-a]pyrimidine, l,2,4-triazolo[4,3-a]pyrimidine, lH-l,2.3-triazolo[4,5-d]pyrimidine), cinnoline, quinazoline, quinoline, pyridopyridazine (e.g. pyrido[2,3-c]pyridazine), pyridopyrazine (e.g. pyrido[2,3-b]pyrazine), pyridopyrimidine (e.g. pyrido[2,3- d]pyrimidine, pyrido[3,2-d]pyrimidine), pyrimidopyrimidine (e.g. pyrimido[4,5- djpyrimidine, pyrimido[5,4-d]pyrimidine), pyrazinopyrimidine (e.g. pyrazino[2,3- djpyrimidine), naphthyridine (e.g. 1,8-naphthyridine), tetrazolopyrimidine (e.g. tetrazolo[l ,5-a]pyrimidine), thienopyridine (e.g. thieno[2,3-b]pyιidine), thienopyrimidine (e.g. [2,3-d]pyrimidine), thiazolopyridine (e.g. thiazolo[4,5- bjpyridine, thiazolo[5,4-b]pyridine), thiazolopyrimidine (e.g, thiazolo[4,5- d]pyrimidine, thiazolo[5,4-d]pyrimidine), oxazolopyridine (e.g. oxazolo[4,5- bjpyridine, oxazolo[5,4-b]pyridine), oxazolopyrimidine (e.g. oxazolo[4,5- djpyrimidine, oxazolo[5,4-d]pyrimidine), furopyridine (e.g, furo[2,3-b]pyridine, furo[3,2-b]pyridine), furopyrimidine (e.g. furo[2,3-d]pyrimidine, furo[3,2- d]pyrimidine), 2,3-dihydropyrrolopyridine (e.g, 2,3-dihydro-lH-pyιτolo[2,3- bjpyridine, 2,3-dihydro-lFI-pyrrolo[3,2-b]pyridine), 2,3-dihydropyrrolopyrimidine (e.g, 2,3-dihydro-lH-pyrrolo[2,3-d]p)rimidine, 2,3-dihydro-lH-pyrrolo[3,2- djpyrimidine), 5,6,7,8-tetrahydropyrido[2,3-d]pyrimidine, 5, 6,7, 8-tetrahydro- 1,8- naphthyridine or 5, 6, 7, 8-tetrahydro quinoline. When these rings form hydrogenated aromatic rings, the carbon atom in the ring may be carbonyl and, for example, 2,3- dihydro-2-oxopyrrolopyridine, 2,3-dihydro-2,3-dioxopyrrolopyridine, 7,8-dihydro-7- oxonaphthyridine and 5,6,7,8-tetrahydro-7-oxonaphthyridine are included, These rings may be substituted by substituent such as halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, -NRcRd, cyano, formyl, acyl, aminoalkyl, mono- or dialkylammoalkyl, azido, -COOR10, -CONRnR12 or optionally substituted hydrazino,
Pharmaceutically acceptable acid addition salts of a compound of formula (III) or (V) include salts with an inorganic acid or organic acid. The compound may talce the foπn of a hydrate or solvate. When the compound has a carboxyl group, the salt may be a metal salt such as a sodium salt, potassium salt, calcium salt or aluminum salt or a salt with an amino acid such as lysine and ornithine. The compound may take the form of a cis- or trans-geometrical isomer or a mixture thereof, When the compound of formula (III) or (V) has an asymmetric carbon, it may be an optical isomer or a racemate.
Further Rho-kinase inhibitors for use in the invention are disclosed in WO 02/076977, These inhibitors are compounds of formula (VI) to (XI):
Figure imgf000043_0001
Figure imgf000043_0002
Figure imgf000044_0001
Figure imgf000044_0002
and pharmaceutically acceptable salts thereof. Suitable pharmaceutically acceptable salts include basic salts of inorganic and organic acids, such as hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid, methanesulphonic acid, sulphonic acid, acetic acid, trifiuoroacetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicyclic acid, phenyl acetic acid and mandelic acid, ln addition, pharmaceutically acceptable salts include acid salts of inorganic bases, such as salts containing alkaline cations (e.g, Li-, Na+ or K~), alkaline earth cations (e.g, Mg+, Ca+ or Ba~), the ammonium cation, as well as acid salts of organic bases, including aliphatic and aromatic substituted ammonium, and quaternary ammonium cations, such as those arising from protonation or peralkylation of triethylamine, N,N-dimethylamine, N,N- dicyclohexylamine, pyridine, N,N-dimethylaminopyridine (DMAP), 1,4- diazabicyclo[2.2.2]octane (DABCO), l,5-diazabicyclo[4.3.0]non-5-ene (DBN) and l,8-diazabicyclo[5.4.0]undec-7-ene (DBU). A number of the compounds of formulae (VI) to (XI) possess asymmetric carbons and can therefore exist in racemic and optically active forms, Yet further, Rho-kinase inhibitors are disclosed in EP-A-0187371. A particularly prefeπed Rho-kinase inhibitor is (-)-(R)-/ra«-s-4-(l-aminoethyl)-N-(4- pyridyl)cyclohexanecarboxamide or a pharmaceutically acceptable acid addition salt thereof, especially the dihydrochloride which has the formula:
Figure imgf000045_0001
.2HC1
This compound is typically provided as a monohydrate.
