WO2000035435A9 - Technique de prevention du rejet de greffe par utilisation d'un inhibiteur du mek - Google Patents

Technique de prevention du rejet de greffe par utilisation d'un inhibiteur du mek

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Publication number
WO2000035435A9
WO2000035435A9 PCT/US1999/029591 US9929591W WO0035435A9 WO 2000035435 A9 WO2000035435 A9 WO 2000035435A9 US 9929591 W US9929591 W US 9929591W WO 0035435 A9 WO0035435 A9 WO 0035435A9
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WO
WIPO (PCT)
Prior art keywords
methyl
phenylamino
iodo
benzamide
bromo
Prior art date
Application number
PCT/US1999/029591
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English (en)
Other versions
WO2000035435A1 (fr
Inventor
Richard Buell Gilbertsen
Original Assignee
Warner Lambert Co
Richard Buell Gilbertsen
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Warner Lambert Co, Richard Buell Gilbertsen filed Critical Warner Lambert Co
Priority to HU0104607A priority Critical patent/HUP0104607A3/hu
Priority to IL14323199A priority patent/IL143231A0/xx
Priority to AU21805/00A priority patent/AU2180500A/en
Priority to KR1020017007397A priority patent/KR20010101203A/ko
Priority to JP2000587756A priority patent/JP2002532414A/ja
Priority to EP99966203A priority patent/EP1140046A1/fr
Priority to CA002346684A priority patent/CA2346684A1/fr
Publication of WO2000035435A1 publication Critical patent/WO2000035435A1/fr
Publication of WO2000035435A9 publication Critical patent/WO2000035435A9/fr

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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
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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
    • 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
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/136—Amines having aromatic rings, e.g. ketamine, nortriptyline having the amino group directly attached to the aromatic ring, e.g. benzeneamine
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/18—Sulfonamides
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/196—Carboxylic acids, e.g. valproic acid having an amino group the amino group being directly attached to a ring, e.g. anthranilic acid, mefenamic acid, diclofenac, chlorambucil
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
    • 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
    • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
    • A61K31/381—Heterocyclic compounds having sulfur as a ring hetero atom having five-membered rings
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • 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
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • 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
    • 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
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4453—Non condensed piperidines, e.g. piperocaine only substituted in position 1, e.g. propipocaine, diperodon
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • This invention relates to a method for preventing mammals that have undergone an organ, tissue, cell, or limb transplant from rejecting the transplant.
  • the method comprises administering an effective amount of a MEK inhibitor, ideally a phenyl amine derivative.
  • Transplantation of organs and limbs has become a common procedure to treat mammals that have diseased organs, or have been the victims of accidents or other traumas that have resulted in loss of organ function or limbs.
  • Routinely transplanted organs include the liver, kidney, pancreas, and lung.
  • Other types of transplantation are also common, such as skin, bone marrow, and small intestine.
  • Limb transplantation includes fingers, toes, and larger limbs such as arms.
  • Transplant rejection involves both humoral immunity and a cell-mediated immune reaction, or a delayed type hypersensitivity response in a mammal patient.
  • the patient receives an immunosuppressant agent to control or at least diminish the rejection response.
  • immunosuppressants are currently available for clinical use, each is associated with adverse side effects.
  • cyclosporine is a cyclic peptide which inhibits the T-cell production of several cytokines, including IL-2 (interleukin-2), IL-3, IL-4, IL-5, IFN- ⁇ , and probably other lymphokines. Cyclosporine is used extensively for the prophylaxis of organ rejection in allogeneic kidney, liver, and heart transplants.
  • Cyclosporine is often used in combination with other immunosuppressant agents such as corticosteroids or azathioprine. Unfortunate side effects associated with cyclosporine include nephrotoxicity, hepatotoxicity, severe renal dysfunction, tremor, hirsutism, and hypertension.
  • Another immunosuppressive agent is mycophenolate mofetil, the 2-morpholinoethyl ester of mycophenolic acid that is frequently used by patients receiving allogeneic renal transplants.
  • This agent is often used in combination with other immunosuppressive agents, including cyclosporine and corticosteroids.
  • mycophenolate mofetil can cause side effects, most notably the increased risk of developing lymphomas and other malignancies, particularly concerning the skin. Adverse effects on fetal development have also been noted.
  • immunosuppressant agents not only useful for treating or preventing transplant rejection but also with less severe side effects than those associated with existing therapy.
  • compounds that are MEK inhibitors are useful for preventing rejection of transplants in mammals. Moreover, these potent immunosuppressive agents may have fewer or no adverse side effects.
  • the compounds to be administered according to this invention are described in US Patent No. 5,525,625, and in WO 98/37881, both of which are incorporated herein by reference.
  • This invention provides a method for the prevention of rejection in a mammal of transplanted organs, tissues, and limbs, said method including administering an effective immunosuppressive amount of a selective MEK inhibitor to a subject who has undergone a transplant or is scheduled to undergo a transplant.
  • the MEK inhibitor administered is 2-(2-amino-3-methoxyphenyl)-4-oxo-4H-[l]benzopyran, also known as "98059", as described in US 5,525,625.
  • the immunosuppressive agent administered is a phenyl amine compound of Formula I or II:
  • Ri is hydrogen, hydroxy, Cj-Cg alkyl, Ci -Cg alkoxy, halo, trifluoromethyl, or CN.
  • R2 is hydrogen.
  • R3, R4, and R5 are independently selected from hydrogen, hydroxy, halo, trifluoromethyl, Cj-Cg alkyl,
  • n - 9- 9 is hydrogen, hydroxy, COOH, or NR1 Q I 1 ; n is 0-4; m is 0 or 1.
  • Each of R ⁇ Q and R ⁇ ⁇ is independently selected from hydrogen and C1 -Cg alkyl, or taken together with the nitrogen to which they are attached can complete a 3-10 member cyclic ring optionally containing 1, 2, or 3 additional heteroatoms selected from O, S, NH, orN-(C ⁇ -Cg alkyl).
  • Z is COOR7, tetrazolyl, CONRgR , CONHNRIQRI 1, or CH2OR7.
  • R6 and R7 independently are hydrogen, Ci -Cg alkyl, C2-Cg alkenyl,
  • C3-C10 (cycloalkyl optionally containing one, two, or three heteroatoms selected from O, S, NH, or N alkyl); or Rg and R7 together with the nitrogen to which they are attached complete a 3-10 member cyclic ring optionally containing 1, 2, or 3 additional heteroatoms selected from O, S, NH, or N alkyl.
