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EP1140046A1 - Use of a mek inhibitor for preventing transplant rejection - Google Patents

Use of a mek inhibitor for preventing transplant rejection

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Publication number
EP1140046A1
EP1140046A1 EP99966203A EP99966203A EP1140046A1 EP 1140046 A1 EP1140046 A1 EP 1140046A1 EP 99966203 A EP99966203 A EP 99966203A EP 99966203 A EP99966203 A EP 99966203A EP 1140046 A1 EP1140046 A1 EP 1140046A1
Authority
EP
European Patent Office
Prior art keywords
methyl
phenylamino
iodo
benzamide
difluoro
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP99966203A
Other languages
German (de)
French (fr)
Inventor
Richard Buell Gilbertsen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Warner Lambert Co LLC
Original Assignee
Warner Lambert Co LLC
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 LLC filed Critical Warner Lambert Co LLC
Publication of EP1140046A1 publication Critical patent/EP1140046A1/en
Withdrawn legal-status Critical Current

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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:
  • R ⁇ is hydrogen, hydroxy, CJ-CQ, alkyl, Cj-Cg alkoxy, halo, trifluoromethyl, or CN.
  • R2 is hydrogen.
  • R3, R4, and R5 are independently selected from hydrogen, hydroxy, halo, trifluoromethyl, Cj-Cg alkyl,
  • R9 is hydrogen, hydroxy, COOH, or NR1 Q I 1 ; n is 0-4; m is 0 or 1.
  • Each of Ri Q and R ⁇ ⁇ is independently selected from hydrogen and Cj-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, or N-(Cj-Cg alkyl).
  • Z is COOR7, tetrazolyl, CONRgR ⁇ , CONHNRioRi 1, or CH2OR7.
  • R and R7 independently are hydrogen, Ci -Cg alkyl, C2-C8 alkenyl,
  • C3-C10 (cycloalkyl optionally containing one, two, or three heteroatoms selected from O, S, NH, or N alkyl); or R ⁇ 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, heterocyclic, and alkynyl groups can be unsubstituted or substituted by halo, hydroxy, C]-C alkoxy, amino, nitro, d-C 4 alkylamino, di(C ⁇ -C 4 )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 , R 4 , and R 5 independently are hydrogen, fluoro, chloro, bromo, iodo, methyl, methoxy, or nitro; (d) R 10 and R ⁇ independently are hydrogen or methyl; (e) Z is COOR 7 , tetrazolyl, CONReR 7 , CONHNR 10 R ⁇ , or CH 2 OR 7 ; Re and R 7 independently are hydrogen, C alkyl, heteroaryl, or C 3-5 cycloalkyl optionally containing one or two heteroatoms selected from O, S, or NH; or R$ and R together with the nitrogen to which they are attached complete a 5-6 member cyclic ring optionally containing 1 or 2 additional heteroatoms selected from O, NH or N-alkyl; and wherein any of the foregoing
  • 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, Cj-Cg alkyl, Ci -Cg alkoxy, halo, trifluoromethyl, or CN.
  • R2 a is hydrogen.
  • Each ofR3 a , R4 a , and R5 a is independently selected from hydrogen, hydroxy, halo, trifluoromethyl, Ci-Cg alkyl, Cj-Cg alkoxy, nitro, CN, and (O or NH) m -(CH2) n -R9a- R 9a is hydrogen, hydroxy, CO2H or NRiOa ⁇ l la» n * s 0-4; and m is 0 or 1.
  • Each of Ri 0 a and R ⁇ 1 a is independently hydrogen or C1 -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).
  • R6 a is hydrogen, C ⁇ Cg alkyl, (CO)-(C ⁇ -Cg alkyl), aryl, aralkyl, or C3-C10 cycloalkyl.
  • R7 a is hydrogen, C1 -Cg alkyl, C2-Cg alkenyl, C2-Cg alkynyl, C3-C10 (cycloalkyl or cycloalkyl optionally containing a heteroatom selected from O, S, or NRc; ⁇ ).
  • any of the alkyl, alkenyl, aryl, heterocyclic, and alkynyl groups can be unsubstituted or substituted by halo, hydroxy, Cj-C 6 alkoxy, amino, nitro, C ⁇ -C alkylamino, di(C]- C 4 )alkylamino, C 3 -C 6 cycloalkyl, phenyl, phenoxy, C 3 -C 5 heteroaryl, or C 3 -C 5 heteroaryloxy; or R 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 NRi o a Ri i a .
  • the invention also encompasses pharmaceutically acceptable salts, esters, amides or prodrugs of each of the disclosed compounds.
  • Preferred embodiments of Formula (II) are those structures wherein: (a) R la is H, methyl, fluoro, or chloro; (b) R 2a is H; R 3a , Rig, 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) ig is F at the 4 position, para to the CO-N-R6a-OR a group and meta to the bridging nitrogen; (f) R 3a or R 5a is F; (g) at least one of R 3a , ia, and R 5a is F; (h) R ⁇ a is methyl or chloro
  • 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 of the 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 of the 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 of the 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 of the MAP Kinase Pathway," column 6, line 36 to column 7, line 4 of the 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 of the 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, l,l-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, morpholinyi, piperazinyl, pyrrolidinyl, 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-hydroxycyclohexyl, 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 of the above-named other enzymes.
  • a selective inhibitor has an IC 0 that is at least 1/100, more preferably 1/500, and even more preferably 1/1000, 1/5000, or less than that of its IC 50 or one or more of the above- named enzymes.
  • the MEK inhibitors of the 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 of the 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 of the 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 of the 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 of the 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 of the 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-butyleneglycol, 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 of the 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 of the 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 of the patient, the severity of the condition being treated, and the pharmacological activity of the 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 of the compounds of the 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, of the compounds of the invention.
  • salts refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention.
  • salts can be prepared in situ during the final isolation and purification of the 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 of the compounds of this invention examples include Cj-C ⁇ 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. C1-C4 alkyl esters are preferred. Esters of the compounds of the present invention may be prepared according to conventional methods.
  • Examples of pharmaceutically acceptable, non-toxic amides of the compounds of this invention include amides derived from ammonia, primary C ⁇ -C alkyl amines and secondary Cj-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 of the compounds of the invention may be prepared according to conventional methods.
  • prodrug refers to compounds that are rapidly transformed in vivo to yield the parent compound of the above formula, for example, by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and
  • the compounds of the 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 of the 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 of the formula HOR7
  • reaction of the benzoic acid with an alcohol can be carried out in the presence of a coupling agent.
  • Typical coupling reagents include
  • 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 of the coupling reagent is added.
  • a base such as triethylamine or diisopropylethylamine 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 of the invention are readily prepared by reacting the foregoing benzoic acids with an amine of the formula HNR6R7.
  • the reaction is carried out by reacting approximately equimolar quantities of the 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 of the invention compounds of Formula (I) where Z is CH2OR6 and R6 is hydrogen, are readily prepared by reduction of the 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-l 8, 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
  • Step b Preparation of 5-chloro-2-fluoro-benzaldehvde 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 OR7 a in the presence of a peptide coupling reagent.
  • Hydroxylamine derivatives that can be employed include methoxylamine,
  • N-ethyl-isopropoxy amine, and tetrahydro-oxazine 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 benzotriazolyloxytripyrrolidino phosphonium hexa
  • 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 of the coupling reagent is added.
  • a base such as triethylamine or diisopropylethylamine 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 reaction mixture was stirred 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) and 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 Inhibition of IL-2 Production Induced by Concanavalin A
  • Con A Several of the 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 human 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 carried out by adding 50 ⁇ L of the diluted test compounds to appropriate wells of a plate. To the wells were added 100 ⁇ L of the 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.
  • 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 of the 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 2 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 IC50 of 47 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 of the 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
  • 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 of the assay plate was labeled with the 3 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 of the 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 of the 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 of the 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 IC50 was determined to be 1.9 ⁇ M in this assay.
  • the data further establish the ability of the 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 of the 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 of the phenyl amine MEK inhibitors when evaluated in the MTT assay.
  • Figure 8 shows the relative activities of several of the phenyl amine MEK inhibitors of Formulas I and II, compared with the activities of rolipram and dexamethasone, in a number of the 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 of the measured parameter (the IC50), except in the case of the 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 l,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 (*) IC5 QS or % inhibition at the concentration given, except toxicity data
  • 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 of the 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 of the 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 of the 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 of the 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.

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Abstract

This invention provides a method for the prophylaxis or maintenance of rejection of transplants of organs, cells, limbs, and tissues in mammals, comprising administering a selective MEK inhibitor, preferably a compound of formulas (I) and (II).

Description

E OF A MEK INHIBITOR FOR PREVENTING TRANSPLANT REJECTION
FIELD OF THE INVENTION
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.
BACKGROUND OF THE INVENTION
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. As a result, the patient receives an immunosuppressant agent to control or at least diminish the rejection response. While several immunosuppressants are currently available for clinical use, each is associated with adverse side effects. For example, 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-δ, 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. Like cyclosporine, 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. In view of the above, there is a continuing need for immunosuppressant agents not only useful for treating or preventing transplant rejection but also with less severe side effects than those associated with existing therapy. According to the present invention, 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.
SUMMARY OF THE INVENTION
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. In a preferred embodiment, 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. In another preferred embodiment, the immunosuppressive agent administered is a phenyl amine compound of Formula I or II:
In formula (I), R\ is hydrogen, hydroxy, CJ-CQ, alkyl, Cj-Cg alkoxy, halo, trifluoromethyl, or CN. R2 is hydrogen. R3, R4, and R5 are independently selected from hydrogen, hydroxy, halo, trifluoromethyl, Cj-Cg alkyl,
C^-Cg alkoxy, nitro, CN, and -(O or NH)m-(CH2)n-RQ. R9 is hydrogen, hydroxy, COOH, or NR1 Q I 1 ; n is 0-4; m is 0 or 1. Each of Ri Q and R\ \ is independently selected from hydrogen and Cj-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, or N-(Cj-Cg alkyl). Z is COOR7, tetrazolyl, CONRgRγ, CONHNRioRi 1, or CH2OR7. R and R7 independently are hydrogen, Ci -Cg alkyl, C2-C8 alkenyl,
C2-C8 alkynyl, (CO)-Cj-C8 alkyl, aryl, heteroaryl, C3-C1 Q cycloalkyl, or
C3-C10 (cycloalkyl optionally containing one, two, or three heteroatoms selected from O, S, NH, or N alkyl); or Rβ 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. In formula (I), any of the foregoing alkyl, alkenyl, aryl, heteroaryl, heterocyclic, and alkynyl groups can be unsubstituted or substituted by halo, hydroxy, C]-C alkoxy, amino, nitro, d-C4 alkylamino, di(Cι-C4)alkylamino, C3-C6 cycloalkyl, phenyl, phenoxy, C3-C5 heteroaryl, or C3-C5 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) R2 is hydrogen; (c) R3, R4, and R5 independently are hydrogen, fluoro, chloro, bromo, iodo, methyl, methoxy, or nitro; (d) R10 and Rπ independently are hydrogen or methyl; (e) Z is COOR7, tetrazolyl, CONReR7, CONHNR10Rπ, or CH2OR7; Re and R7 independently are hydrogen, C alkyl, heteroaryl, or C 3-5 cycloalkyl optionally containing one or two heteroatoms selected from O, S, or NH; or R$ and R together with the nitrogen to which they are attached complete a 5-6 member cyclic ring optionally containing 1 or 2 additional heteroatoms selected from O, NH or N-alkyl; and wherein any of the foregoing alkyl or aryl groups can be unsubstituted or substituted by halo, hydroxy, methoxy, ethoxy, or heteroaryloxy (such as the synthetic intermediate 2,3,4,5,6-pentafluorophenyl); (f) Z is COOR ; (g) R7 is H, pentafluorophenyl, or tetrazolyl; (h) R3, t, and R5 are independently H, fluoro, or chloro; (i) Ri is fluoro; (j) two of R3, R^ and R5 are fluoro; or (k) combinations of the above. In another preferred embodiment of Formula (I), Rj is methyl, fluoro, chloro, or bromo.