The Rho-kinase inhibitors are used to treat benign prostatic hyperplasia (BPH). An effective amount of a Rho-kinase inhibitor is administered to a man suffering from BPH, The condition of the patient can thus be improved. The symptoms of BPFI can be alleviated. Generally, an inhibitor is provided in a pharmaceutical pack together with instructions for use in treating BPH. Administration of an inhibitor may be in a variety of dosage forms, Thus, an inhibitor may be administered orally, for example as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules. An inhibitor may also be administered parenterally, either subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques. An inhibitor may be administered locally using a catheter (intraurethrally), An inhibitor may be administered as a suppository, The dosage form and route of administration will depend largely on the nature of the particular inhibitor.
An inhibitor modulator is typically formulated for administration in the present invention with a pharmaceutically acceptable carrier or diluent. The phaπnaceutical carrier or diluent may be, for example, an isotonic solution. For example, solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g, silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, gum arabic, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film-coating processes.
Liquid dispersions for oral administration may be syrups, emulsions or suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.
Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate,- pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g, sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.
Solutions for intravenous administration or infusion may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions. A therapeutically effective amount of an inhibitor is administered to man having BPH. The dose of inhibitor may be determined according to various parameters, especially according to the particular inhibitor used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.1 to 50 mg per kg of body weight, according to the activity of the specific inhibitor, the age, weight and conditions of the subject to be treated, the severity of the BPH and the frequency and route of administration. Preferably, daily dosage levels are from 5 mg to 2 g. A daily dose may be given in one administration or more than one administration, for example 2, 3 or 4 administrations.
As described, an inhibitor may be administered as a polynucleotide. The polynucleotide may comprise coding sequence for a molecule with inhibitory activity, for example a protein or mRNA. The polynucleotide is generally constructed so as to permit expression of the inhibitory molecule in a target cell. For example, the coding sequence may be operably linked to a suitable promoter sequence, which will direct expression in a target cell. Alternatively the polynucleotide may itself have inhibitory activity. For example, an inhibitor}' polynucleotide may encode an inhibitory protein or mRNA, or the polynucleotide may itself be an inhibitor, for example, an antisense molecule or iRNA, The polynucleotide may be administered as a naked nucleic acid construct,
Uptake of naked nucleic acid constructs by mammalian cells is enhanced by several known transfection techniques for example those including the use of transfection agents, Examples of these transfection agents include cationic agents (for example calcium phosphate and DEAE-dextran) and lipofectants (for example Lipofectam™ and Transfectam™), A nucleic acid may also be administered using a viral vector. A polynucleotide composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, transdermal or intraurethral administration, Typically, a polynucleotide inhibitor is mixed with a transfection agent to produce a composition. Preferably the naked polynucleotide construct, viral vector comprising the polynucleotide or polynucleotide composition is combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical composition, Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline,
A polynucleotide is administered in such a way that the polynucleotide, for example viral vector can be incorporated into cells of the prostate. When the polynucleotide is delivered to cells by a viral vector, the amount of virus administered is in the range of from 106 to 10:o pfu, preferably from 107 to 109 pfu, more preferably about 108 pfu for adenoviral vectors. When injected, typically 1-2 ml of virus in a pharmaceutically acceptable suitable carrier or diluent is administered. When the polynucleotide is administered as a naked nucleic acid, the amount of nucleic acid administered is typically in the range of from l μg to 10 mg,
The routes of administration and dosages described above are intended only as a guide since a skilled physician will be able to determine readily the optimum route of administration and dosage for any particular patient and condition.
The following Example illustrates the invention.
Example
Materials and Methods Human Tissue Collection
Human prostate tissue was obtained from four patients subjected to transurethral resection of the prostate (TURP) due to symptomatic BPH, Specimens were resected from normal appearing, non-adenomatous parts of the prostate close to tlϊe bladder neck. The biopsy specimens were immediately placed in sterile tubes containing phosphate buffered saline solution (PBS) and transported on ice to the laboratory. Specimens not used immediately were frozen in liquid nitrogen and stored at -80=C.