  • any of the foregoing alkyl, alkenyl, aryl, heteroaryl, heterocychc, and alkynyl groups can be unsubstituted or substituted by halo, hydroxy, C ⁇ -C 6 alkoxy, amino, nitro, d-C 4 alkylamino, di(C]-C )alkylamino, C 3 -C 6 cycloalkyl, phenyl, phenoxy, C 3 -C 5 heteroaryl, or C 3 -C 5 heteroaryloxy; or a pharmaceutically acceptable salt, ester, amide, or prodrug thereof.
  • Preferred embodiments of Formula (I) have a structure wherein: (a) Ri is hydrogen, methyl, methoxy, fluoro, chloro, or bromo; (b) R 2 is hydrogen; (c) R 3 , P , and R 5 independently are hydrogen, fluoro, chloro, bromo, iodo, methyl, methoxy, or nitro; (d) R 10 and Rn independently are hydrogen or methyl; (e) Z is COOR 7 , tetrazolyl, CO FER ? , CONHNR 10 R] 1 , or CH 2 OR 7 ; Re and R 7 independently are hydrogen, C 1 -4 alkyl, heteroaryl, or C 3 .
  • Examples of preferred embodiments include methods comprising a MEK inhibitor selected from Formula (I) Compound Table below.
  • the MEK inhibitor is a compound of Formula II
  • Ri a is hydrogen, hydroxy, Ci -Cg alkyl, Ci -Cg alkoxy, halo, trifluoromethyl, or CN.
  • R2 a is hydrogen.
  • Each ofR3 a , R ⁇ a , and R5 a is independently selected from hydrogen, hydroxy, halo, trifluoromethyl, Ci-Cg alkyl, Ci-Cg alkoxy, nitro, CN, and (O or NH) m -(CH2) n -R9 a .
  • R9a is hydrogen, hydroxy, CO2H or NRj Q a ⁇ ⁇ a ; n is 0-4; and m is 0 or 1.
  • Each of Ri 0a and Rl la * s independently hydrogen or Cj-Cg alkyl, or taken together with the nitrogen to which they are attached can complete a 3- to 10-member cyclic ring optionally containing one, two, or three additional heteroatoms selected from O, S, NH, or N-(C ⁇ -Cg alkyl).
  • R 6a is hydrogen, C ⁇ Cg alkyl, (CO)-(Ci -Cg alkyl), aryl, aralkyl, or C3-C10 cycloalkyl.
  • R7 a is hydrogen, Cj-Cg alkyl,
  • any ofthe alkyl, alkenyl, aryl, heterocychc, and alkynyl groups can be unsubstituted or substituted by halo, hydroxy, C C ⁇ alkoxy, amino, nitro, C]-C alkylamino, di(Cj- C 4 )alkylamino, C 3 -C 6 cycloalkyl, phenyl, phenoxy, C 3 -C 5 heteroaryl, or C 3 -C 5 heteroaryloxy; or Rg a and R7 a taken together with the N to which they are attached can complete a 5- to 10-membered cyclic ring, optionally containing one, two, or three additional heteroatoms selected from O, S, or NR1 Oa l
  • Preferred embodiments of Formula (II) are those structures wherein: (a) R la is H, methyl, fluoro, or chloro; (b) R a is H; R 3a , ia, and R 5a are each H, Cl, nitro, or F; (c) R a is H; (d) R a is methyl, ethyl, 2-propenyl, propyl, butyl, pentyl, hexyl, cyclopropylmethyl, cyclobutylmethyl, cyclopropylmethyl, or cyclopropylethyl; (e) the 4' position is I, rather than Br; (f) ⁇ a is F at the 4 position, para to the CO-N-R f ⁇ -OR ⁇ group and meta to the bridging nitrogen; (f) R3a or R 5a is F; (g) at least one of R3 a , R- ⁇ a , and R 5a is F; (h) R la is
  • the MEK inhibitor is a compound selected from Formula (II) Compound Table below.
  • a compound selected from the following is administered to a patient (ie, a mammal) in an amount that is effective to prevent or treat transplant rejection:
  • the benzoic acid derivative of PD 198306 is 2-(2 -Methyl -4-iodophenylamino)-3,4,5-trifluorobenzoic acid.
  • Additional preferred compounds include 2-(2-chloro-4- iodophenylamino)-5-chloro-N-cyclopropylmethoxy -3,4-difluorobenzamide (PD 297189), 2-(4-iodophenylamino)-N-cyclopropylmethoxy-5-chloro-3,4- difluorobenzamide (PD 297190), 2-(4-iodophenylamino)-5-chloro-3,4- difluorobenzoic acid (PD 296771), 2-(2-chloro-4-iodophenylamino)-5-chloro- 3,4-difluorobenzoic acid (PD 296770), 5-chloro-3,4-difluoro-2-(4-iodo-2- methyl
  • Another preferred method according to this invention comprises administering to a mammal that has undergone a transplant, or is about to undergo a transplant, the immunosuppressive agent which is 2-(2-chloro- 4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide.
  • Still another preferred method according to this invention employs the compound which is 2-(2-methyl-4-iodophenylamino)-N-cyclopropylmethoxy- 3,4,5-trifluorobenzamide.
  • the invention further provides methods of synthesis and synthetic intermediates.
  • Figure 1 shows the dose response ability of 2-(2-chloro-4- iodophenylamino)-N-cycloproplymethoxy-3,4-difluorobenzamide (PD 184352) to inhibit the cellular production of interleukin-2 (IL-2) in human peripheral blood mononuclear cells stimulated with concanavalin A (Con A).
  • Figure 2 shows the dose response ability of PD 184352 to inhibit the cellular production of IL-2 in human peripheral blood mononuclear cells stimulated with anti-CD3 plus anti-CD28.
  • Figure 3 shows the dose response ability of PD 184352 to inhibit cellular production of interferon- ⁇ (IFN- ⁇ ) in cells stimulated with Con A.
  • Figure 4 shows the ability of PD 184352 to suppress the human mixed lymphocyte reaction (MLR) as measured by the uptake of tritiated thymidine (3H-TDR).
  • MLR human mixed lymphocyte reaction
  • Figure 5 shows the dose response ability of PD 184352 to inhibit Con A induced T-cell proliferation.
  • Figure 6 shows the dose response ability of PD 184352 to inhibit T-cell proliferation induced by phytohemagglutinin (PHA).
  • PHA phytohemagglutinin
  • Figure 7 shows the lack of toxicity of PD 184352 in cells.
  • Figure 8 shows the inhibitory activity of several MEK inhibitors against MLR, IFN-gamma, and IL-2, and the ability ofthe compounds to inhibit PHA and
  • Figure 9 shows the relative IL-2 suppressive activity of several phenyl amine compounds compared to rolipram and to dexamethasone (Dex).
  • Figure 10 shows the comparative activity of several phenyl amines, rolipram, and dexamethasone to suppress production of IFN- ⁇ .