Examples of preferred embodiments include methods comprising a MEK inhibitor selected from Formula (I) Compound Table below.
FORMULA (I) COMPOUND TABLE (page 1 of 10)
[4-Chloro-2-(lH-tetrazol-5-yl)-phenyl-(4-iodo-2-methyl-phenyl)-amine
(4-iodo-2-methyl-phenyl)-[2-(lH-tetrazol-5-yl)-phenyl]amine [4-nitro-2-(lH-tetrazol-5-yl)-phenyl-(4-iodo-2-methyl-phenyl)-amine
4-Fluoro-2-(4-iodo-2-methylphenylamino)-benzoic acid
3,4,5-Trifluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid
3 ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid
5-Bromo-3 ,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid 5-Chloro-2-(4-iodo-2 -methyl -phenylamino)-benzoic acid
Sodium 5-Chloro-2-(4-iodo-2-methyl-phenylamino)-benzoate 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-benzoic acid 2-(4-Iodo-2-methyl-phenylamino)-5-nitro-benzoic acid 4-Chloro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid 2-(4-Iodo-2-methyl-phenylamino)-benzoic acid
5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid
5-Iodo-2-(4-iodo-2-methyl-phenylamino)-benzoic acid
2,3 ,5-Trifluoro-4-(4-iodo-2-methyl-phenylamino)-benzoic acid
2-(4-Iodo-phenylamino)-5-methoxy-benzoic acid 5-Methyl-2-(4-iodo-2-methyl-phenylamino)-benzoic acid
2-(4-Iodo-2-methyl-phenylamino)-4-nitro-benzoic acid 2-(4-Bromo-2-methyl-phenylamino)-4-fluoro-benzoic acid 2-(2-Bromo-4-iodo-phenylamino)-5 -nitro-benzoic acid 2-(4-Bromo-2-methyl-phenylamino)-3 ,4-difluoro-benzoic acid 5-Chloro-N-(2-hydroxyethyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-benzamide 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-methyl-benzamide N-Ethyl-4-fluoro-2-(4-iodo-2-methyl-phenylamino)-benzamide 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N,N-dimethyl-benzamide
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(lH-tetrazol-5-yl)- benzamide FORMULA (I) COMPOUND TABLE (continued, page 2 of 10) 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-benzamide
5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N,N-dimethyl-benzamide [5-Chloro-2-(4-iodo-2-methyl-phenylamino)-benzoylamino]-acetic acid 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-propyl-benzamide 5-Bromo-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide
N,N-Diethyl-4-fluoro-2-(4-iodo-2-methyl-phenylamino)-benzamide 4-Fluoro-N- { 3 - [4-(2-hydroxy-ethyl)-piperazin- 1 -y 1] -propyl } -2-(4-iodo-
2-methyl-phenylamino)-benzamide N,N-Diethyl-2-(4-iodo-2-methyl-phenylamino)-5-nitro-benzamide N-Butyl-4-fluoro-2-(4-iodo-2-methyl-phenylamino)-benzamide
5-Chloro-N,N-diethyl-2-(4-iodo-2-methyl-phenylamino)-benzamide 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N,N-dimethyl-benzamide 5-Bromo-3,4-difluoro-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl- phenylamino)-benzamide N-(2,3-Dihydroxy-propyl)-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)- benzamide 5-Bromo-3 ,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-piperidin- 1 - yl-ethyl)-benzamide 3,4-Difluoro-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide
N-(2,3-Dihydroxy-propyl)-4-fluoro-2-(4-iodo-2-methyl-phenylamino)- benzamide 3,4-Difluoro-N-(3-hydroxy-propyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide 5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-pyrrolidin-
1 -yl-ethyl)-benzamide 5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-pyridin-4- yl-ethyl)-benzamide FORMULA (I) COMPOUND TABLE (continued, page 3 of 10)
4-Fluoro-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide
5-Bromo-N-(3-dimethylamino-propyl)-3,4-difluoro-2-(4-iodo-2-methyl- phenylamino)-benzamide 5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-morpholin- 4-yl-ethyl)-benzamide 3 ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-morpholin-4-yl- ethyl)-benzamide 3 ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-pyrrolidin- 1 -yl- ethyl)-benzamide 3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-pyridin-4-yl-ethyl)- benzamide
N-(3-Dimethylamino-propyl)-3,4-difluoro-2-(4-iodo-2-methyl- phenylamino)-benzamide N-Benzyl-4-fluoro-2-(4-iodo-2-methyl-phenylamino)-benzamide 2-(4-Bromo-2-methyl-phenylamino)-3,4-difluoro-N-(2-hydroxy-ethyl)- benzamide
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-morpholin-4-yl-ethyl)- benzamide 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(3 -piperidin- 1 -yl-propyl)- benzamide 3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(3-piperidin-l-yl- propyl)-benzamide 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-thiophen-2-yl-ethyl)- benzamide 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-pyrrolidin-l-yl-ethyl)- benzamide
2-(4-Bromo-2-methyl-phenylamino)-3,4-difluoro-N-(2-morpholin-4-yl- ethyl)-benzamide FORMULA (I) COMPOUND TABLE (continued, page 4 of 10) -Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N-pyridin-4- ylmethyl-benzamide ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-pyridin-4-ylmethyl- benzamide -(4-Bromo-2-methyl-phenylamino)-N-(3-dimethylamino- propyl)-3 ,4-difluoro-benzamide -Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-pyridin-4-ylmethyl- benzamide -Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-pyridin-4-yl-ethyl)- benzamide -(4-Bromo-2-methyl-phenylamino)-3,4-difluoro-N-(2-pyridin-4-yl-ethyl)- benzamide -(4-Bromo-2-methyl-phenylamino)-3,4-difluoro-N-(3-hydroxy-propyl)- benzamide -(4-Bromo-2-methyl-phenylamino)-3 ,4-difluoro-N-(2-pyrrolidin- 1 -yl- ethyl)-benzamide -Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-phenethyl-benzamide -(4-Bromo-2-methyl-phenylamino)-3,4-difluoro-N-(2-thiophen-2-yl- ethyl)-benzamide -(4-Bromo-2-methyl-phenylamino)-3,4-difluoro-N-pyridin-4-ylmethyl- benzamide -(4-Bromo-2-methyl-phenylamino)-3,4-difluoro-N-phenethyl-benzamide -(4-Bromo-2-methyl-phenylamino)-3 ,4-difluoro-N-(2-piperidin- 1 -yl- ethyl)-benzamide -Chloro-N- { 3 - [4-(2-hydroxy-ethyl)-piperazin- 1 -yl] -propyl } -2-(4-iodo-2- methyl- phenylamino)-benzamide -Fluoro-N- { 3 - [4-(2-hydroxy-ethyl)-piperazin- 1 -yl] -propyl } -2-(4-iodo-2- methyl-phenylamino)- benzamide -(4-Iodo-2-methyl-phenylamino)-5-nitro-N-pyridin-4-yl methyl- benzamide FORMULA (I) COMPOUND TABLE (continued, page 5 of 10)
5 -Bromo-N- { 3 - [4-(2-hydroxy-ethyl)-piperazin- 1 -yl] -propyl } -2-(4-iodo-2- methyl- phenylamino)- benzamide
5-Chloro-N-(2-diethylamino-ethyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide 5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N-(2-piperidin-l-yl-ethyl)- benzamide (3-Hydroxy-pyrrolidin- 1 -yl)-[5-nitro-2-(4-iodo-2-methyl-phenylamino)- phenyl] -methanone 5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N-(2-pyrrolidin-l-yl-ethyl)- benzamide 5-Bromo-N-(2-diethylamino-ethyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide
N-{2-[Bis-(2-hydroxy-ethyl)-amino]-ethyl}-5-chloro-2-(4-iodo-2-methyl- phenylamino)- benzamide N- { 2- [Bis-(2-hydroxy-ethyl)-amino] -ethyl } -5 -bromo-2-(4-iodo-2-methyl- phenylamino)- benzamide N-{3-[4-(2-Hydroxy-ethyl)-piperazin-l-yl]-ρropyl}-2-(4-iodo-2-methyl- phenylamino)- benzamide 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-pyridin-4-ylmethyl- benzamide 5-Bromo-2-(4-iodo-2-ethyl-phenylamino)-N-(2-pyrrolidin-l-yl-ethyl)- benzamide
5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-(2-piperidin-l-yl-ethyl)- benzamide 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-pyrrolidin-l-yl-ethyl)- benzamide 5-Chloro-N-(3-dimethylamino-propyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide N-{2-[Bis-(2-hydroxy-ethyl)-amino]-ethyl}-5-fluoro-2-(4-iodo-2-methyl- phenylamino)-benzamide FORMULA (I) COMPOUND TABLE (continued, page 6 of 10)
5-Chloro-N-(3-hydroxy-propyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide
5-Chloro-N-(3-diethylamino-2-hydroxy-propyl)-2-(4-iodo-2-methyl- phenylamino)- benzamide 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-piperidin-l-yl-ethyl)- benzamide 5-Bromo-N-(3-hydroxy-propyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-(3-piperidin-l-yl-propyl)- benzamide N-{2-[Bis-(2-hydroxy-ethyl)-amino]-ethyl}-2-(4-iodo-2-methyl- phenylamino)-5-nitro-benzamide