Establishment of Smooth Muscle Cell Culture from Human and Rat Prostate
Tissue specimens were transferred into sterile culture medium (MCDB-131 with 10% fetal calf serum, 1% antibiotics, lOmM HEPES, 1% penicillin- streptomycin solution, 1% non-essential aminoacids, 5μg/ml insulin, lOμg/ml transfeπin, 5ng/ml sodium-selenite, 0.1 μM dexamethasone and 0.1 μM β-estradiol). This medium has been previously shown to stimulate the growth of smooth muscle cells and to suppress the growth of fibroblasts from prostatic explants (Corvin et al, Prostate 37, 209-214, 1998). The specimens were then cut into 1 mm3 pieces using a Mcllwain tissue chopper (MLE, UK). 5-10 small pieces were seeded into each well of 6- well culture plates (NUNC, Gibco, UK) each containing 0.5 ml of medium. After incubating the pieces at 37'C in 5% C02 in a humidified incubator for
24 hours, 2.5 ml of medium was added to each well. The tissue pieces were kept in the incubator for another 5 days, avoiding any movement or agitation. On the 5th day the tissue explants were removed from the wells by gentle agitation, leaving 10-20 smooth muscle cells migrated out from each explant and attached firmly on the surface. Once the cells in each well reached 100% confluency, they were transfeπed into 10cm or 15cm diameter culture dishes using trypsin-EDTA (0,05% trypsin and 0.53mM EDTA). During the next passage, some of the cells were seeded on 22x22 mm glass cover slips for immunofluorescence. Any cell population containing fibroblasts (negative smooth muscle specific α-actin immunostaining) were discarded.
Im mini oflu orescence
0.5-1.0x105 cells on each 22x22mm coverslip were fixed in methanol at -20°C and washed in PBS containing 1% bovine serum albumin. The coverslips were incubated with Rho-kinase (1 : 100, AutogenBioclear, UK) and smooth muscle specific α-actin (1 :200, Sigma, UK) antibodies overnight at 4°C. They were then incubated with biotinylated anti-goat IgG (1 :200, Vector Laboratories, USA) for 2 hours followed by FITC avidin-D (1 :200) and anti-mouse IgG-Texas Red (1 :200, Vector Laboratories, USA) for 1 hour at room temperature. The cover slips were mounted on glass-slides with a drop of Vectashield mounting medium (Vector
Laboratories, USA) containing 4!,6-diamidino-2-phenylindole (DAPI) to stain the nuclei, The fluorescence was visualized using a confocal laser-scanning microscope (Leica, Model TCS-SP). The excitation wavelengths used for FITC, Texas Red and DAPI were (in nm) 488, 543 and 190-370 respectively. The acquired images were overlayed using the Leica TCS-SP Software.
Immunoblotting
2.0xl07 cells on a 15 cm dish were collected using trypsin-EDTA, washed three times with ice cold PBS and then resuspended with 1 ml of homogenising buffer (20mM HEPES, ImM EDTA, 0.2 M sucrose, 20μg/ml soyabean trypsin inhibitor, 20μg/ml leupeptin, 5μg/ml pepstatin A, 5mM DL-dithiothreitol (DTT), 5μg/ml E-64, 5μg/ml bestatin, 5μg/ml aprotinin, lOμg/ml 3-4-DCL, pFI: 7,2, 4"C). The cell membranes were disrupted using a sonicator at 20μm for 5 s at 4°C. The lysate was centrifuged at 13,000 g for 15 min at 4°C. Tissue specimens (human and rat prostate) were homogenised in dry ice using a stainless steel pestle and mortar. The homogenate was resuspended in 1 ml homogenising buffer (composition as above), vortexed vigorously for 20 s and centrifuged at 13,000 g for 15 min at 4°C.
The protein content of the supernatant of cell and tissue lysates λvere determined using protein assay reagent (BIO-RAD, UK) based on the dye-binding procedure of Bradford (Anal, Biochem. 72, 248-254, 1976). lOOμg of each sample was run on 7.5% polyacrylamide SDS gel and then transfeπed on to nitrocellulose membranes. The blots were incubated overnight with polyclonal Rho-kinase antibody (1 :100, AutogenBioclear, UK) at 4°C. A final incubation was carried out for 2 hours at room temperature with horse-radish peroxidase-conjugated anti-goat IgG (1 :4000, AutogenBioclear, UK), The reactive bands were detected with a luminol-based kit (ECL, Amersham, UK). A7r5 rat vascular smooth muscle cells and rat brain were used as positive control.