  • Figure 11 shows the human MLR suppressive activity of several phenyl amine MEK inhibitors compared to dexamethasone.
  • Figure 12 shows the ability of several phenyl amine MEK inhibitors to suppress human T-cell proliferation, compared to dexamethasone.
  • Figure 13 shows the percent cell death caused by several phenyl amine MEK inhibitors in the human MTT test.
  • This invention provides a method for the prophylaxis of rejection of transplants in mammals, as well as control and maintenance of grafts.
  • the invention is practiced by administering to a mammal that has undergone a transplant, or to a patient who is scheduled to undergo a transplant, an effective immunosuppressive amount of a selective MEK inhibitor to prevent or control the rejection ofthe transplanted organ, limb, cell(s), or tissue.
  • a selective MEK inhibitor that is described in WO 98/37881.
  • the method is ideally suited to prevent and control of rejection of kidney, liver, lung, and limb transplants.
  • the mammals to be treated according to this invention are patients who have undergone a transplant of an organ, a tissue, a limb, or cells, or who are about to undergo such transplant.
  • Those skilled in the medical art are readily able to identify individual patients who are in need of an immunosuppressive agent in order to prevent or control the rejection of a foreign organ, limb, cell, or tissue.
  • the compounds ofthe present invention are MEK inhibitors.
  • a MEK inhibitor is a compound that shows MEK inhibition when tested in the assays titled "Enzyme Assays" in United States Patent Number 5,525,625, column 6, beginning at line 35.
  • a MEK inhibitor is 2-(2-amino-3-methoxyphenyl)-4-oxo-4H-[l]benzopyran.
  • a compound is a MEK inhibitor if a compound shows activity in the assay titled "Cascade Assay for Inhibitors ofthe MAP Kinase Pathway," column 6, line 36 to column 7, line 4 ofthe United States Patent Number 5,525,625 and/or shows activity in the assay titled "In Vitro MEK Assay” at column 7, lines 4 to 27 ofthe above-referenced patent.
  • aryl means a cyclic, bicyclic, or tricyclic aromatic ring moiety having from five to twelve carbon atoms.
  • typical aryl groups include phenyl, naphthyl, and fluorenyl.
  • the aryl may be substituted by one, two, or three groups selected from fluoro, chloro, bromo, iodo, alkyl, hydroxy, alkoxy, nitro, amino, alkylamino, or dialkylamino.
  • Typical substituted aryl groups include 3-fluorophenyl, 3,5-dimethoxyphenyl, 4-nitronaphthyl, 2-methyl-4-chloro-7-aminofluorenyl, and the like.
  • aryloxy means an aryl group bonded through an oxygen atom, for example phenoxy, 3-bromophenoxy, naphthyloxy, and 4-methyl- 1 -fluorenyloxy.
  • Heteroaryl means a cyclic, bicyclic, or tricyclic aromatic ring moiety having from four to eleven carbon atoms and one, two, or three heteroatoms selected from O, S, or N. Examples include furyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, thiazolyl, oxazolyl, xanthenyl, pyronyl, indolyl, pyrimidyl, naphthyridyl, pyridyl, benzinnidazolyl, and triazinyl.
  • heteroaryl groups can be unsubstituted or substituted by one, two, or three groups selected from fluoro, chloro, bromo, iodo, alkyl, hydroxy, alkoxy, nitro, amino, alkylamino, or dialkylamino.
  • substituted heteroaryl groups include chloropyranyl, methylthienyl, fluoropyridyl, amino- 1,4-benzisoxazinyl, nitroisoquinolinyl, and hydroxyindolyl.
  • heteroaryl groups can be bonded through oxygen to make heteroaryloxy groups, for example thienyloxy, isothiazolyloxy, benzofuranyloxy, pyridyloxy, and 4-methylisoquinolinyloxy.
  • alkyl means straight and branched chain aliphatic groups. Typical alkyl groups include methyl, ethyl, isopropyl, tert.-butyl,
  • alkyl groups can be unsubstituted or substituted by halo, hydroxy, alkoxy, amino, alkylamino, dialkylamino, cycloalkyl, aryl, aryloxy, heteroaryl, or heteroaryloxy, as those terms are defined herein.
  • Typical substituted alkyl groups include chloromethyl, 3-hydroxypropyl, 2-dimethylaminobutyl, and 2-(hydroxymethylamino)ethyl.
  • Examples of aryl and aryloxy substituted alkyl groups include phenylmethyl, 2-phenylethyl, 3 -chlorophenylmethyl, 1 , 1 -dimethyl-3 -(2-nitrophenoxy)butyl, and 3,4,5-trifluoronaphthylmethyl.
  • Examples of alkyl groups substituted by a heteroaryl or heteroaryloxy group include thienylmethyl, 2-furylethyl, 6-furyloxyoctyl, 4-methylquinolyloxymethyl, and 6-isothiazolylhexyl.
  • Cycloalkyl substituted alkyl groups include cyclopropylmethyl, 2-cyclohexyethyl, piperidyl- 2-methyl, 2-(piperidin-l-yl)-ethyl, 3-(morpholin-4-yl)propyl.
  • Alkenyl means a straight or branched carbon chain having one or more double bonds. Examples include but-2-enyl, 2-methyl-prop-2-enyl, 1,1-dimethyl- hex-4-enyl, 3-ethyl-4-methyl-pent-2-enyl, and 3-isopropyl-pent-4-enyl.
  • alkenyl groups can be substituted with halo, hydroxy, alkoxy, amino, alkylamino, dialkylamino, aryl, aryloxy, heteroaryl, or heteroyloxy, for example 2-bromoethenyl, 3-hydroxy-2-butenyl, 1-aminoethenyl, 3-phenylprop-2-enyl,
  • 6-thienyl-hex-2-enyl 2-furyloxy-but-2-enyl, and 4-naphthyloxy-hex-2-enyl.
  • Alkynyl means a straight or branched carbon chain having at least one triple bond. Typical alkynyl groups include prop-2-ynyl, 2-methyl-hex-5-ynyl, 3,4-dimethyl-hex-5-ynyl, and 2-ethyl-but-3-ynyl.
  • the alkynyl groups can be substituted as the alkyl and alkenyl groups, for example, by aryl, aryloxy, heteroaryl, or heteroaryloxy, for example 4-(2-fluorophenyl)-but-3-ynyl, 3-methyl-5-thienylpent-4-ynyl, 3-phenoxy-hex-4-ynyl, and 2-furyloxy-3-methyl- hex-4-ynyl.
  • the alkenyl and alkynyl groups can have one or more double bonds or triple bonds, respectively, or a combination of double and triple bonds.