5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N-(2-morpholin-4-yl-ethyl)- benzamide 5-Chloro-N-(3-diethylamino-propyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide 5-Chloro-N-(2-diisopropylamino-ethyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide 5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N-(3-piperidin-l-yl-propyl)- benzamide 2-(4-Iodo-2-methyl-phenylamino)-5-nitro-N-(2-piperidin-1-yl-ethyl)- benzamide
5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-(2-piperazin-l-yl-ethyl)- benzamide N-(2-Diethylamino-ethyl)-5-fluoro-2-(4-iodo-2-methyl-phenylamino)- benzamide 5-Bromo-N-(3-dimethylamino-propyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide N-(3-Hydroxy-propyl)-2-(4-iodo-2-methyl-phenylamino)-5-nitro- benzamide FORMULA (I) COMPOUND TABLE (continued, page 7 of 10)
5-Fluoro-N-(3-hydroxy-propyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide
N-(3-Diethylamino-propyl)-5-fluoro-2-(4-iodo-2-methyl-phenylamino)- benzamide N-(3-Diethylamino-propyl)-2-(4-iodo-2-methyl-phenylamino)-5-nitro- benzamide 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-(2-morpholin-4-yl-ethyl)- benzamide 2-(4-Iodo-2-methyl-phenylamino)-5-nitro-N-(3-piperidin-l-yl-propyl)- benzamide [5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-phenyl]-(2 or 3-hydroxy- pyrrolidin-l-yl)-methanone
5-Bromo-N-(2-diisopropylamino-ethyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-morpholin-4-yl-ethyl)- benzamide 5 -Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(3 -piperidin- 1 -yl-propyl)- benzamide [5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-phenyl]-[4-(2-hydroxy-ethyl)- piperazin- 1 -yl)-methanone N-(3-Diethylamino-2-hydroxy-propyl)-5-fluoro-2-(4-iodo-2-methyl- phenylamino)- benzamide
N-Cyclopropyl-5-fluoro-2-(4-iodo-2-methyl-phenylamino)-benzamide; 5-Chloro-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide 5-Fluoro-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide
N-Benzyloxy-5-fluoro-2-(4-iodo-2-methyl-phenylamino)-benzamide N-Benzyloxy-5-bromo-2-(4-iodo-2-methyl-phenylamino)-benzamide FORMULA (I) COMPOUND TABLE (continued, page 8 of 10)
2-(4-Iodo-2-methyl-phenylamino)-5-nitro-N-(4-sulfamoyl-benzyl)- benzamide
5-Bromo-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide N-(2-Hydroxy-ethyl)-5-iodo-2-(4-iodo-2-methyl-phenylamino)-benzamide N-(2-Hydroxy-ethyl)-2-(4-iodo-2-ethyl-phenylamino)-5-nitro-benzamide 2-(4-Iodo-2-methyl-phenylamino)-N-methyl-5-nitro-N-phenyl-benzamide
5-Chloro-N-cyclopropyl-2-(4-iodo-2-methyl-phenylamino)-benzamide 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-methyl-N-phenyl- benzamide N-Allyl-5-fluoro-2-(4-iodo-2-methyl-phenylamino)-benzamide N-Benzyloxy-5-iodo-2-(4-iodo-2-methyl-phenylamino)-benzamide
5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(4-sulfamoyl-benzyl)- benzamide N-Allyl-5-chloro-2-(4-iodo-2-methyl-phenylamino)-benzamide N-Cyclopropyl-2-(4-iodo-2-methyl-phenylamino)-5-nitro-benzamide 5-Bromo-N-cyclopropyl-2-(4-iodo-2-methyl-phenylamino)-benzamide
5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N-methyl-N-phenyl- benzamide 5-Iodo-2-(4-iodo-2-methyl-phenylamino)-N-(4-sulfamoyl-benzyl)- benzamide 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-(4-sulfamoyl-benzyl)- benzamide N-Allyl-2-(4-iodo-2-methyl-phenylamino)-5-nitro-benzamide 2-(4-Iodo-2-methyl-phenylamino)-5-nitro-N-(4-sulfamoyl-benzyl)- benzamide N-Allyl-5-bromo-2-(4-iodo-2-methyl-phenylamino)-benzamide
5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(3-methyl-benzyl)- benzamide N-Cyclopropyl-5-iodo-2-(4-iodo-2-methyl-phenylamino)-benzamide FORMULA (I) COMPOUND TABLE (continued, page 9 of 10)
5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-methyl-N-phenyl- benzamide
N-Benzyloxy-2-(4-iodo-2-methyl-phenylamino)-5-nitro-benzamide N-Cyclohexyl-5-iodo-2-(4-iodo-2-methyl-phenylamino)-benzamide N-AUyl-5-iodo-2-(4-iodo-2-methyl-phenylamino)-benzamide 5-Iodo-2-(4-iodo-2-methyl-phenylamino)-N-(3-methyl-benzyl)-benzamide 2-(4-Iodo-2-methyl-phenylamino)-N-(3-methyl-benzyl)-5-nitro-benzamide
5-Iodo-2-(4-iodo-2-methyl-phenylamino)-N-methyl-N-phenyl-benzamide N-Cyclohexyl-5-fluoro-2-(4-iodo-2-methyl-phenylamino)-benzamide 5-Chloro-N-cyclohexyl-2-(4-iodo-2-methyl-phenylamino)-benzamide 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-(3-methyl-benzyl)- benzamide
5-Bromo-N-cyclohexyl-2-(4-iodo-2-methyl-phenylamino)-benzamide 5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N-(3-methyl-benzyl)- benzamide N-Cyclohexyl-2-(4-iodo-2-methyl-phenylamino)-5-niuO-benzamide N-Benzyloxy-5-bromo-2-(4-iodo-2-methyl-phenylamino)-benzamide
N-Benzyloxy-5-fluoro-2-(4-iodo-2-methyl-phenylamino)-benzamide 5-Chloro-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide 5-Bromo-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide
2-(4-Iodo-2-methyl-phenylamino)-N-methyl-5-nitro-N-phenyl-benzamide 5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N-methyl-N-phenyl- benzamide N-(2-Hydroxy-ethyl)-5-iodo-2-(4-iodo-2-methyl-phenylamino)-benzamide 5-Chloro-N-cyclopropyl-2-(4-iodo-2-methyl-phenylamino)-benzamide
N-Allyl-5-chloro-2-(4-iodo-2-methyl-phenylamino)-benzamide 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-methyl-N-phenyl- benzamide FORMULA (I) COMPOUND TABLE (continued, page 10 of 10)
N-(2-Hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)-5-nitro-benzamide 5 -Fluoro-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide 5-Bromo-N-cyclopropyl-2-(4-iodo-2-methyl-phenylamino)-benzamide N-Cyclopropyl-5-fluoro-2-(4-iodo-2-methyl-phenylamino)-benzamide 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(4-sulfamoyl-benzyl)- benzamide
N-Cyclopropyl-2-(4-iodo-2-methyl-phenylamino)-5-nitro-benzamide N-Allyl-5-fluoro-2-(4-iodo-2-methyl-phenylamino)-benzamide N-Benzyloxy-5-iodo-2-(4-iodo-2-methyl-phenylamino)-benzamide N-Allyl-5-bromo-2-(4-iodo-2-methyl-phenylamino)-benzamide 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-(4-sulfamoyl-benzyl)- benzamide 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-methyl-N-phenyl- benzamide N-Allyl-2-(4-iodo-2-methyl-phenylamino)-5-nitro-benzamide 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-benzyl alcohol
[5-Chloro-2-(4-iodo-2-methyl-phenylamino)-phenyl]-methanol [2-(4-Iodo-2-methyl-phenylamino)-5 -nitro-phenyl] -methanol [5-Bromo-2-(4-iodo-2-methyl-phenylamino)-phenyl]-methanol N-Allyl-2-(4-iodo-2-methyl-phenylamino)-5-nitro-benzamide.
In another preferred embodiment, the MEK inhibitor is a compound of Formula II
In Formula (II), Ri a is hydrogen, hydroxy, Cj-Cg alkyl, Ci -Cg alkoxy, halo, trifluoromethyl, or CN. R2a is hydrogen. Each ofR3a, R4a, and R5a is independently selected from hydrogen, hydroxy, halo, trifluoromethyl, Ci-Cg alkyl, Cj-Cg alkoxy, nitro, CN, and (O or NH)m-(CH2)n-R9a- R9a is hydrogen, hydroxy, CO2H or NRiOa^l la» n *s 0-4; and m is 0 or 1. Each of Ri 0a and R\ 1 a is independently hydrogen or C1 -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-(Cι-Cg alkyl). R6a is hydrogen, C^Cg alkyl, (CO)-(Cι-Cg alkyl), aryl, aralkyl, or C3-C10 cycloalkyl. R7a is hydrogen, C1 -Cg alkyl, C2-Cg alkenyl, C2-Cg alkynyl, C3-C10 (cycloalkyl or cycloalkyl optionally containing a heteroatom selected from O, S, or NRc;^). In Formula (II), any of the alkyl, alkenyl, aryl, heterocyclic, and alkynyl groups can be unsubstituted or substituted by halo, hydroxy, Cj-C6 alkoxy, amino, nitro, Cι-C alkylamino, di(C]- C4)alkylamino, C3-C6 cycloalkyl, phenyl, phenoxy, C3-C5 heteroaryl, or C3-C5 heteroaryloxy; or R a and R7a 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 NRi oaRi ia. The invention also encompasses pharmaceutically acceptable salts, esters, amides or prodrugs of each of the disclosed compounds.
Preferred embodiments of Formula (II) are those structures wherein: (a) Rla is H, methyl, fluoro, or chloro; (b) R2a is H; R3a, Rig, and R5aare each H, Cl, nitro, or F; (c) Rόa 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) ig is F at the 4 position, para to the CO-N-R6a-OR a group and meta to the bridging nitrogen; (f) R3a or R5a is F; (g) at least one of R3a, ia, and R5a is F; (h) Rιa is methyl or chloro; or (i) a combination of the above.
In a more preferred embodiment the MEK inhibitor is a compound selected from Formula (II) Compound Table below.