Cell proliferation assay
Human prostatic smooth muscle cells were cultured in 96-well plates. After serum starvation for 24 hours, cells were incubated in the presence or absence of Rho-kinase inhibitor, Y-27632 ((+)-(R)-trans-4-(l-aminoethyl)-N-(4- pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate; Calbiochem, UK; 0.01-100μM) in the culture medium as above but containing only 1% FCS for 24, 48 and 72 hours. 5-bromo-2'-deoxyuridine (BrdL ) labelling (Roche Diagnostics, UK) was performed according to the protocol provided by the manufacturer. Briefly, during the labelling period, the pyrimidine analogue of BrdU is incorporated in place of thymidine into the DNA of proliferating cells. After removing the culture medium the cells are fixed and the DNA is denatured, Incorporated BrdU is then detected by immunoassay.
Cell viability assay
The viability of human prostatic smooth muscle cells was assessed using the neutral red and MTT assays as described previously (Morgan, J. Immunol, Methods 145. 259-262, 1991 ; Mosmann, Immunol. Methods 65, 55-63, 19S3). The cells were cultured in 6-well plates. After serum starvation for 24 hours, the cells were incubated in the presence or absence of Y-27632 (0.01-100μM) in the culture medium as above but containing only 1% FCS for 24, 48 and 72 hours. At the end of the incubation period the medium was removed and the cells were washed twice with warm PBS, The cells were then incubated with neutral red (O.lmg/ml) or MTT (0.5mg/ml) in RPMI-1640 (without phenol red) at 37°C for 90 minutes. The cells were then washed with warm PBS and lyzed in DMSO with mild agitation at room temperature for 30 minutes. The developed colour was then quantified in a microplate reader at 550 and 570nm for neutral red and MTT respectively. Detection of apoptosis
After serum starvation for 24 hours, the cells in 10cm dishes were restimulated with 1% serum in the presence or absence of Y-27632 (0.01-100μM) for 48 hours. The cells were collected and incubated in the presence of TRIS (10 mM), NaCl (ImM), RNase (100 μg ml), Tween 20 (0, 1%) and propidium iodide (0.004%) at 37°C for 25 min. Apoptotic cells were distinguished from non-apoptotic cells by their decreased DNA content, as determined by their lower propidium iodide staining intensity in the presence of RNase. The cells were counted using a flow cytometer (Becton Dickinson, FACSCalibur, UK), Human prostatic smooth muscle cells treated with 0.1 μM staurosporine for 4 hours were used as positive controls,
Functional Studies Using Rat Prostate
Rat prostate was obtained from male Wistar rats (250-275g), The ventral part of the prostate was dissected, cleaned from adherent tissue and divided vertically into three equally sized strips, The prostatic strips were placed horizontally between two ring electrodes in tissue chambers at 37°C. The tissues were superfused at a constant flow of 1 ml/min by means of peristaltic pumps (Miniplus 2, Gilson, UK) with a medium of the following composition (mM): NaCl 136.9, KC1 2.7, CaCl2 1 ,8, MgSO, 0.6, NaHC03 11.9, KH2PO40.5, glucose 11.5, indomethacin 0.01, dexamethasone 0.01 and gassed with 5% CO2 in 02 (pH 7.4-7.6).
One end of the preparation was tied to a Grass FT 03 C force-displacement transducer connected to a Linearcorder WR 3101 (Graphtec, USA) for registration of isometric changes in tension. The preparations were stretched (0.2 to 0.5g) until they reached approximately the in situ length and allowed to equilibrate for 90 min, The preparations were stimulated electrically (electrical field stimulation; EFS) with 5 s trains of rectangular pulses of 50 V, 0,3 ms pulse duration and frequencies ranging from 1 to 50 Hz, delivered by Grass S88 stimulators every 120 s throughout the experiment. Drugs were applied into the medium reservoir,
Chemicals Y-27632 was obtained from Calbiochem, UK, All other reagents were from Sigma, UK unless otherwise stated. NUNC cell culture dishes and cell culture medium were obtained from Life Technologies, UK, FCS was endotoxin-free and was obtained from Insight Biotechnology, UK.