  • typical groups having both double and triple bonds include hex-2-en- 4-ynyl, 3-methyl-5-phenylpent-2-en-4-ynyl, and 3-thienyloxy-hex-3-en-5-ynyl.
  • cycloalkyl means a nonaromatic ring or fused rings. Examples include cyclopropyl, cyclobutyl, cyclopenyl, cyclooctyl, bicycloheptyl, adamantyl, and cyclohexyl.
  • the ring can optionally contain one, two, or three heteroatoms selected from O, S, or N.
  • Such groups include tetrahydrofuryl, tetrahydropyrrolyl, octahydrobenzofuranyl, morpholinyl, piperazinyl, pyrrohdinyl, piperidinyl, octahydroindolyl, and octahydrobenzothiofuranyl.
  • the cycloalkyl groups can be substituted with the same substituents as an alkyl and alkenyl groups, for example, halo, hydroxy, aryl, and heteroaryloxy. Examples include 3 -hydroxy cyclohexyl, 2-aminocyclopropyl, 2-phenylpyrrolidinyl, and 3-thienylmorpholin-l-yl.
  • the term "maintenance" means controlling the tendency of a mammal to reject a cell, organ, limb, or tissue that has been transplanted into or onto the mammals body.
  • the method is practiced by administering an amount of a selective MEK inhibitor that is effective to prevent or control the rejection.
  • a selective MEK 1 or MEK 2 inhibitor has an IC 50 for MEK 1 or MEK 2 that is at least one-fiftieth (1/50) that of its IC 50 for one ofthe above-named other enzymes.
  • a selective inhibitor has an IC 50 that is at least 1/100, more preferably 1/500, and even more preferably 1/1000, 1/5000, or less than that of its ICso or one or more ofthe above- named enzymes.
  • the MEK inhibitors ofthe present method can be administered to a patient as part of a pharmaceutically acceptable composition.
  • the compositions can be administered to humans and animals either orally, rectally, parenterally (intravenously, intramuscularly, or subcutaneously), intracisternally, intravaginally, intraperitoneally, intravesically, locally (powders, ointments, or drops), or as a buccal or nasal spray.
  • compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • suitable aqueous and nonaqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersions and by the use of surfactants.
  • compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents.
  • adjuvants such as preserving, wetting, emulsifying, and dispensing agents.
  • Prevention ofthe action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.
  • isotonic agents for example sugars, sodium chloride, and the like.
  • Prolonged absorption ofthe injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or
  • fillers or extenders as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid
  • binders as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia
  • humectants as for example, glycerol
  • disintegrating agents as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate
  • solution retarders as for example paraffin
  • absorption accelerators as for example, quaternary ammonium compounds
  • wetting agents such as sodium citrate or dicalcium phosphate
  • fillers or extenders as for example
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethyleneglycols, and the like.
  • Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and others well- known in the art. They may contain opacifying agents, and can also be of such composition that they release the active compound or compounds in a certain part ofthe intestinal tract in a delayed manner. Examples of embedding compositions which can be used are polymeric substances and waxes.
  • the active compounds can also be in micro-encapsulated form, if appropriate, with one or more ofthe above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3 -butyl eneglycol, dimethylformamide, oils, in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol, polyethyleneglycols, and fatty acid esters of sorbitan or mixtures of these substances, and the like.
  • the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • Suspensions in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.
  • Compositions for rectal administrations are preferably suppositories which can be prepared by mixing the compounds ofthe present invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethyleneglycol, or a suppository wax, which are solid at ordinary temperatures but liquid at body temperature and therefore, melt in the rectum or vaginal cavity and release the active component.
  • Dosage forms for topical administration of a compound of this invention include ointments, powders, sprays, and inhalants.
  • the active component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants as may be required.
  • Ophthalamic formulations, eye ointments, powders, and solutions are also contemplated as being within the scope of this invention.
  • the compounds ofthe present method can be administered to a patient at dosage levels in the range of about 0.1 to about 1000 mg per day. For a normal human adult having a body weight of about 70 kg, a dosage in the range of about 0.01 to about 100 mg per kg of body weight per day is preferable. The specific dosage used, however, can vary.
  • the dosage can depend on a numbers of factors including the requirements ofthe patient, the severity ofthe condition being treated, and the pharmacological activity ofthe compound being used. The determination of optimum dosages for a particular patient is well- known to those skilled in the art.
  • the compounds of the present method can be administered as pharmaceutically acceptable salts, esters, amides, or prodrugs.
  • salts refers to those carboxylate salts, amino acid addition salts, esters, amides, and prodrugs ofthe compounds ofthe present invention which are, within the scope of sound medical judgment, suitable for contact with the tissues of patients without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, ofthe compounds ofthe invention.
  • salts refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds ofthe present invention.
  • salts can be prepared in situ during the final isolation and purification ofthe compounds or by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed.
  • Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactiobionate and laurylsulphonate salts, and the like.
  • alkali and alkaline earth metals such as sodium, lithium, potassium, calcium, magnesium and the like
  • nontoxic ammonium, quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
  • ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like See, for example, S.M. Berge, et al., "Pharmaceutical Salts," J Pharm. Sci.. 1977;66:1-19 which is incorporated herein by reference.)
  • esters ofthe compounds of this invention examples include Ci -Cg alkyl esters wherein the alkyl group is a straight or branched chain. Acceptable esters also include C5-C7 cycloalkyl esters as well as arylalkyl esters such as, but not limited to benzyl. Ci -C4 alkyl esters are preferred. Esters ofthe compounds ofthe present invention may be prepared according to conventional methods.
  • Examples of pharmaceutically acceptable, non-toxic amides ofthe compounds of this invention include amides derived from ammonia, primary C ⁇ -Cg alkyl amines and secondary C 1 -Cg dialkyl amines wherein the alkyl groups are straight or branched chain. In the case of secondary amines the amine may also be in the form of a 5 or 6 membered heterocycle containing one nitrogen atom. Amides derived from ammonia, C1 -C3 alkyl primary amines and C1-C2 dialkyl secondary amines are preferred. Amides ofthe compounds ofthe invention may be prepared according to conventional methods.
  • prodrug refers to compounds that are rapidly transformed in vivo to yield the parent compound ofthe above formula, for example, by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and
  • the compounds ofthe present method can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like.
  • the solvated forms are considered equivalent to the unsolvated forms for the purposes ofthe present invention.
  • the 2-(4-bromo and 4-iodo phenylamino)-benzoic acid derivatives of Formula (I ) can be prepared from commercially available starting materials utilizing synthetic methodologies well-known to those skilled in organic chemistry. A typical synthesis is carried out by reacting a 4-bromo or 4-iodo aniline with a benzoic acid having a leaving group at the 2-position to give a 2-(phenylamino)-benzoic acid. This process is depicted in Scheme 1.