FORMULA (II) COMPOUND TABLE (page 1 of 7)
-Fluoro-N-hydroxy-2-(4-iodo-2-methyl-phenylamino)-benzamide -Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(methoxy)-benzamide -Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(prop-2-ynyloxy)- benzamide -Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-phenoxyethoxy)- benzamide -Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-thienylmethoxy)- benzamide -Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(prop-2-enyloxy)- benzamide -Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(cyclopropylmethoxy)- benzamide -Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(cyclopentoxy)-benzamide ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(3-furylmethoxy)- benzamide ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-ethoxy-benzamide ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(but-2-enyloxy)- benzamide ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(cyclopropylmethoxy)- benzamide ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-( 1 -methylprop- 2-ynyloxy)-benzamide ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(3-phenylprop-
2-ynyloxy)-benzamide ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(3-methyl- 5-phenylpent-2-en-4-ynyloxy)-benzamide ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(prop-2-yny loxy)- benzamide ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(propoxy)-benzamide FORMULA (II) COMPOUND TABLE (continued, page 2 of 7) ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(cyclobutyloxy)- benzamide ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-thienylmethoxy)- benzamide ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-methyl-prop-
2-enyloxy)-benzamide ,4-Difluoro-2-(4-iodo-2 -methyl -phenylamino)-N-(2-phenoxyethoxy)- benzamide ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(but-2-enyloxy)- benzamide ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(but-3 -ynyloxy)- benzamide ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(cyclopentyloxy)- benzamide ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(3-(2-fluorophenyl)- prop-2-ynyloxy)-benzamide -Bromo-3,4-difluoro-N-hydroxy-2-(4-iodo-2-methyl-phenylamino)- benzamide -Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(n-propoxy)- benzamide -Bromo-3,4-difluoro-N-(furan-3-ylmethoxy)-2-(4-iodo-2-methyl- phenylamino)-benzamide -Bromo-N-(but-2-enyloxy)-3,4-difluoro-2-(4-iodo-2-methyl- phenylamino)-benzamide -Bromo-N-butoxy-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)- benzamide -Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(3-methyl-but-
2-enyloxy)-benzamide -Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(3-methyl- pent-2-en-4-ynyloxy)-benzamide FORMULA (II) COMPOUND TABLE (continued, page 3 of 7) -Bromo-3,4-difluoro-2-(4-iodo-2-methyl-benzyl)-N-[5-(3-methoxy- phenyl)-3 -methyl-pent-2-en-4-ynyloxy] -benzamide -Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(prop-
2-ynyloxy)-benzamide -Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N-
[3-(3-methoxy-phenyl)-prop-2-ynyloxy]-benzamide -Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(thiopen-
2-ylmethoxy)-benzamide -Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(pyridin-
3 -ylmethoxy)-benzamide -Bromo-3-4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N- (3 -(2-fluorophenyl)-prop-2-ynyloxy)-benzamide -Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(ethoxy)- benzamide -Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N-
(cyclopropylmethoxy)-benzamide -Bromo-3 ,4-difluoro-2-(4-iodo-2-methy l-phenylamino)-N-(isopropoxy)- benzamide -Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N-but-
3 -ynyloxy)-benzamide -Chloro-N-hydroxy-2-(4-iodo-2-methyl-phenylamino)-benzamide -Chloro-2-(4-iodo-2-methyl-phenylamino)-N-(tetrahydro-pyran-2-yloxy)- benzamide -Chloro-2-(4-iodo-2-methyl-phenylamino)-N-methoxy-benzamide -Bromo-2-(4-iodo-2-methyl-phenylamino)-N-phenylmethoxy-benzamide -Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-phenylmethoxy-benzamide -Fluoro-N-hydroxy-2-(4-iodo-2-methyl-phenylamino)-benzamide -Iodo-2-(4-iodo-2-methyl-phenylamino)-N-phenylmethoxy-benzamide -Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(tetrahydropyran-2-yloxy)- benzamide FORMULA (II) COMPOUND TABLE (continued, page 4 of 7) ,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N-(3-phenylprop- 2-ynyloxy)-benzamide ,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N-(3-furylmethoxy)- benzamide ,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N-(2-thienylmethoxy)- benzamide ,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N-(but-3-ynyloxy)- benzamide ,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N-(2-methyl-prop-
2-enyloxy)-benzamide ,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N-(but-2-enyloxy)- benzamide ,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N-(methoxy)-benzamide ,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N-(ethoxy)-benzamide ,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N-(cyclobutoxy)- benzamide ,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N-(isopropoxy)- benzamide ,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N-(2-phenoxyethoxy)- benzamide ,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N-(cyclopropyl- methoxy)-benzamide ,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N-(n-propoxy)- benzamide ,4-Difluoro-2-(4-bromo-2 -methyl -phenylamino)-N-( 1 -methyl-prop-
2-ynyloxy)-benzamide ,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N-(3-(3-fluorophenyl)- prop-2-ynyloxy)-benzamide ,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N-(4,4-dimethylpent-
2-ynyloxy)-benzamide FORMULA (II) COMPOUND TABLE (continued, page 5 of 7)
3,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N-(cyclopentoxy)- benzamide
3,4,5-Trifluoro-N-hydroxy-2-(4-iodo-2-methyl-phenylamino)-benzamide 5-Chloro-3,4-difluoro-N-hydroxy-2-(4-iodo-2-methyl-phenylamino)- benzamide 5-Bromo-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-N-hydroxy- benzamide
N-Hydroxy-2-(4-iodo-2-methyl-phenylamino)-4-nitro-benzamide 3,4,5-Trifluoro-2-(2-fluoro-4-iodo-phenylamino)-N-hydroxy-benzamide 5-Chloro-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-N-hydroxy- benzamide 5-Bromo-2-(2-chloro-4-iodo-phenylamino)-3,4-difluoro-N-hydroxy- benzamide 2-(2-Fluoro-4-iodo-phenylamino)-N-hydroxy-4-nitro-benzamide 2-(2-Chloro-4-iodo-phenylamino)-3,4,5-trifluoro-N-hydroxy-benzamide 5-Chloro-2-(2-chloro-4-iodo-phenylamino)-3,4-difluoro-N-hydroxy- benzamide
5-Bromo-2-(2-bromo-4-iodo-phenylamino)-3,4-difluoro-N-hydroxy- benzamide 2-(2-Chloro-4-iodo-phenylamino)-N-hydroxy-4-methyl-benzamide 2-(2-Bromo-4-iodo-phenylamino)-3,4,5-trifluoro-N-hydroxy-benzamide 2-(2-Bromo-4-iodo-phenylamino)-5-chloro-3,4-difluoro-N-hydroxy- benzamide 2-(2-Bromo-4-iodo-phenylamino)-N-hydroxy-4-nitro-benzamide 4-Fluoro-2-(2-fluoro-4-iodo-phenylamino)-N-hydroxy-benzamide 3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-N-hydroxy-benzamide 2-(2-Chloro-4-iodo-phenylamino)-4-fluoro-N-hydroxy-benzamide
2-(2-Chloro-4-iodo-phenylamino)-3,4-difluoro-N-hydroxy-benzamide
2-(2-Bromo-4-iodo-phenylamino)-4-fluoro-N-hydroxy-benzamide
2-(2-Bromo-4-iodo-phenylamino)-3,4-difluoro-N-hydroxy-benzamide FORMULA (II) COMPOUND TABLE (continued, page 6 of 7)
N-Cyclopropylmethoxy-3,4,5-trifluoro-2-(4-iodo-2-methyl-phenylamino)- benzamide
5-Chloro-N-cyclopropylmethoxy-3,4-difluoro-2-(4-iodo-2-methyl- phenylamino)-benzamide 5-Bromo-N-cyclopropylmethoxy-3,4-difluoro-2-(2-fluoro-4-iodo- phenylamino)-benzamide N-Cyclopropylmethoxy-2-(4-iodo-2-methyl-phenylamino)-4-nitro- benzamide N-Cyclopropylmethoxy-3,4,5-trifluoro-2-(2-fluoro-4-iodo-phenylamino)- benzamide 5 -Chloro-N-cyclopropylmethoxy-3 ,4-difluoro-2-(2-fluoro-4-iodo- phenylamino)-benzamide
5-Bromo-2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-
3 ,4-difluoro-benzamide N-Cyclopropylmethoxy-2-(2-fluoro-4-iodo-phenylamino)-4-nitro- benzamide 2-(2-Chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4,5-trifluoro- benzamide 5-Chloro-2-(2-chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-
3 ,4-difluoro-benzamide 5-Bromo-2-(2-bromo-4-iodo-phenylamino)-N-ethoxy-3,4-difluoro- benzamide
2-(2-Chloro-4-iodo-phenylamino)-N-ethoxy-4-nitro-benzamide 2-(2-Bromo-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4,5-trifluoro- benzamide 2-(2-Bromo-4-iodo-phenylamino)-5-chloro-N-cyclopropylmethoxy- 3,4-difluoro-benzamide
2-(2-Bromo-4-iodo-phenylamino)-N-cyclopropylmethoxy-4-nitro- benzamide FORMULA (II) COMPOUND TABLE (continued, page 7 of 7)
N-Cyclopropylmethoxy-4-fluoro-2-(2-fluoro-4-iodo-phenylamino)- benzamide
N-Cyclopropylmethoxy-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)- benzamide 2-(2-Chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-4-fluoro- benzamide 2-(2-Chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3 ,4-difluoro- benzamide 2-(2-Bromo-4-iodo-phenylamino)-N-cyclopropylmethoxy-4-fluoro- benzamide 2-(2-Bromo-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro- benzamide.
In the most preferred embodiment of this invention, 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:
2-(2-Chloro-4-iodophenylamino)-N-cyclopropylmethoxy- 3,4-difluorobenzamide (PD 184352); 2-(2-Methyl-4-iodoρhenylamino)-N- hydroxy-4-fluorobenzamide (PD 170611); 2-(2-Methyl-4-iodophenylamino)-N- hydroxy-3,4-difluoro-5-bromobenzamide (PD171984); 2-(2-Methyl- 4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluoro-5-bromobenzamide
(PD 177168); 2-(2-Methyl-4-iodophenylamino)-N-cyclobutylmethoxy- 3,4-difluoro-5-bromobenzamide (PD 180841); 2-(2-Chloro- 4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluoro-5-bromobenzamide (PD 184161); 2-(2-Chloro-4-iodophenylamino)-N-hydroxy-3,4-difluoro- 5-bromobenzamide (PD184386); 2-(2-Chloro-4-iodophenylamino)-N- cyclobutylmethoxy-3,4-difluorobenzamide (PD 185625); 2-(2-Chloro- 4-iodophenylamino)-N-hydroxy-4-fluorobenzamide (PD 185848); 2-(2-Methyl-4-iodophenylamino)-N-hydroxy-3,4-difluorobenzamide (PD 188563); 2-(2-Methyl-4-iodophenylamino)-N-cyclopropylmethoxy- 3,4,5-trifluorobenzamide (PD 198306); and 2-(2-Chloro-4-iodophenylamino)- N-cyclopropylmethoxy-4-fluorobenzamide (PD 203311); and the benzoic acid derivatives thereof. For example, 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- methylphenylamino)-benzoic acid (PD 296767); and 5-chloro-N- cyclopropylmethoxy -3,4-difluoro-2-(4-iodo-2-methylphenylamino)-benzamide (PD 298127).
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.
Other features and advantages of the invention are apparent from the detailed description, examples, and claims set forth.
BRIEF DESCRIPTION OF THE FIGURES
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-δ (IFN-δ) 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).
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).
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 of the compounds to inhibit PHA and
Con A-induced proliferation with little or no toxicity (MTT). The compounds tested were PD 184352;
2-(2-methyl-4-iodophenylamino)-N-hydroxy-3,4-difluoro-5- bromobenzamide (PD 171984); 2-(2-methyl-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluoro-
5-bromobenzamide (PD 177168);
2-(2-chloro-4-iodophenylamino)-N-cycloproplymethoxy-3,4-difluoro- 5-bromobenzamide (PD 184161); and
2-(2-chloro-4-iodophenylamino)-N-hydroxy-3,4-difluoro- 5-bromobenzamide (PD 184386).
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-δ. 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.