Results
Im m u n oflu o rescen ce
It has been demonstrated previously that MCDB-131 medium with insulin, selenite and estradiol stimulates the growth of smooth muscle cells and to suppress the growth of fibroblasts from prostatic explants (Corvin et al, 1998). Using this medium we have established successful migration of smooth muscle cells from rat and human prostatic explants by day 5. Culture morphology was characterized by the formation of nodules of cells (not shown), which has been described as "hill and valley appearance" specific to smooth muscle cells (Chamley-Campbell et al, J. Physiol. 59, 1-61, 1979). Immunostaining using antibodies against smooth muscle specific α-actin and Rho-kinase showed clearly that the cells have smooth muscle actin filaments (Figure 1). Rho-kinase was located in the perinuclear cytosol suggesting a location in or around endoplasmic reticulum (Figure 1),
Immunoblotting
A band coπesponding to Rho-kinase at -180 kDa was observed in the Western blotting of crude extracts of human and rat prostate tissue and prostatic smooth muscle cells (Figure le). Extracts of A7r5 rat pulmonary artery smooth muscle cells and rat brain were used as positive controls in which similar bands were observed (Figure le),
Effect of Y-27632 on cell viability and proliferation
Human and rat prostatic smooth muscle cells were made quiescent in serum- free medium for 24 hours before restimulation by addition of 1% serum. Neutral red, MTT and BrdU assays were performed 24, 48 and 72 hours after switching back to 1% serum. During this period some of the dishes were exposed to Y-27632 (0.01- lOOμM), As shown in Figure 2a and 2b, Y-27632 reduced number of the live cells as measured with neutral red and MTT assays. The proliferation of the cells was also reduced with Y-27632 as measured by BrdU assay (Figure 2c).
Apoptotic index
Y-27632 (0.01-100μM) did not change the apoptotic index of human and rat prostatic smooth muscle cells (Figure 2d).
Effect of Y-27632 on adrenergic contractions of rat prostatic strips
We examined the effect of Y-27632 on contractile responses of the rat prostate. In the first set of experiments, we investigated the effect of Y-27632 on contractions elicited by endogenous noradrenaline released from sympathetic nerves in the prostate. Noradrenergic contractions were produced by electrical field stimulation (EFS) of strips of rat prostate. Y-27632 (0.1-500 μM) inhibited EFS- induced noradrenergic contractions in a concentration-dependent manner (IC50 = 17.8+4.8 μM; n=4; Figure 3). After Y-27632 was washed, the noradrenergic contractions returned to their control levels (Figure 3). Application of guanethidine (10 μM) or prazosin (5 μM) completely abolished the responses (Figure 3, prazosin not shown).
In the second set of experiments, we tested Y-27632 on the elevated tone of prostatic strips. The tone of prostatic strips was elevated with application of exogenous phenylephrine (10 μM, ECS0; n=4). In the presence of Y-27632 (0.1-300 μM), phenylephrine-induced tone was reduced in a concentration-dependent manner (IC50 = 7.8±2.1 μM; n=4; Figure 3). After Y-27632 was washed, the same concentration of phenylephrine produced contraction at a magnitude as the control (Figure 3).

Claims

1. Use of a Rho-kinase inhibitor for the manufacture of a medicament for use in the treatment of benign prostatic hyperplasia.
2. Use according to claim 1, wherein the Rho-kinase inhibitor is a compound of formula (I):
0 Rb
II I Ra— C-N-Rc ( I )
wherein Ra is a group of the formula:
Figure imgf000055_0001
in which, in the formulae (a) and (b):
R is hydrogen, alkyl or cycloalkyl, cycloalkylalkyl, phenyl or aralkyl, which optionally have a substituent on the ring, or a group of the formula:
NR'
J ( d )
X
wherein R6 is hydrogen, alkyl or formula : -NR8N"R9 wherein R8 and R9 are the same or different and each is hydrogen, alkyl, aralkyl or phenyl, R7 is hydrogen, alkyl, aralkyl, phenyl, nitro or cyano, or R6 and R' in combination form a heterocyclic group optionally having, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom,
R1 is hydrogen, alkyl or cycloalkyl, cycloalkylalkyl, phenyl or aralkyl, which optionally have a substituent on the ring, or
R and R1 in combination form, together with the adjacent nitrogen atom, a heterocyclic group optionally having, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom, R2 is hydrogen or alkyl, R3 and R4 are the same or different and each is hydrogen, alkyl, aralkyl, halogen, nitro, amino, alkylammo, acylamino, hydroxy, alkoxy, aralkyloxy, cyano, acyl, mercapto, alkylthio, aralkylthio, carboxy, alkoxycarbonyl, carbamoyl, alkylcarbamoyl or azide, and A is a group of the formula:
( e )
Figure imgf000056_0001
wherein R10 and R11 are the same or different and each is hydrogen, alkyl, haloalkyl, aralkyl, hydroxyalkyl, carboxy or alkoxycarbonyl, or R10 and R11 in combination foπ a cycloalkyl group and 1, m and n are each 0 or an integer of 1-3, and in which, in the formula (c): a broken line is a single bond or a double bond,
R5 is hydrogen, hydroxy, alkoxy, alkoxycarbonyloxy, alkanoyloxy or aralkyloxycarbonyloxy, and
L is hydrogen, alkyl, aminoalkyl, mono or dialkylammoalkyl, tetrahydrofurfuryl, carbamoyl alkyl, phthalimidoalkyl, amidino or a group of the formula: 0
I I B-C — ( f)
wherein B is hydrogen, alkyl, alkoxy, aralkyl, aralkyloxy, aminoalkyl, hydroxyalkyl, alkanoyloxyalkyl, alkoxycarbonylalkyl, alpha-aminobenzyl, furyl, pyridyl, phenyl, phenylamino, styryl or imidazopyridyl,
Q1 is hydrogen, halogen, hydroxy, aralkyloxy or thienylmethyl,
W is alkylene,
Q2 is hydrogen, halogen, hydroxy or aralkyloxy, X is alkylene, and
Q3 is hydrogen, halogen, hydroxy, alkoxy, nitro, amino, 2,3-dihydrofuryl or 5-methyl-
3-oxo-2, 3, 4,5-tetrahydropyridazin-6-yl; and Y is a single bond, alkylene or alkenylene; and
Rb is a hydrogen, an alkyl, ah aralkyl, an aminoalkyl or a mono- or di alkyl amino alkyl; and
Re is an optionally substituted heterocycle containing nitrogen; or an isomer thereof; or a pharmaceutically acceptable acid addition salt thereof. 3. Use according to claim 1, wherein th Rho-kinase inhibitor is a compound of formula (FI):
Figure imgf000058_0001
wherein
R12 is hydrogen, chlorine or hydroxy, and when R12 is a hydrogen,
Alk is an alkylene group having 2 to 6 carbon atoms, which optionally has alkyl having 1 to 10 carbon atoms, aryl or aralkyl as a substituent;
R13 is hydrogen;
R is hydrogen, or linear or branched alkyl having 1 to 6 carbon atoms, aryl or aralkyl;
R15 is hydrogen, linear or branched alkyl having 1 to 6 carbon atoms, aryl or aralkyl, or benzoyl, cinnamyl, cinnamoyl, furoyl or a group of the following formula
Figure imgf000058_0002
wherein R is linear or branched alkyl having 1 to 6 carbon atoms or a group of the following formula
NR 17
( k )
NHR1
wherein R17 and R18 are hydrogen or directly bonded to form alkylene having 2 to 4 carbon atoms; or R13 and Rl4 are directly bonded to form alkylene having 4 or less carbon atoms, which is optionally substituted by alkyl having 1 to 10 carbon atoms, phenyl or benzyl, or
R14 and R15 directly or in combination via an oxygen atom form a heterocyclic group together with the adjacent nitrogen atom, and when R12 is chlorine or hydroxy, Alk is an alkylene having 2 to 6 carbon atoms which is optionally substituted by alkyl having 1 to 6 carbon atoms,
R13 and R14 are each hydrogen, linear or branched alkyl having 1 to 6 carbon atoms or directly bonded to each other to foπn ethylene or trimethylene, wherein said alkyl, ethylene or trimethylene group is optionally substituted by alkyl having 1 to 6 carbon atoms; or
R15 is hydrogen, linear or branched alkyl having 1 to 6 carbon atoms or an amidino group; or an isomer thereof; or a phannaceutically acceptable acid addition salt thereof,
4, LTse according to claim 1 , wherein the Rho-kinase inhibitor is a compound of the formula (111):
Figure imgf000059_0001
wherein R1 and R2 are the same or different and each is hydrogen, alkyl, or cycloalkyl, cycloalkylalkyl, phenyl, aralkyl, piperidyl or pyrrolidinyl, any of which may have a substituent on the ring, or a group of the fonmila: NRυ
-C ( IV )
R
wherein R is hydrogen, alkyl, -NR'R" (where R' and R" are the same or different and each is hydrogen, alkyl, aralkyl or phenyl) and R° is hydrogen, alkyl, aralkyl, phenyl, nitro or cyano, or R and R° may in combination form a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom, or R1 and R2 in combination represent alkylidene or phenylalkylidene, or
R1 and R2 form, together with the nitrogen atom, a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom; R3 and R4 are each hydrogen or alkyl; A is a single bond or alkylene; X is =C(R7)- or =N-;
R5 and Rδ together form a group of the formula:
(a) -CRa=CRb-,
(b) -NRa-C(-Rb)-,
(c) -N=CRb-, (d) -C(=Ra)-NRb-,
(e) -CRa=N-, or