  • L is a leaving group, for example halo such as fluoro.
  • the reaction of aniline and the benzoic acid derivative generally is accomplished by mixing the benzoic acid with an equimolar quantity or excess of the aniline in an unreactive organic solvent such as tetrahydrofuran or toluene, in the presence of a base such as lithium diisopropylamide, n-butyl lithium, sodium hydride, triethylamine, and Hunig's base.
  • a base such as lithium diisopropylamide, n-butyl lithium, sodium hydride, triethylamine, and Hunig's base.
  • the reaction generally is carried out at a temperature of about -78°C to about 100°C, and normally is complete within about 2 hours to about 4 days.
  • the product can be isolated by removing the solvent, for example by evaporation under reduced pressure, and further purified, if desired, by standard methods such as chromatography, crystallization, or distillation.
  • the 2-(phenylamino)-benzoic acid (e.g., Formula I, where R7 is hydrogen) can be reacted with an organic or inorganic base such as pyridine, triethylamine, calcium carbonate, or sodium hydroxide to produce a pharmaceutically acceptable salt.
  • organic or inorganic base such as pyridine, triethylamine, calcium carbonate, or sodium hydroxide.
  • the free acids can also be reacted with an alcohol ofthe formula HOR7
  • EEDQ 2-ethoxy- 1 -ethoxycarbonyl- 1 ,2-dihydroquinoline
  • DCC 1,3 -dicyclohexylcarbodiimide
  • PyBrOP bromo-tris(pyrrolidino)- phosphonium hexafluorophosphate
  • PyBOP benzotriazolyloxy tripyrrolidino phosphonium hexafluorophosphate
  • the phenylamino benzoic acid and alcohol derivative normally are mixed in approximately equimolar quantities in an unreactive organic solvent such as dichloromethane, tetrahydrofuran, chloroform, or xylene, and an equimolar quantity ofthe coupling reagent is added.
  • a base such as triethylamine or dusopropylethylamine can be added to act as an acid scavenger if desired.
  • the coupling reaction generally is complete after about 10 minutes to 2 hours, and the product is readily isolated by removing the reaction solvent, for instance by evaporation under reduced pressure, and purifying the product by standard methods such as chromatography or crystallizations from solvents such as acetone, diethyl ether, or ethanol.
  • the benzamides ofthe invention are readily prepared by reacting the foregoing benzoic acids with an amine ofthe formula HNR6R7.
  • the reaction is carried out by reacting approximately equimolar quantities ofthe benzoic acid and amine in an unreactive organic solvent in the presence of a coupling reagent.
  • Typical solvents are chloroform, dichloromethane, tetrahydrofuran, benzene, toluene, and xylene.
  • Typical coupling reagents include DCC, EEDQ, PyBrOP, and PyBOP. The reaction is generally complete after about 10 minutes to about 2 hours when carried out at a temperature of about 0°C to about 60°C.
  • the product amide is readily isolated by removing the reaction solvent, for instance by evaporation, and further purification can be accomplished by normal methods such as chromatography, crystallization, or distillation.
  • benzyl alcohols ofthe invention compounds of Formula (I) where Z is CH2OR6 and Rg is hydrogen, are readily prepared by reduction ofthe corresponding benzoic acid according to the following Scheme 2.
  • Typical reducing agents commonly employed include borane in tetrahydrofuran.
  • the reduction normally is carried out in an unreactive organic solvent such as tetrahydrofuran, and generally is complete within about 2 hours to about 24 hours when conducted at a temperature of about 0°C to about 40°C.
  • Aqueous HCl (10%) was added to the concentrate, and the solution was extracted with dichloromethane.
  • the organic phase was dried (MgSO4) and then boiled over a steambath to low volume and cooled to room temperature.
  • the off-white fibers were collected by vacuum filtration, rinsed with hexanes, and vacuum-oven dried. (76°C; ca.
  • Example 2-30 By following the general procedure of Example 1, the following benzoic acids and salts of Formula (I) were prepared.
  • the reaction mixture was transferred to a 2-dram vial and diluted with 2 mL of ethyl acetate.
  • the organic layer was washed with 3 mL of distilled water and the water layer washed again with 2 mL of ethyl acetate.
  • the combined organic layers were allowed to evaporate to dryness in an open fume hood.
  • the residue was taken up in 2 mL of 50% acetonitrile in water and injected on a semi-prep reversed phase column (10 mm x 25 cm, 5 ⁇ M spherical silica, pore size 115 A derivatized with C-18, the sample was eluted at 4.7 mL/min with a linear ramp to 100% acetonitrile over 8.5 minutes. Elution with 100% acetonitrile continued for 8 minutes). Fractions were collected by monitoring at 214 nM. The residue was dissolved in chloroform and transferred to a preweighed vial, evaporated, and weighed again to determine
  • Step b Preparation of 5-chloro-2-fluoro-benzaldehyde oxime
  • the 4-bromo and 4-iodo phenylamino benzhydroxamic acid derivatives of Formula II can be prepared from commercially available starting materials utilizing synthetic methodologies well-known to those skilled in organic chemistry. A typical synthesis is carried out by reacting a 4-bromo or 4-iodo aniline with a benzoic acid having a leaving group at the 2-position to give a phenylamino benzoic acid, and then reacting the benzoic acid phenylamino derivative with a hydroxylamine derivative (Scheme 3).
  • L is a leaving group, for example halo such as fluoro, chloro, bromo or iodo, or an activated hydroxy group such as a diethylphosphate, trimethylsilyloxy, p-nitrophenoxy, or phenylsulfonoxy.
  • halo such as fluoro, chloro, bromo or iodo
  • an activated hydroxy group such as a diethylphosphate, trimethylsilyloxy, p-nitrophenoxy, or phenylsulfonoxy.
  • the reaction of aniline and the benzoic acid derivative generally is accomplished by mixing the benzoic acid with an equimolar quantity or excess of the aniline in an unreactive organic solvent such as tetrahydrofuran, or toluene, in the presence of a base such as lithium diisopropylamide, n-butyl lithium, sodium hydride, and sodium amide.
  • the reaction generally is carried out at a temperature of about -78°C to about 25 °C, and normally is complete within about 2 hours to about 4 days.
  • the product can be isolated by removing the solvent, for example by evaporation under reduced pressure, and further purified, if desired, by standard methods such as chromatography, crystallization, or distillation.
  • phenylamino benzoic acid next is reacted with a hydroxylamine derivative HNRg a ' 7a m the presence of a peptide coupling reagent.