DETAILED DESCRIPTION OF THE INVENTION
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 of the transplanted organ, limb, cell(s), or tissue. For example, the method is practiced by administering a phenyl amine MEK inhibitor that is described in WO 98/37881. These are selective MEK inhibitors, namely they inhibit MEK 1 and MEK 2 without substantial inhibition of other enzymes. 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 of the 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. The complete disclosure of United States Patent Number 5,525,625 is hereby incorporated by reference. An example of a MEK inhibitor is 2-(2-amino-3-methoxyphenyl)-4-oxo-4H-[l]benzopyran. Specifically, a compound is a MEK inhibitor if a compound shows activity in the assay titled "Cascade Assay for Inhibitors of the MAP Kinase Pathway," column 6, line 36 to column 7, line 4 of the 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 of the above-referenced patent. A. Terms
Some of the terms used herein are defined below in combination with their usage throughout this disclosure.
As used herein, the term "aryl" means a cyclic, bicyclic, or tricyclic aromatic ring moiety having from five to twelve carbon atoms. Examples of 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.
The term "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. The 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. Examples of substituted heteroaryl groups include chloropyranyl, methylthienyl, fluoropyridyl, amino- 1,4-benzisoxazinyl, nitroisoquinolinyl, and hydroxyindolyl.
The heteroaryl groups can be bonded through oxygen to make heteroaryloxy groups, for example thienyloxy, isothiazolyloxy, benzofuranyloxy, pyridyloxy, and 4-methylisoquinolinyloxy.
The term "alkyl" means straight and branched chain aliphatic groups. Typical alkyl groups include methyl, ethyl, isopropyl, tert.-butyl,
2,3-dimethylhexyl, and 1,1-dimethylpentyl. The 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, l,l-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. The 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. For example, 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.
The term "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, morpholinyi, piperazinyl, pyrrolidinyl, 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-hydroxycyclohexyl, 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.
The term "patient" means all animals including humans. Examples of patients include humans, cows, dogs, cats, goats, sheep, horses, and pigs. Selective MEK 1 or MEK 2 inhibitors are those compounds which inhibit the MEK 1 or MEK 2 enzymes, respectively, without substantially inhibiting other enzymes such as MKK3, PKC, Cdk2A, phosphorylase kinase, EGF, and PDGF receptor kinases, and C-src. In general, a selective MEK 1 or MEK 2 inhibitor has an IC50 for MEK 1 or MEK 2 that is at least one-fiftieth (1/50) that of its IC50 for one of the above-named other enzymes. Preferably, a selective inhibitor has an IC 0 that is at least 1/100, more preferably 1/500, and even more preferably 1/1000, 1/5000, or less than that of its IC50or one or more of the above- named enzymes.
B. Administration and Formulation
The MEK inhibitors of the 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. Examples of 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 of the required particle size in the case of dispersions and by the use of surfactants.
These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride, and the like. Prolonged absorption of the 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. In such solid dosage forms, the active compound is admixed with at least one inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a) fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, as for example, carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as for example, glycerol, (d) disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate, (e) solution retarders, as for example paraffin, (f) absorption accelerators, as for example, quaternary ammonium compounds, (g) wetting agents, as for example, cetyl alcohol and glycerol monostearate, (h) adsorbents, as for example, kaolin and bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents.
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 of the 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 of the above-mentioned excipients. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs. In addition to the active compounds, 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-butyleneglycol, 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. Besides such inert diluents, 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 of the 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 of the 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. For example, the dosage can depend on a numbers of factors including the requirements of the patient, the severity of the condition being treated, and the pharmacological activity of the 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. The term "pharmaceutically acceptable salts, esters, amides, and prodrugs" as used herein refers to those carboxylate salts, amino acid addition salts, esters, amides, and prodrugs of the compounds of the 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, of the compounds of the invention. The term "salts" refers to the relatively non-toxic, inorganic and organic acid addition salts of compounds of the present invention. These salts can be prepared in situ during the final isolation and purification of the 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. These may include cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations including, but not limited to 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.)
Examples of pharmaceutically acceptable, non-toxic esters of the compounds of this invention include Cj-Cβ 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. C1-C4 alkyl esters are preferred. Esters of the compounds of the present invention may be prepared according to conventional methods.
Examples of pharmaceutically acceptable, non-toxic amides of the compounds of this invention include amides derived from ammonia, primary C\ -C alkyl amines and secondary Cj-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 of the compounds of the invention may be prepared according to conventional methods.
The term "prodrug" refers to compounds that are rapidly transformed in vivo to yield the parent compound of the above formula, for example, by hydrolysis in blood. A thorough discussion is provided in T. Higuchi and
V. Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drus Design, ed. Edward B.
Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference.
In addition, the compounds of the present method can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. In general, the solvated forms are considered equivalent to the unsolvated forms for the purposes of the present invention.
Some of the compounds of the present method can exist in different stereoisometric forms by virtue of the presence of chiral centers. It is contemplated that all stereoisometric forms of the compounds as well as mixtures thereof, including racemic mixtures, form part of this invention. C. Synthesis
The examples presented below are intended to illustrate particular embodiments of the invention and are not intended to limit the scope of the specification, including the claims, in any way. After the priority date of the present disclosure, related syntheses and MEK inhibition data were also published in WO 99/01421 and WO 99/01426, hereby incorporated by reference.
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.
Scheme 1
base
where 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. 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. The free acids can also be reacted with an alcohol of the formula HOR7
(where R7 is other than hydrogen, for example methyl) to produce the corresponding ester. Reaction of the benzoic acid with an alcohol can be carried out in the presence of a coupling agent. Typical coupling reagents include
2-ethoxy- 1 -ethoxycarbonyl- 1 ,2-dihydroquinoline (EEDQ), 1,3-dicyclohexylcarbodiimide (DCC), bromo-tris(pyrrolidino)- phosphonium hexafluorophosphate (PyBrOP), and (benzotriazolyloxy) tripyrrolidino phosphonium hexafluorophosphate (PyBOP). 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 of the coupling reagent is added. A base such as triethylamine or diisopropylethylamine 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 of the invention, Formula (I )where Z is CONR6R7, are readily prepared by reacting the foregoing benzoic acids with an amine of the formula HNR6R7. The reaction is carried out by reacting approximately equimolar quantities of the 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. The hydrazides (z = CONHNRj QR\ \) are similarly prepared by coupling a benzoic acid with a hydrazine of the formula H2HNR10Rn.
The benzyl alcohols of the invention, compounds of Formula (I) where Z is CH2OR6 and R6 is hydrogen, are readily prepared by reduction of the corresponding benzoic acid according to the following Scheme 2.
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.
The following detailed examples illustrate specific compounds provided by this invention. EXAMPLE 1 4-Fluoro-2-(4-iodo-2-methylphenylamino)benzoic acid
To a stirring solution comprised of 3.16 g (0.0133 mol) of 2-amino-5- iodotoluene in 5 mL of tetrahydrofuran at -78°C was added 10 mL (0.020 mol) of a 2.0 M lithium diisopropylamide in tetrahydrofuran heptane/ethenylbenzene
(Aldrich) solution. The resulting green suspension was stirred vigorously for 15 minutes, after which time a solution of 1.00 g (0.00632 mol) of 2,4-difluorobenzoic acid in 10 mL of tetrahydrofuran was added. The reaction temperature was allowed to increase slowly to room temperature, at which temperature it was stirred for 2 days. The reaction mixture was concentrated.
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. 10 mm of Hg) to afford 1.10 g (47%) of the desired material; mp 224-229.5°C; lH NMR (400 MHz; DMSO): δ 9.72 (s, IH), 7.97 (dd, IH, J = 7.0, 8.7 Hz), 7.70 (d, IH, J = 1.5 Hz), 7.57 (dd, IH, J = 8.4, 1.9 Hz), 7.17 (d, IH, J = 8.2 Hz), 6.61-6.53 (m, 2H), 2.18 (s, 3H); 13C NMR (100 MHz; DMSO): δ 169.87, 167.60, 165.12, 150.17, 150.05, 139.83,
138.49, 136.07, 135.31, 135.20, 135.07, 125.60, 109.32, 105.09, 104.87, 99.72, 99.46, 89.43, 17.52;
19F NMR (376 MHz; DMSO): δ -104.00 to -104.07 (m);
IR (KBr) 1670 (C = O stretch) cm'1; MS (Cl) M+l = 372.
Analysis calculated for C1 H11FINO2:
C, 45.31; H, 2.99; N, 3.77. Found: C, 45.21; H, 2.77; N, 3.64.
EXAMPLES 2-30 By following the general procedure of Example 1 , the following benzoic acids and salts of Formula (I) were prepared. Example Compound MP °C No.
2 3,4,5-Trifluoro-2-(4-iodo-2-methyl-phenylamino)- 206-210 benzoic acid
3 3 ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic 240.5 -244.5 acid
4 5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl- 259.5-262 phenylamino)-benzoic acid
5 5-Chloro-2-(2-chloro-4-iodo-phenylamino)-benzoic acid 255-260
6 5-Chloro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid 234-238
7 Sodium 5-Chloro-2-(4-iodo-2-methyl-phenylamino)- 310-320 DEC benzoate
8 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-benzoic acid 239.5-240
9 2-(2-Chloro-4-iodo-phenylamino)-5-nitro-benzoic acid 289-293
10 4-Fluoro-2-(3-fluoro-4-iodo-2-methyl-phenylamino)- 233-235 benzoic acid
11 2-(4-lodo-2-methyl-phenylamino)-5-nitro-benzoic acid 264-267
12 2-(2-Fluoro-4-iodo-phenylamino)-5-nitro-benzoic acid 256-258
13 2-(4-Bromo-2-methyl-phenylamino)-4-fluoro-benzoic 218.5-220 acid
14 2-(2-Bromo-4-iodo-phenylamino)-5-nitro-benzoic acid 285-288 DEC
15 2-(4-Bromo-2-methyl-phenylamino)-3,4-difluoro- 230-234 benzoic acid
16 3-Fluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid 218-221
17 3,4-Difluoro-2-(4-iodo-2-methoxy-phenylamino)- 230-233 benzoic acid
18 4-Chloro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid 245-255 DEC
Example Compound MP °C No.