(f) -NRa-, wherein Ra and Rb are the same or different and each is hydrogen, halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, -NRcRd [wherein Re and Rd are the same or different and each is hydrogen, alkyl, -COR9, -COOR9' or -S02R9' (where R9 is hydrogen, alkyl, phenyl or aralkyl and R9' is alkyl, phenyl or aralkyl) or Re and Rd form, together with the nitrogen atom to which they are attached, a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom], cyano, azide, optionally substituted hydrazino, -COOR10, or -CONRπR12 (wherein R10"12 are each hydrogen, alkyl, phenyl or aralkyl), or Ra and Rb in combination form an optionally hydrogenated 5- or 6-membered aromatic ring which may have at least one of a nitrogen atom, sulfur atom and oxygen atom provided that, when R5 and Rδ are of the foπnula (b) or (d), Ra and Rb together form an optionally hydrogenated 5- or 6-membered aromatic ring which may have at least one of a nitrogen atom, sulfur atom and oxygen atom;
R7 and R8 are the same or different and each is hydrogen, halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, -NReRf [wherein Re and Rf are the same or different and each is hydrogen, alkyl, -COR9, -COOR9' or -S02R9' (where R9 is hydrogen, alkyl, phenyl or aralkyl and R9' is alkyl, phenyl or aralkyl) or Re and Rf form, together with the nitrogen atom to which they are attached, a heterocyclic ring which may have, in the ring, oxygen atom, sulfur atom or optionally substituted nitrogen atom], cyano, azido, optionally substituted hydrazino, -COOR10 or -CONRπR12 (wherein R10"12 are each hydrogen, alkyl, phenyl or aralkyl); and_ n is 0 or 1 ; with the proviso that, when R2 and Rδ are of the formula (a), X is =C(R7)- and either one of Ra, Rb, R7 and R8 is -NRcRd, -NReRf, azido, optionally substituted hydrazino, -COOR10 or -CONR^'R12, or Ra and Rb together foπn an optionally hydrogenated 5- or 6-membered aromatic ring which may have, in the ring, at least one of nitrogen atom, sulfur atom and oxygen atom; or an isomer thereof; or a pharmaceutically acceptable acid addition salt thereof.
5. Use according to claim 1, wherein the Rho-kinase inhibitor is a compound of formula (III)
Figure imgf000061_0001
wherein R1 and R2 are the same or different and each is hydrogen, alkyl having 1 to 10 carbon atoms, cycloalkyl having 3 to 7 carbon atoms, cycloalkylalkyl, phenyl, aralkyl, piperidyl or pyrrolidinyl which may have substituent on the ring, or R1 and R2 in combination represent alkylidene or phenylalkylidene, or R1 and R2 form, together with the nitrogen atom to which they are attached, a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom;
R3 and R4 are each hydrogen or all y 1; A is a single bond or alkylene; X is =C(R7)- or =N-;
R5 and R" together form a group of the formula (a) -CRa=CRb-,
(b) -NRa-C(=Rb)-,
(c) -N=CRb-,
(d) -C(=Ra)-NRb-,
(e) -CRa=N- or (f) -NRa-, wherein Ra and Rb are the same or different and each is hydrogen, halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, -NRcRd [wherein Re and Rd are the same or different and each is hydrogen, alkyl, -COR9, -COOR9' or -S02R9' (where R9 is hydrogen, alkyl, phenyl or aralkyl and R9' is alkyl, phenyl or aralkyl) or Re and Rd form, together with the nitrogen atom to which they are attached, a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom], cyano, azido, optionally substituted hydrazino, -COOR10 or -CONR! 1R12 (wherein R1(M2 are each hydrogen, alkyl, phenyl or aralkyl), or Ra and Rb in combination form an optionally hydrogenated 5- or 6-membered aromatic ring which may have, in the ring, at least one of a nitrogen atom, sulfur atom and oxygen atom provided that, when R5 and Rδ are of the formula (b) or (d), Ra and Rb together form an optionally hydrogenated 5- or 6-membered aromatic ring which may have at least one of a nitrogen atom, sulfur atom and oxygen atom; R7 and R8 are the same or different and each is hydrogen, halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, -NReRf [wherein Re and Rf are the same or different are each is hydrogen, alkyl, -COR9, -COOR9' or -S02R9' (where R9 is hydrogen, alkyl, phenyl or aralkyl and R9' is alkyl, phenyl or aralkyl) or Re and Rf form, together with the nitrogen atom to which they are attached, a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom], cyano, azido, optionally substituted hydrazino, -COOR10 or -CONR: ιR12 (wherein R10'12 are each hydrogen, alkyl, phenyl or aralkyl); and n is 0 or 1 ; with the proviso that, when R5 and Rδ are of the formula (a), X is =C(R7)- and either one of Ra, Rb, R7 and R8 is -NRcRd, -NReRf, azido, optionally substituted hydrazino, -COOR10 or -CONRnR12, or Ra and Rb together form an optionally hydrogenated 5- or 6-membered aromatic ring which may have, in the ring, at least one of a nitrogen atom, sulfur atom and oxygen atom; or an isomer thereof; or a pharmaceutically acceptable acid addition salt thereof.