  • Hydroxylamine derivatives that can be employed include methoxylamine,
  • Typical coupling reagents include 2-ethoxy-l-ethoxycarbonyl-l,2-dihydroquinoline (EEDQ), 1 ,3 -dicyclohexylcarbodiimide (DCC), bromo-tris(pyrrolidino)-phosphonium hexafluorophosphate (PyBrOP) and (benzotriazolyloxy)tripyrrolidino phosphonium hexafluorophosphate (PyBOP).
  • EEDQ 2-ethoxy-l-ethoxycarbonyl-l,2-dihydroquinoline
  • DCC 1 ,3 -dicyclohexylcarbodiimide
  • PyBrOP bromo-tris(pyrrolidino)-phosphonium hexafluorophosphate
  • PyBOP benzotriazolyloxy
  • the phenylamino benzoic acid and hydroxylamino derivative normally are mixed in approximately equimolar quantities in an unreactive organic solvent such as dichloromethane, tetrahydrofuran, chloroform, or xylene, and an equimolar quantity ofthe coupling reagent is added.
  • a base such as triethylamine or dusopropylethylamine can be added to act as an acid scavenger if desired.
  • the coupling reaction generally is complete after about 10 minutes to 2 hours, and the product is readily isolated by removing the reaction solvent, for instance by evaporation under reduced pressure, and purifying the product by standard methods such as chromatography or crystallizations from solvents such as acetone, diethyl ether, or ethanol.
  • An alternative method for making the invention compounds involves first converting a benzoic acid to a hydroxamic acid derivative, and then reacting the hydroxamic acid derivative with an aniline. This synthetic sequence is depicted in Scheme 4.
  • Yet another method for making invention compounds comprises reacting a phenylamino benzhydroxamic acid with an ester forming group as depicted in Scheme 5.
  • L is a leaving group such as halo
  • a base is triethylamine or diisopropylamine.
  • Aqueous HCl (10%) was added to the concentrate, and the solution was extracted with dichloromethane.
  • the organic phase was dried (MgSO4) and then concentrated over a steambath to low volume (10 mL) and cooled to room temperature.
  • the off-white fibers which formed were collected by vacuum filtration, rinsed with hexane, and dried in a vacuum-oven (76°C; ca.
  • This intermediate was dissolved in 25 mL of ethanolic hydrogen chloride, and the solution was allowed to stand at room temperature for 15 minutes.
  • the reaction mixture was concentrated in vacuo to a brown oil that was purified by flash silica chromatography. Elution with a gradient (100 % dichloromethane to 0.6 % methanol in dichloromethane) afforded 0.2284 g of a light-brown viscous oil.
  • the solid product was partitioned between diethyl ether (150 mL) and aq. HCl (330 mL, pH 0).
  • the aqueous phase was extracted with a second portion (100 mL) of diethyl ether, and the combined ethereal extracts were washed with 5% aqueous sodium hydroxide (200 mL) and water (100 mL, pH 12).
  • These combined alkaline aqueous extractions were acidified to pH 0 with concentrated aqueous hydrochloric acid.
  • the resulting suspension was extracted with ether (2 x 200 mL).
  • the cold bath was subsequently removed, and the reaction mixture stined for 18 hours.
  • the mixture was concentrated, and the concentrate was treated with 100 mL of dilute (10%) aqueous hydrochloric acid.
  • the resulting suspension was extracted with ether (2 x 150 mL), and the combined organic extractions were dried (MgSO4) and concentrated in vacuo to give an orange solid.
  • the solid was triturated with boiling dichloromethane, cooled to ambient temperature, and collected by filtration.
  • the reaction mixture was stined at 24°C for 10 minutes, and then was concentrated to dryness in vacuo.
  • the concentrate was suspended in 100 mL of 10% aqueous hydrochloric acid.
  • the suspension was extracted with 125 mL of diethyl ether.
  • the ether layer was separated, washed with 75 mL of 10% aqueous sodium hydroxide, and then with 100 mL of dilute acid.
  • the ether solution was dried (MgSO4) nd concentrated in vacuo to afford
  • Examples 3 a to 12a in the table below were prepared by the general procedure of Examples la and 2a.
  • the reaction mixture was transferred to a 2-dram vial and diluted with 2 mL of ethyl acetate.
  • the organic layer was washed with 3 mL of distilled water and the water layer washed again with 2 mL of ethyl acetate.
  • the combined organic layers were allowed to evaporate to dryness in an open fume hood.
  • Concanavalin A Several ofthe phenyl amine MEK inhibitors described above have been evaluated in a number of assays which establish their utility in preventing the rejection of transplants in mammals.
  • One such assay measured the ability of a test compound to inhibit the production of IL-2 from T cells (T lymphocytes) present in human peripheral blood mononuclear cells (HPBMC).
  • T lymphocytes T lymphocytes
  • HPBMC peripheral blood mononuclear cells
  • the cells (HPBMC) were prepared by first centrifuging tubes of heparinized blood
  • PBMC peripheral blood mononuclear cells
  • Concanavalin A was purchased from CalBiochem (Catalog No. 234567). A stock solution was prepared by dissolving 250 mg of Con A in 10 mL of sterile water (25 mg/mL). The assay was canied out by adding 50 ⁇ L ofthe diluted test compounds to appropriate wells of a plate. To the wells were added 100 ⁇ L ofthe PBMC cell solution (2.5 x 10" cells/mL). The mixtures were pre-incubated for 15 minutes at 37°C, in a 5% carbon dioxide incubator.
  • the HPBMC assay 50 ⁇ L ofthe Con A solution (80 ⁇ g/mL Con A in RPMI-1640) were added to the appropriate wells.
  • 50 ⁇ L of a Con A solution 800 ⁇ g/mL Con A in RPMI-1640
  • Control wells contained medium plus 50 ⁇ L of RPMI-1640.
  • the well plates were incubated for 2 days at 37°C in a 5% carbon dioxide incubator. At the end of Day 2, the plates were centrifuged at 2200 rpm for 5 minutes at 0-4°C. Samples of supernatant (150 ⁇ L) were removed from each well and stored at -20°C until analyzed. Each sample was analyzed by an IL-2 ELISA kit (No. D2050 from R & D Systems, Minneapolis, MN) to measure the content of IL-2.
  • PD 184352 4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide, also known as PD 184352.
  • Figure 1 shows that no IL-2 is produced by unstimulated cells, but large amounts are produced in the presence of Con A.
  • Figure 9 shows the inhibition of IL-2 production in cells caused by several ofthe phenyl amine MEK inhibitors of Formulas I and II, compared to known immunosuppressive agents dexamethasone, a steroid, which is 9-fluoro- 1 l ⁇ ,17,21-trihydroxy-16 ⁇ -methylpregna-l,4-diene-3,20-dione, and rolipram, a phosphodiesterase-4 inhibitor which is 4-[3-(cyclopentyloxy)-4-methoxyphenyl]-
  • T cell receptor is a complex, multi- protein receptor comprised in part of a set up proteins collectively called CD3. In order for T cells to produce IL-2, they must also be activated by a co-receptor.