19 2-(4-Iodo-2-methyl-phenylamino)-benzoic acid 218-223
20 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid 243-46
21 5-Iodo-2-(4-iodo-2-methyl-phenylamino)-benzoic acid 241-245
22 2,3,5-Trifluoro-4-(4-iodo-2-methyl-phenylamino)- 218-222 benzoic acid
23 4-Fluoro-2-(3-chloro-4-iodo-2-methyl-phenylamino)- 248-252.5 benzoic acid
24 2-(4-Iodo-phenylamino)-5-methoxy-benzoic acid 208-211
25 3-Chloro-2-(2-chloro-4-iodo-phenylamino)-benzoic acid 232-233
26 2-Fluoro-6-(4-iodo-2-methyl-phenylamino)-benzoic acid 179-182
27 4-Fluoro2-(2,3-dimethyl-4-iodo-2-methyl- 258-261 phenylamino)benzoic acid
28 5-Methyl-2-(4-iodo-2-methyl-phenylamino)-benzoic 209.5-211 acid
29 2-Chloro-6-(4-iodo-2-methyl-phenylamino)-benzoic acid 171 - 175
30 2-(4-Iodo-2-methyl-phenylamino)-4-nitro-benzoic acid 251-263
EXAMPLE 31 5-Chloro-N-(2-hvdroxyethyl)-2-(4-iodo-2-methyl-phenylamino -benzamide
To a stirring solution comprised of 0.1020 g (0.2632 mmol) of 5-chloro- 2-(4-iodo-2-methyl-phenylamino)-benzoic acid, 0.1 mL (1.7 mmol) of ethanolamine, and 0.05 mL (0.29 mmol) of diisopropylethylamine in 5 mL of a
1:1 (v/v) tetrahydrofuran-dichloromethane solution was added 0.15 g (0.29 mmol) of solid PyBOP powder directly. The reaction mixture was stirred at room temperature overnight. The solvent was removed in vacuo. The crude residue was partitioned between ether (50 mL) and 10% aqueous hydrochloric acid (50 mL). The organic phase was washed with 10% aqueous sodium hydroxide (50 mL), dried (MgSO_ι) and concentrated in vacuo to afford a yellow-brown oil which was crystallized from hexanes-ether to afford 0.0831 g (73%) of a green-yellow powder; mp l 20-121 °C; lH NMR (400 MHz; CDCI3): δ 9.11 (s, IH), 7.56 (d, IH, J = 1.4 Hz), 7.46-7.41
(m, 2H), 7.20 (dd, IH, J = 8.9, 2.4 Hz), 7.00 (t, 2H, J = 9.6 Hz), 6.55 (broad t, IH), 3.86 (t, 2H, J = 5.0 Hz), 3.61 (dd, 2H, J = 10.1, 5.5 Hz), 2.23 (s, 3H), 1.56 (broad s, IH); IR (KBr) 3297 (O-H stretch), 1627 (C = O stretch) cm" l ;
MS (CI) M+1 = 431. Analysis calculated for C16H16CIIN2O2:
C, 44.62; H, 3.74; N, 6.50. Found: 44.63; H, 3.67; N, 6.30.
EXAMPLES 32-48
By following the general procedure of Example 31, the following benzamides were prepared by reacting the corresponding benzoic acid with the corresponding amine.
Example Compound MP °C
No.
32 4-Methoxy-N-(4-methoxy-phenyl)-3-nitro- 153.5-156 benzamide
33 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)- 158 benzamide
34 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N- 102.5-104.5 methyl-benzamide
35 N-Ethyl-4-fluoro-2-(4-iodo-2-methyl-phenylamino)- 90-91 benzamide
36 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N,N- oil dimethyl-benzamide
37 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(lH- 285-288 DEC tetrazol-5-yl)-benzamide
38 5-Bromo-2-(4-iodo-2-methyl-phenylamino)- 180-182 benzamide
39 5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N,N- 137-138 dimethyl-benzamide Example Compound MP °C
No.
40 [5-Chloro-2-(4-iodo-2-methyl-phenylamino 170-173 benzoylamino] -acetic acid
41 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N- 69-71 propyl-benzamide
42 5-Bromo-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl- 132-133.4 phenylamino)-benzamide
43 N,N-Diethyl-4-fluoro-2-(4-iodo-2-methyl- oil phenylamino)-benzamide
44 4-Fluoro-N-{3-[4-(2-hydroxy-ethyl)-piperazin-l-yl]- 122-124 propyl } -2-(4-iodo-2-methyl-phenylamino)- benzamide
45 N,N-Diethyl-2-(4-iodo-2-methyl-phenylamino)-5- 91-93 nitro-benzamide
46 N-Butyl-4-fluoro-2-(4-iodo-2-methyl-phenylamino)- 97-99 benzamide
47 5-Chloro-N,N-diethyl-2-(4-iodo-2-methyl- 118-120 phenylamino)-benzamide
48 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N,N- 142.5-144 dimethyl-benzamide
EXAMPLE 49 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-benzyl alcohol
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid (0.50 g, 1.35 mmol) was dissolved in 6 mL (6 mmol) of cold 1.0 M borane- tetrahydrofuran complex in tetrahydrofuran solution. The reaction mixture was stirred under nitrogen atmosphere at room temperature overnight. The reaction was quenched with 80 mL of methanol. Concentration in vacuo produced a clear tan oil which was purified by MPLC. Elution with dichloromethane afforded 0.4285 g (89%) of a white solid; mp 99-100.5°C; lH NMR (400 MHz; DMSO): δ 7.57 (d, IH, J=1.7 Hz), 7.45 (dd, IH, J=8.4,
1.9 Hz), 7.39 (s, IH), 7.29 (t, IH, J=7.5 Hz), 6.89 (d, IH, J=8.4 Hz), 6.67-6.60 (m, IH), 5.47 (t, IH, J=5.5 Hz), 4.49 (d, 2H, 5.1 Hz), 2.14 (s, 3H); IR (KBr) 3372 (O-H stretch) cm"1;
MS (Cl) M+l = 358.
Analysis calculated for C14H13FINO:
C, 47.08; H, 3.67; N, 3.92. Found: C, 47.17; H, 3.75; N, 3.72.
EXAMPLE 50-52 The following benzyl alcohols were prepared by the general procedure of Example 49.
Example No. Compound MP °C
50 [5-Chloro-2-(4-iodo-2-methyl-phenylamino)- 82-85 phenyl] -methanol
51 [2-(4-Iodo-2-methyl-phenylamino)-5-nitro-phenyl]- 126.5-128.5 methanol
52 [5-Bromo-2-(4-iodo-2-methyl-phenylamino)- 60.5-63.5 phenyl] -methanol
Several invention compounds of Formula (I) were prepared utilizing combinatorial synthetic techniques. The general procedure is as follows:
To a 0.8-mL autosampler vial in a metal block was added 40 μL of a 0.5 M solution of the acid in DMF and 40 μL of the reagent amine (2 M solution in Hunig's base and 1 M in amine in DMF). A 0.5 M solution of PyBrop was freshly prepared and 50 μL were added to the autosampler vial. The reaction was allowed to stand for 24 hours.
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 μM spherical silica, pore size 115 A derivatized with C-l 8, 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 the yield.
EXAMPLES 53-206 The following compounds of Formula I were prepared by combinatorial methodology:
Example Compound MS
No. M-H
53 5-Bromo-3,4-difluoro-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl- 510 phenylamino)-benzamide
54 N-(2,3-Dihydroxy-propyl)-3,4-difluoro-2-(4-iodo-2-methyl- 462 phenylamino)-benzamide
55 5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2- 577 piperidin- 1 -y 1-ethy l)-benzamide
56 3,4-Difluoro-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl- 432 phenylamino)-benzamide
57 N-(2,3-Dihydroxy-propyl)-4-fluoro-2-(4-iodo-2-methyl- 444 phenylamino)-benzamide
58 3,4-Difluoro-N-(3-hydroxy-propyl)-2-(4-iodo-2-methyl- 446 phenylamino)-benzamide
59 5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N- 564 (2-pyrrolidin- 1 -yl-ethyl)-benzamide
60 5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N- 571 (2-pyridin-4-yl-ethyl)-benzamide
61 4-Fluoro-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- 414 benzamide
62 5-Bromo-N-(3-dimethylamino-propyl)-3,4-difluoro-2-(4-iodo- 551 2-methyl-ρhenylamino)-benzamide Example Compound MS
No. M-H
63 5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N- 580 (2-morpholin-4-yl-ethyl)-benzamide
64 3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-morpholin- 501 4-yl-ethyl)-benzamide
65 3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-pyrrolidin- 485 1 -yl-ethyl)-benzamide
66 3 ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-pyridin-4-yl- 493 ethyl)-benzamide
67 N-(3-Dimethylamino-propyl)-3,4-difluoro-2-(4-iodo-2-methyl- 473 phenylamino)-benzamide
68 N-Benzyl-4-fluoro-2-(4-iodo-2-methyl-phenylamino)-benzamide 460
69 2-(4-Bromo-2-methyl-phenylamino)-3 ,4-difluoro-N-(2-hydroxy- 384 ethyl)-benzamide
70 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-morpholin-4-yl- 483 ethyl)-benzamide
71 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(3 -piperidin- 1 -yl- 495 propyl)-benzamide
72 3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(3-piperidin- 513 1 -yl-propyl)-benzamide
73 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-thiophen-2-yl- 480 ethyl)-benzamide
74 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-pyrrolidin- 1 -yl- 467 ethyl)-benzamide
75 2-(4-Bromo-2-methyl-phenylamino)-3,4-difluoro-N-(2-morpholin- 453 4-yl-ethyl)-benzamide
76 5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N- 557 pyridin-4-ylmethyl-benzamide
77 3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-pyridin- 479 4-ylmethyl-benzamide
78 2-(4-Bromo-2-methyl-phenylamino)-N-(3-dimethylamino-propyl)- 425 3 ,4-difluoro-benzamide Example Compound MS
No. M-H
79 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-pyridin-4-ylmethyl- 461 benzamide
80 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-pyridin-4-yl- 475 ethyl)-benzamide
81 2-(4-Bromo-2-methyl-phenylamino)-3,4-difluoro-N-(2-pyridin- 445 4-yl-ethyl)-benzamide
82 2-(4-Bromo-2-methyl-phenylamino)-3,4-difluoro-N-(3-hydroxy- 400 propyl)-benzamide
83 2-(4-Bromo-2-methyl-phenylamino)-3,4-difluoro-N-(2-pyrrolidin- 437 1 -yl-ethyl)-benzamide
84 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-phenethyl- 474 benzamide
85 2-(4-Bromo-2-methyl-phenylamino)-3,4-difluoro-N-(2-thiophen- 450 2-yl-ethyl)-benzamide
86 2-(4-Bromo-2-methyl-phenylamino)-3,4-difluoro-N-pyridin- 431 4-ylmethyl-benzamide
87 2-(4-Bromo-2-methyl-phenylamino)-3,4-difluoro-N-phenethyl- 444 benzamide
88 2-(4-Bromo-2-methyl-phenylamino)-3,4-difluoro-N-(2 -piperidin- 451 1 -yl-ethyl)-benzamide
89 5-Chloro-N-{3-[4-(2-hydroxy-ethyl)-piperazin-l -yl]-propyl}- 557* 2-(4-iodo-2-methyl- phenylamino)- benzamide
90 5-Fluoro-N-{3-[4-(2-hydroxy-ethyl)-piperazin-l-yl]-propyl}- 541 * 2-(4-iodo-2-methyl- phenylamino)- benzamide
91 2-(4-Iodo-2-methyl-phenylamino)-5-nitro-N-pyridin-4-yl methyl- 487 benzamide
92 5-Bromo-N-{3-[4-(2-hydroxy-ethyl)-piperazin-l-yl]-propyl}- 601 * 2-(4-iodo-2-methyl- phenylamino)- benzamide
93 5-Chloro-N-(2-diethylamino-ethyl)-2-(4-iodo-2-methyl- 486* phenylamino)- benzamide Example Compound MS
No. M-H
94 5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N-(2-piperidin-l-yl- 497* ethyl)-benzamide
95 (3-Hydroxy-pyrrolidin- 1 -yl)-[2-(4-iodo-2-methyl-phenylamino)- 466 5-nitro-phenyl]-methanone
96 5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N-(2-pyrrolidin- 1 -yl- 484* ethyl)-benzamide
97 5-Bromo-N-(2-diethylamino-ethyl)-2-(4-iodo-2-methyl- 530* phenylamino)- benzamide