6. Use according to claim 1, wherein the Rho-kinase inhibitor is a compound of the formula (V) :
Figure imgf000063_0001
wherein R is hydrogen, alkyl or -NR'R" (wherein R' and R" are the same or different and each is hydrogen, alkyl, aralkyl or phenyl) and R° is hydrogen, alkyl, aralkyl, phenyl, nitro or cyano, or R and R° in combination form a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom;
R2 is hydrogen, alkyl or aralkyl;
R3 and R4 are each hydrogen or alkyl;
A is a single bond or alkylene; X is =C(R7)- or =N-; Rs and Rδ together form a group of the formula
(a) -CRa=CRb-,
(b) -NRa-C(=Rb)-,
(c) -N=CRb-, (d) -C(=Ra)-NRb-,
(e) -CRa=N-, or
(f) or -NRa-, wherein Ra and Rb are the same or different and each is hydrogen, halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, -NRcRd [wherein Re and Rd are the same or different and each is hydrogen, alkyl, -COR9, -COOR9' or -S02R9' (where R9 is hydrogen, alkyl, phenyl or aralkyl and R9' is alkyl, phenyl or aralkyl) or Re and Rd form, together with the nitrogen atom to which they are attached, a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom], cyano, azido, optionally substituted hydrazino, -COOR10 or -CONRπR12 (wherein R10_!2 are each hydrogen, alkyl, phenyl or aralkyl), or
Ra and Rb in combination form an optionally hydrogenated 5- or 6-membered aromatic ring which may have, in the ring, at least one of a nitrogen atom, sulfur atom and oxygen atom provided that, when R3 and Rδ are of the formula (b) or (d), Ra and Rb together form an optionally hydrogenated 5- or 6-membered aromatic ring which may have at least one of a nitrogen atom, sulfur atom and oxygen atom; R7 and Rs are the same or different and each is hydrogen, halogen, alkyl, alkoxy, aralkyl, haloalkyl, nitro, -NReRf [wherein Re and Rf are the same or different are each is hydrogen, alkyl, -COR9, -COOR9' or -S02R9' (where R9 is hydrogen, alkyl, phenyl or aralkyl and R9' is alkyl, phenyl or aralkyl) or Re and Rf form, together with the nitrogen atom to which they are attached, a heterocyclic ring which may have, in the ring, an oxygen atom, sulfur atom or optionally substituted nitrogen atom], cyano, azido, optionally substituted hydrazino, -COOR10 or -CONR! 1R12 (wherein R10"12 are each hydrogen, alkyl, phenyl or aralkyl); and n is 0 or 1 ; with the proviso that, when R5 and Rδ are of the formula (a), X is =C(R7)- and either one of Ra, Rb, R7 and R8 is -NRcRd, -NReRf, azido, optionally substituted hydrazino, -COOR10 or -CONRnR12, or Ra and Rb together form an optionally hydrogenated 5- or 6-membered aromatic ring which may have, in the ring, at least one of a nitrogen atom, sulfur atom and oxygen atom; or an isomer thereof; or a pharmaceutically acceptable acid addition salt thereof.
7. Use according to claim 1, wherein the Rho-kinase inhibitor is a compound of any one of formulae (VI) to (XI):
Figure imgf000065_0001
Figure imgf000065_0002
Figure imgf000066_0001
or is a pharmaceutically acceptable salt thereof,
8. Use according to claim 1, wherein the Rho-kinase inhibitor is (÷)-(R)- ran5-4-(l-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride.
9. A method of treating benign prostatic hyperplasia in a man suffering therefrom, which method comprises the step of administering thereto a therapeutically effective amount of a Rho-kinase inhibitor.
10. An agent for treating benign prostatic hyperplasia, comprising a Rho- kinase inhibitor.
PCT/GB2004/000139 2003-01-16 2004-01-16 Use of rho-kinase inhibitorrs for treatment of benign prostatic hyperplasia WO2004062666A2 (en)

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