  • CD28 The most prominent and best-characterized T cell co-receptor is CD28.
  • Anti-CD3 was purchased from BioSource Int. (catalog #AHS2812). A working solution was prepared containing 10 ⁇ g/mL of anti-CD3 in PBS. A
  • Anti-CD28 was purchased from BioSource Int. (catalog #AH0312) and was added as a solution (0.5 ⁇ g/mL) to appropriate wells after addition of HPBMC and MEK inhibitor.
  • HPBMC were prepared as described in Example 1 and stimulated with concentrations of anti-CD3 and anti-CD28 determined from pilot studies to provide a high degree of T cell activation, and hence IL-2 release. After a 2-day culture period in a humidified 37°C incubator containing 5% CO in air, supernatant was collected and assayed for IL-2 as described in Example 1.
  • a preferred compound to be used in accordance with this invention is PD 184352.
  • the Figure shows that no IL-2 is produced by unstimulated cells, but large amounts are produced in the presence of anti-3 plus anti-CD28.
  • the Figure shows that PD 184352 causes a dramatic dose dependent inhibition of IL-2 production, and has an IC5o of47 nM.
  • Figure 10 shows the dose dependent inhibition of IFN-gamma caused by various phenyl amine MEK inhibitors of Formulas I and II, and the activity of known immunosuppressive agents rolipram and dexamethasone.
  • the data establish that the phenyl amine MEK inhibitors are much more potent that rolipram, and cause almost 100% inhibition at concentrations of 1 ⁇ M or higher.
  • the ability ofthe MEK inhibitors of Formulas I and II to inhibit IFN-gamma production establishes that they can be used for the prophylaxis of transplants of organs, limbs, cells, and tissues in mammals.
  • lymphocytes or leukocytes
  • leukocytes from one donor (eg, the potential recipient of a transplant) are cultured in the presence of leukocytes from another donor (eg, the potential transplant donor, generally a living related donor, not cadaveric donors).
  • the assay is a mixed lymphocyte (or leukocyte) reaction, and is referred to as the "MLR”.
  • MLR mixed lymphocyte (or leukocyte) reaction
  • inhibition of tritiated thymidine ( 3 H-TDR) incorporation is measured. Tritiated thymidine was supplied from Amersham (Catalog No. TRK.758, 250 ⁇ Ci).
  • the commercial product was diluted in RPMI-1640 in a 50 mL conical centrifuge tube to provide a working stock solution of 5-10 ⁇ Ci/mL.
  • Cells and test compounds were prepared as described above. The compounds and cells were incubated at 37°C in a 5% carbon dioxide incubator. On Day 6, each well ofthe assay plate was labeled with the H-TDR working stock solution (total of 0.1 - 0.5 ⁇ Ci per well). The plates were incubated an additional 6 hours following labeling. The plate samples were harvested using a multichannel harvester, and the radioactivity of each sample was counted using a betaplate Wallace 1205 counter.
  • Figure 4 shows the activity of PD 184352 in the human MLR assay.
  • the activity is measured as counts per minute (CPM) of tritiated thymidine ( 3 H-TDR) uptake.
  • CPM counts per minute
  • 3 H-TDR tritiated thymidine
  • the Figure shows that untreated MLR values are in excess of 4500 CPM, whereas the test compound causes a dose dependent inhibition of 3 H-TDR uptake, with almost total inhibition occurring at 10 ⁇ M.
  • the IC50 for PD 184352 was established as 186 nM.
  • Figure 11 shows the activity of several phenyl amine MEK inhibitors in the MLR assay, compared to dexamethasone.
  • Another measure of immunosuppressive activity is a compound's ability to block the growth of T cells. Uncontrolled proliferation of T cells leads to rejection of transplanted organs, tissues, cells, and limbs in mammals. Immunological studies have established that cyclosporine A blocks activation of T cells, and that this is partly the result of inhibition ofthe synthesis of interleukin-2, the main growth factor for T cells.
  • the assay was carried out by following the general procedure described above for preparing cells and test compounds, and 3 H-TDR inhibition was measured.
  • Con A was used to induce T-cell proliferation.
  • Figure 5 shows the degree to which PD 184352 inhibits T-cell proliferation. Namely, the compound causes about 50% inhibition ofthe Con A induced proliferation at the lowest dose tested (0.12 ⁇ M), and causes almost total inhibition at the highest dose tested (10.0 ⁇ M).
  • the IC50 for the compound was determined to be 340 nM.
  • Figure 12 shows that all ofthe phenyl amine MEK inhibitors that were tested caused a dramatic and dose dependent inhibition of T-cell proliferation.
  • the T-cell inhibition study was carried out using the agent PHA to induce the proliferation.
  • Figure 6 shows the effects of PD 184352.
  • the test compound failed to cause inhibition at the low dose (0.12 ⁇ M), but caused a measurable inhibition at all other doses, with almost total inhibition at the high dose (10 ⁇ M).
  • the IC5Q was determined to be 1.9 ⁇ M in this assay.
  • the data further establish the ability ofthe phenyl amine MEK inhibitor to inhibit T-cell proliferation, and thereby to be useful in the prophylaxis of transplant rejections in mammals.
  • the MEK inhibitors to be used in the method of this invention are potent inhibitors of transplant rejection, while at the same time have little or no toxicity, a feature which severely limits the clinical usefulness of commercial immunosuppressive agents.
  • the toxic effects ofthe compounds were evaluated in an assay using MTT, which is a chemical substance known as 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide. MTT changes color when it is activated by a cell, and that color change can be measured by routine methods. Only living cells can change the color of MTT. For this assay, living U-937 cells were obtained from American Type Culture Collection (Rockville, MD).
  • PD 184352 was added to the cells in plate wells, and the cells were incubated as described above. Following the incubation period, the color change of MTT was measured using a spectrophotometer.
  • Figure 7 shows that PD 184352 caused no toxicity at concentrations below 33 ⁇ M, and caused only slight color change even at concentrations as high as 100 ⁇ M. The dose of PD 184352 required to cause cell death of one-half of the cells (the TC50) was thus determined to be greater than 100 ⁇ M.
  • Figure 13 shows the toxicity of several ofthe phenyl amine MEK inhibitors when evaluated in the MTT assay. The data establish that all ofthe compounds evaluated have a very favorable therapeutic index, ie, biological efficacy for prophylaxis of transplant rejection vs toxicity. Thus, the compounds will find widespread use in the clinical setting for preventing and controlling transplant rejection in mammals.