98 N-{2-[Bis-(2-hydroxy-ethyl)-amino]-ethyl}-5-chloro-2-(4-iodo- 518* 2-methyl- phenylamino)- benzamide
99 N-{2-[Bis-(2-hydroxy-ethyl)-amino]-ethyl}-5-bromo-2-(4-iodo- 562* 2-methyl- phenylamino)- benzamide
100 [5-Bromo-2-(4-iodo-2-methyl-phenylamino)-phenyl]-(3-hydroxy- 499 pyrrolidin- 1 -yl)-methanone
101 2-(4-Iodo-2-methyl-phenylamino)-5-nitro-benzoic acid phenethyl 501 ester
102 N-{3-[4-(2-Hydroxy-ethyl)-piperazin-l-yl]-propyl}-2-(4-iodo- 568* 2-methyl-phenylamino)- benzamide
103 [5-Chloro-2-(4-iodo-2-methyl-phenylamino)-phenyl]-(3-hydroxy- 455 pyrrolidin- 1 -yl)-methanone
104 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-pyridin-4-ylmethyl- 460 benzamide
105 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-(2-pyrrolidin-l-yl- 528* ethyl)-benzamide
106 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-(2-piperidin-l-yl- 542* ethyl)-benzamide
107 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-pyrrolidin-l -yl- 468* ethyl)-benzamide
108 5-Chloro-N-(3-dimethylamino-propyl)-2-(4-iodo-2-methyl- 472* phenylamino)-benzamide
109 N-{2-[Bis-(2-hydroxy-ethyl)-amino]-ethyl}-5-fluoro-2-(4-iodo- 502* 2-methyl- phenylamino)- benzamide Example Compound MS
No. M-H
110 5-Chloro-N-(3-hydroxy-propyl)-2-(4-iodo-2-methyl- 445* phenylamino)-benzamide
111 5-Chloro-N-(3-diethylamino-2-hydroxy-propyl)-2-(4-iodo- 516* 2-methyl-phenylamino)- benzamide
112 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-piperidin-l-yl- 482* ethyl)-benzamide
113 5-Bromo-N-(3-hydroxy-propyl)-2-(4-iodo-2-methyl- 489* phenylamino)-benzamide
114 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-(3-piperidin-l-yl- 556* propyl)-benzamide
115 N-{2-[Bis-(2-hydroxy-ethyl)-amino]-ethyl}-2-(4-iodo-2-methyl- 529* phenylamino)-5-nitro- benzamide
116 5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N-(2-morpholin-4-yl- 500* ethyl)-benzamide
117 5-Chloro-N-(3-diethylamino-propyl)-2-(4-iodo-2-methyl- 500* phenylamino)-benzamide
118 5-Chloro-N-(2-diisopropylamino-ethyl)-2-(4-iodo-2-methyl- 514* phenylamino)-benzamide
119 5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N-(3-piperidin- 1 -yl- 512* propyl)-benzamide
120 2-(4-Iodo-2-methyl-phenylamino)-5-nitro-N-(2-piperidin-l-yl- 509* ethyl)-benzamide
121 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-(2 -piperazin- 1-yl- 544* ethyl)-benzamide
122 N-(2-Diethylamino-ethyl)-5-fluoro-2-(4-iodo-2-methyl- 470* phenylamino)-benzamide
123 5-Bromo-N-(3-dimethylamino-propyl)-2-(4-iodo-2-methyl- 516* phenylamino)-benzamide
124 N-(3-Hydroxy-propyl)-2-(4-iodo-2-methyl-phenylamino)-5-nitro- 456* benzamide
125 5-Fluoro-N-(3-hydroxy-propyl)-2-(4-iodo-2-methyl- 429* phenylamino)-benzamide Example Compound MS
No. M-H
126 N-(3-Diethylamino-propyl)-5-fluoro-2-(4-iodo-2-methyl- 484* phenylamino)-benzamide
127 N-(3-Diethylamino-propyl)-2-(4-iodo-2-methyl-phenylamino)- 511 * 5-nitro-benzamide
128 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-(2-morpholin-4-yl- 544* ethyl)-benzamide
129 2-(4-Iodo-2-methyl-phenylamino)-5-nitro-N-(3-piperidin- 1 -yl- 523 * propyl)-benzamide
130 [5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-phenyl]-(3-hydroxy- 439 pyrrolidin- 1 -yl)-methanone
131 5-Bromo-N-(2-diisopropylamino-ethyl)-2-(4-iodo-2-methyl- 558* phenylamino)-benzamide
132 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-moφholin-4-yl- 484* ethyl)-benzamide
133 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(3-piperidin-l-yl- 496* propyl)-benzamide
134 [5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-phenyl]- 482 [4-(2-hydroxy-ethyl)-piperazin- 1 -
135 N-(3-Diethylamino-2-hydroxy-propyl)-5-fluoro-2-(4-iodo- 500* 2-methyl-phenylamino)- benzamide
136 [5-Chloro-2-(4-iodo-2-methyl-phenylamino)-benzoylamino]- 443 acetic acid
137 2-(4-Iodo-2-methyl-phenylamino)-5-nitro-N-(2-pyrrolidin-l -yl- 495* ethyl)-benzamide
138 N-(3-Dimethylamino-propyl)-2-(4-iodo-2-methyl-phenylamino)- 483 * 5-nitro-benzamide
139 N-(2-Diisopropylamino-ethyl)-5-fluoro-2-(4-iodo-2-methyl- 498* phenylamino)- benzamide
140 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-thiobenzoic acid S- 490 phenethyl ester
141 5-Chloro-2-(4-iodo-2-methyl-phenylamino)-thiobenzoic acid S- 506 phenethyl ester Example Compound MS
No. M-H
142 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-thiobenzoic acid S- 536 benzyl ester
143 2-(4-Iodo-2-methyl-phenylamino)-5-nitro-thiobenzoic acid S- 503 benzyl ester
144 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-thiobenzoic acid S- 476 benzyl ester
145 5-Chloro-2-(4-iodo-2-methyl-phenylamino)-thiobenzoic acid S- 492 benzyl ester
146 N-Cyclopropyl-5-fluoro-2-(4-iodo-2-methyl-phenylamino)- 409 benzamide
147 5-Chloro-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- 429 benzamide
148 5-Fluoro-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- 413 benzamide
149 N-Benzyloxy-5-fluoro-2-(4-iodo-2-methyl-phenylamino)- 475 benzamide
150 N-Benzyloxy-5 -bromo-2-(4-iodo-2-methyl-pheny lamino)- 593 * benzamide
151 2-(4-Iodo-2-methyl-phenylamino)-5-nitro-N-(4-sulfamoyl- 567 benzyl)-benzamide
152 5-Bromo-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- 473 benzamide
153 N-(2-Hydroxy-ethyl)-5-iodo-2-(4-iodo-2-methyl-phenylamino)- 521 benzamide
Example Compound MS
No. M-H
154 N-(2-Hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)-5-nitro- 440 benzamide
155 2-(4-Iodo-2-methyl-phenylamino)-N-methyl-5-nitro-N-phenyl- 486 benzamide
156 5-Chloro-N-cyclopropyl-2-(4-iodo-2-methyl-phenylamino)- 425 benzamide
157 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-methyl-N-phenyl- 459 benzamide
158 N-Allyl-5-fluoro-2-(4-iodo-2-methyl-phenylamino)-benzamide 409
159 N-Benzyloxy-5-iodo-2-(4-iodo-2-methyl-phenylamino)- 583 benzamide
160 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(4-sulfamoyl- 538 benzyl)-benzamide
161 N-Allyl-5 -chloro-2-(4-iodo-2-methy 1-pheny lamino)-benzamide 425
162 N-Cyclopropyl-2-(4-iodo-2-methyl-phenylamino)-5-nitro- 436 benzamide
163 5-Bromo-N-cyclopropyl-2-(4-iodo-2-methyl-phenylamino)- 469 benzamide
164 5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N-methyl-N-phenyl- 475 benzamide
165 5-Iodo-2-(4-iodo-2-methyl-phenylamino)-N-(4-sulfamoyl-benzyl)- 646 benzamide
166 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-(4-sulfamoyl- 598 benzyl)-benzamide
167 N-Allyl-2-(4-iodo-2-methyl-phenylamino)-5-nitro-benzamide 436
Example Compound MS
No. M-H
168 2-(4-Iodo-2-methyl-phenylamino)-5-nitro-N-(4-sulfamoyl- 565 benzyl)-benzamide
169 N-Allyl-5-bromo-2-(4-iodo-2-methyl-phenylamino)-benzamide 469
170 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(3-methyl-benzyl)- 473 benzamide
171 N-Cyclopropyl-5-iodo-2-(4-iodo-2-methyl-phenylamino)- 517 benzamide
172 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-methyl-N-phenyl- 519 benzamide
173 N-Benzyloxy-2-(4-iodo-2-methyl-phenylamino)-5-nitro- 502 benzamide
174 N-Cyclohexyl-5-iodo-2-(4-iodo-2-methyl-phenylamino)- 559 benzamide
175 N-Allyl-5-iodo-2-(4-iodo-2-methyl-phenylamino)-benzamide 517
176 5-Iodo-2-(4-iodo-2-methyl-phenylamino)-N-(3-methyl-benzyl)- 581 benzamide
177 2-(4-Iodo-2-methyl-phenylamino)-N-(3-methyl-benzyl)-5-nitro- 500 benzamide
178 5-Iodo-2-(4-iodo-2-methyl-phenylamino)-N-methyl-N-phenyl- 567 benzamide
179 N-Cyclohexy 1-5 -fluoro-2-(4-iodo-2-methy 1-pheny lamino)- 451 benzamide
180 5-Chloro-N-cyclohexyl-2-(4-iodo-2-methyl-phenylamino)- 467 benzamide
181 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-(3-methyl-benzyl)- 533 benzamide
182 5-Bromo-N-cyclohexyl-2-(4-iodo-2-methyl-phenylamino)- 511 benzamide
183 5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N-(3-methyl-benzyl)- 489 benzamide
184 N-Cyclohexyl-2-(4-iodo-2-methyl-phenylamino)-5-nitro- 478 benzamide Example Compound MS
No. M-H
185 N-Benzyloxy-5-bromo-2-(4-iodo-2-methyl-phenylamino)- 538 benzamide
186 N-Benzyloxy-5-fluoro-2-(4-iodo-2-methyl-phenylamino)- 477 benzamide
187 5-Chloro-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- 431 benzamide
188 5-Bromo-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- 475 benzamide
189 2-(4-Iodo-2-methyl-phenylamino)-N-methyl-5-nitro-N-phenyl- 488 benzamide
190 5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N-methyl-N-phenyl- 477 benzamide
191 N-(2-Hydroxy-ethyl)-5-iodo-2-(4-iodo-2-methyl-phenylamino)- 523 benzamide
192 5-Chloro-N-cyclopropyl-2-(4-iodo-2-methyl-phenylamino)- 425 benzamide
193 N-Allyl-5-chloro-2-(4-iodo-2-methyl-phenylamino)-benzamide 427
194 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-methyl-N-phenyl- 461 benzamide
195 N-(2-Hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)-5-nitro- 442 benzamide
196 5-Fluoro-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- 415 benzamide
197 5-Bromo-N-cyclopropyl-2-(4-iodo-2-methyl-phenylamino)- 472 benzamide
198 N-Cyclopropyl-5-fluoro-2-(4-iodo-2-methyl-phenylamino)- 411 benzamide
199 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(4-sulfamoyl- 540 benzyl)-benzamide
200 N-Cyclopropyl-2-(4-iodo-2-methyl-phenylamino)-5-nitro- 438 benzamide
201 N-Allyl-5-fluoro-2-(4-iodo-2-methyl-phenylamino)-benzamide 411 Example Compound MS
No. M-H
202 N-Benzyloxy-5-iodo-2-(4-iodo-2-methyl-phenylamino)- 585 benzamide
203 N-Allyl-5-bromo-2-(4-iodo-2-methyl-phenylamino)-benzarnide 472
204 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-(4-sulfamoyl- 601 benzyl)-benzamide
205 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-methyl-N-phenyl- 522 benzamide
206 N-Allyl-2-(4-iodo-2-methyl-phenylamino)-5-nitro-benzamide 438 _____
EXAMPLE 207 Preparation of 4-Chloro-2-(lH-tetrazol-5-ylV(,4-iodo-2-methyl-phenyl -amine
Step a: Preparation of 5-chloro-2-fluoro-benzaldehvde
To a solution of l-chloro-4-fluorobenzne (13.06 g, 0.1 mol) in THF (180 mL), at -78°C, LDA (2M solution in THF, 50 mL, 0.1 mol) was added drop wise. After stirring at -78°C for 1.5 hours, DMF (8 mL) was added to the reaction mixture and allowed to warm up to room temperature overnight. The reaction mixture was partitioned between water and Et2θ. The Et2θ layer was dried
(MgSO4) and the solvent removed in vacuum to give 14.95 g (94%) yield of crude aldehyde: 1 H NMR (CDC13): δ, 10.3 (s, -C(=O)H).