  • Figure 8 shows the relative activities of several ofthe phenyl amine MEK inhibitors of Formulas I and II, compared with the activities of rolipram and dexamethasone, in a number ofthe assays described above.
  • the Figure establishes that the phenyl amine MEK inhibitors are, in general, as active as or more active than the known agents when evaluated in standard assays which establish utility of compounds in the prophylaxis of transplant rejections in mammals.
  • the Table presents the in vitro effects of several compounds to be used in the method of this invention, together with several comparator immunosuppressive agents, on human leukocytes.
  • the data are concentrations of test compounds required to cause a 50 percent inhibition ofthe measured parameter (the IC50), except in the case ofthe toxicity data, which is presented as
  • APK refers to activity of compounds in a cascade assay, wherein a compound inhibits a MEK enzyme, thereby preventing phosphorylation of another enzyme, namely a MAP (mitogen activated protein) kinase, which otherwise would cause phosphorylation of a substrate, in this assay said substrate being myelin basic protein
  • the comparator agent U0126 (in Pharmacological Table 1) is 1 ,4-diamino-2,3-dicyano-l ,4-bis[2-aminophenylthio]butadiene, an immunosuppressive compound described in US Patent No. 2,779,780.
  • MEK Inhibitors In Vitro Effects on Human Leukocytes (All data are mean (*) IC50 S or % inhibition at the concentration given, except toxicity data
  • IC 50 IL-2 gamma TC 50 PHA MLR Con A (nM) ( ⁇ M) ( ⁇ M) ( ⁇ M) or ( ⁇ M) ( ⁇ M) ( ⁇ M) ( ⁇ M)
  • the MEK inhibitors described above are well-suited to the prophylaxis of transplant rejections in mammals, preferably humans.
  • the MEK inhibitors can be used in combination with other such agents for even better results.
  • the MEK inhibitors can be combined clinically with agents such as cyclosporine A and FK 506, another well-known immunosuppressive agent.
  • the agents can be combined into the same formulation, but are more typically administered in their individual formulated doses, and normally at the dose levels routinely used for the individual agents when used alone; however, lower or higher doses can be used if desired.
  • the individual agents can be packaged together for convenience ofthe medical practitioner, for example in a kit or the like.
  • Example 8 The selective MEK inhibitors to be used in the method of this invention will additionally be evaluated in in vivo assays that establish their ability to prevent and control transplant rejections.
  • a typical in vivo assay is an allogeneic mouse ear-heart model using neonatal or newborn mouse hearts. Mice ofthe BL/6 to C3H strain will be used as test animals. Ten mice will be treated with a MEK inhibitor. Three vehicle control allografts will be included, as well as three isografts, as control animals. Mice will be dosed at 50 mg/kg twice each day, until grafts are rejected, or until there is evidence of a definite anti-rejection effect.
  • the MEK inhibitor being evaluated will be dissolved in a dosing solution which is 10% ethanol, 10% Cremophor EL (Sigma, Cat. No. C-5135), and 80% water (v/v/v).
  • the test animals are dosed orally using a tuberculin syringe and a mouse oral gavage tube.
  • the dosing ratio is 0.1 mL of solution per each 20 g of mouse weight.
  • the MEK inhibitor (300 mg) to be tested is placed in a 50 mL conical tube, and 3.0 mL of ethanol is added. The tube is capped to retard evaporation and vortexed to facilitate dissolution.
  • the Cremophor EL (3.0 mL) is added, followed by the addition of 24.0 mL of water.
  • the 30 mL dosing solution is vortexed, and stored at 5°C until used.
  • All specimens are obtained immediately after sacrificing the animals, and placed in 10-20 mL of buffered formalin. If all allografts survive to the end ofthe study, one-half are placed in the buffered formalin, and the other half are frozen for subsequent analysis. The following tissues are collected for histopathology and phospho-ERK analysis: ear bearing the allograft (or isograft); ipsilateral cervical lymph nodes; contralateral cervical lymph nodes; the spleen; and heparinized blood collected by cardiac puncture for determination of drug concentration. If transplants are still surviving on Day 50, the study is terminated, and the above noted specimens are collected and analyzed.
  • the method of this invention provides for both prophylaxis and maintenance of patients who have undergone a transplant or are scheduled to undergo a transplant.
  • Evaluation of one MEK inhibitor, 2-(2-Methyl- 4-iodophenylamino)-N-cyclopropylmethoxy-3,4,5-trifluorobenzamide (PD 198306)) was performed using the aforementioned protocol, but no enhancement of graft survival was observed (data not shown). This may be the result of any or a combination of several factors, among which is insufficient exposure of target cells to an adequate and sustained concentration ofthe MEK inhibitor. Survival time of isografts in mice treated with PD 198306 was somewhat shortened, which may suggest that MEK inhibitors might be more efficacious for graft maintenance.

Abstract

La présente invention concerne un procédé concernant la prophylaxie ou la maintenance du rejet des transplants d'organes, de cellules, de membres et de tissus chez les mammifères. Ce procédé consiste en une administration d'un inhibiteur sélectif du MEK, de préférence un composé représenté par les formules I et II.
PCT/US1999/029591 1998-12-15 1999-12-14 Technique de prevention du rejet de greffe par utilisation d'un inhibiteur du mek WO2000035435A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
HU0104607A HUP0104607A3 (en) 1998-12-15 1999-12-14 Use of a mek inhibitor for preventing transplant rejection
IL14323199A IL143231A0 (en) 1998-12-15 1999-12-14 Use of a mek inhibitors for preventing transplant rejection
AU21805/00A AU2180500A (en) 1998-12-15 1999-12-14 Use of a mek inhibitor for preventing transplant rejection
KR1020017007397A KR20010101203A (ko) 1998-12-15 1999-12-14 Ｍｅｋ 저해제의 이식 조직 거부를 예방하기 위한 용도
JP2000587756A JP2002532414A (ja) 1998-12-15 1999-12-14 移植片拒絶反応を防止するためのｍｅｋ阻害剤の使用
EP99966203A EP1140046A1 (fr) 1998-12-15 1999-12-14 Technique de prevention du rejet de greffe par utilisation d'un inhibiteur du mek
CA002346684A CA2346684A1 (fr) 1998-12-15 1999-12-14 Technique de prevention du rejet de greffe par utilisation d'un inhibiteur du mek

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US11236998P 1998-12-15 1998-12-15
US60/112,369 1998-12-15

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WO2000035435A1 (fr) 2000-06-22
JP2002532414A (ja) 2002-10-02
EP1140046A1 (fr) 2001-10-10
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ZA200103765B (en) 2002-05-09
AU2180500A (en) 2000-07-03
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