Step b: Preparation of 5-chloro-2-fluoro-benzaldehvde oxime
A solution of 5-chloro-2-fluoro-benzaldehyde (10 g, 0.0631 mol), hydroxylamine hydrochloride (6.57 g, 0.0946 mol) and pyridine (8.3 mL, 0.1010 mol) in EtOH (100 mL) was heated at 75°C (oil bath temperature) for 1 hour and the solvent removed under vacuum to give an oil. The oil was partitioned between water and CH2CI2. The CH2CI2 layer was dried (MgSO4) and the solvent removed under vacuum to give crude aldoxime as a solid. The solid was purified by medium pressure liquid chromatography on silica. Elution with CH2CI2 gave 4.87 g (28%) of the aldoxime as white solid: mp 95-97°C; Analysis calculated for C7H5NOFCI:
C, 48.44; H, 2.90; N, 8.07. Found: C, 48.55; H, 2.69, N, 7.90.
Step c: Preparation of 5-chloro-2-fluoro-benzonirile
A solution of the 5-chloro-2-fluoro-benzaldehyde oxime (3.15 g, 0.0182 mol) in acetic anhydride (150 mL) was refluxed for 16 hours. The reaction mixture was cooled to room temperature and poured into saturated aqueous NaHCO3 (200 mL) solution. The mixture was extracted with Et2θ. The Et2θ layer was dried (K2CO3) and the solvent removed to give the product as an oily solid. The product was used without further purification in the next step.
Step d: Preparation of 5-(5-chloro-2-fluoro-phenyl)-lH-tetrazole
A mixture of 5-chloro-2-fluoro-benzonitrile (2.84 g, 0.01823 mol), butanol (15 mL), sodium azide (1.543 g, 0.0237 mol), acetic acid (1.36 mL, 0.0237 mol) was refluxed for 24 hours. The reaction mixture was cooled to room temperature, additional 1.543 g sodium azide added, and the reaction mixture refluxed for additional 24 hours. After cooling to room temperature, Et2θ (100 mL) and 10% aqueous NaOH (200 mL) were added sequentially. The mixture was vigorously stirred. The aqueous layer was separated, cooled with ice-methanol bath (-15°C) and acidified to pH 1 with cone. HCl. A gray solid precipitated. The solid was dried in vacuum at 50°C to give 1.76 g (49%) of 5-(5-chloro-2-fluoro-phenyl)-lH- tetrazole: mp partial melt at 110°C, complete melting at 124°C); lH (400 Mz, CDCI3): δ 8.19-8.08 (m, IH), 7.77-7.71 (m, IH), 7.61-7.52 (m, IH); 13C (100 Mz, CDCI3): δ 159.00, 156.49, 140.88, 133.02, 132.93, 130.73, 129.23,
129.21, 129.08, 126.05, 118.96, 118.73, 114.50; MS (Cl) M+l = 199 (100), M = 198 (6). Step e: Preparation of |"4-Chloro-2-(l H-tetrazol-5-yl)-(4-iodo-2-methyl-phenyl - amine
To a solution of 2-methyl-4-iodoaniline (3.52 g, 0.0151 mol) in THF (25 mL) at -78°C, LDA (2 molar solution in THF, 11.33 mL, 0.02267 mol) was added dropwise. After stirring for 0.5 hours, a solution of l-(tetrazol-5-yl)-2- fluoro-5-chlorobenzene (1.5 g, 0.00756 mol) in THF (15 mL) was added dropwise. The reaction was stirred for 16 hours as it warmed up to room temperature. The reaction mixture was quenched with aqueous cone. NH4CI solution and extracted with CH2CI2. The organic layer was dried (MgSO4) and the solvent removed giving a crude product as an oil. The oil with CH2CI2-
>CH2Cl2:MeOH (9.7:0.3) gave 1.5 g (48%) of the desired product: mp 205-208°C; ]H (400 Mz, DMSO): δ 9.13 (s, IH), 8.00-7.99 (s, IH), 7.69 (s, IH), 7.55-7.52 (m, IH), 7.43-7.40 (m, IH), 7.12-7.05 (m, IH), 2.24 (s, 3H); 13C (100 Mz, CDCI3): δ 141.87, 139.28, 138.88, 135.47, 133.71, 131.65, 128.15, 123.69, 121.94, 116.68, 87.79, 17.22; MS (Cl) M+2 = 413 (44), M+1 = 412 (85),
M = 411 (100).
Analysis calculated for C14^ ^ClI-O^O:
C, 39.97; H, 2.87; N, 16.65. Found: C, 38.87, H, 2.77; N, 16.47.
The following tetrazole substituted phenylamines were prepared by following the general procedure of Example 207.
EXAMPLE 208 (4-iodo-2-methyl-phenyl)-r2-(lH-tetrazol-5-yl -phenyl]amine, mp 231°C (dec)
EXAMPLE 209 r4-nitro-2-dH-tetrazol-5-yl)-(4-iodo-2-methyl-phenylVamine. mp 205-208°C.
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).
Scheme 3
base
R6a HN-O— R 7, a
where 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.
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.
The phenylamino benzoic acid next is reacted with a hydroxylamine derivative HNRgaOR7a in the presence of a peptide coupling reagent. Hydroxylamine derivatives that can be employed include methoxylamine,
N-ethyl-isopropoxy amine, and tetrahydro-oxazine. 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). 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 of the coupling reagent is added. A base such as triethylamine or diisopropylethylamine 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.
Scheme 4
where L is a leaving group. The general reaction conditions for both of the steps in Scheme 4 are the same as those described above for Scheme 3.
Yet another method for making invention compounds comprises reacting a phenylamino benzhydroxamic acid with an ester forming group as depicted in Scheme 5. Scheme 5
base
where L is a leaving group such as halo, and a base is triethylamine or diisopropylamine. The synthesis of compounds of Formula (II) is further illustrated by the following detailed examples.
EXAMPLE la 4-Fluoro-N-hvdroxy-2-(4-iodo-2-methyl-phenylamino -benzamide (a) Preparation of 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid To a stirred solution containing 3.16 g (0.0133 mol) of 2-amino-
5-iodotoluene in 5 mL of tetrahydrofuran at -78°C was added 10 mL (0.020 mol) of a 2.0 M lithium diisopropylamide in tetrahydrofuran/heptane/ethylbenzene (Aldrich) solution. The resulting green suspension was stirred vigorously for 15 minutes, after which time a solution of 1.00 g (0.00632 mol) of 2,4-difluorobenzoic acid in 10 mL of tetrahydrofuran was added. The reaction temperature was allowed to increase slowly to room temperature, at which temperature the mixture was stirred for 2 days. The reaction mixture was concentrated by evaporation of the solvent under reduced pressure. 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. 10 mm of Hg) to afford 1.10 g (47%o) of the desired material; mp 224-229.5°C; lH NMR (400 MHz, DMSO): δ 9.72 (s, IH), 7.97 (dd, IH, J=7.0, 8.7 Hz), 7.70 (d, IH, J=1.5 Hz), 7.57 (dd, IH, J=8.4, 1.9 Hz), 7.17 (d, IH, J=8.2 Hz),
6.61-6.53 (m, 2H), 2.18 (s, 3H);
13C NMR (100 MHz, DMSO): δ 169.87, 166.36 (d, JC-F=249.4 Hz), 150.11 (d,
JC.F=11.4 Hz), 139.83, 138.49, 136.07, 135.26 (d, Jc-F=U-5 Hz), 135.07,
125.60, 109.32, 104.98 (d, Jc-F=21-1 Hz), 99.54 (d, JC-F=26.0 Hz), 89.43, 17.52; 19F NMR (376 MHz, DMSO): δ -104.00 to -104.07 (m);
IR (KBr) 1670 (C=O stretc^cm"1; MS (Cl) M+1 = 372. Analysis calculated for C14H11 FINO2: C, 45.31; H, 2.99; N, 3.77. Found: C, 45.21 ; H, 2.77; N, 3.64.
(b) Preparation of 4-Fluoro-N-hvdroxy-2-(4-iodo-2-methyl-phenylamino)- benzamide
To a stirred solution of 4-fluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid (0.6495 g, 0.001750 mol), O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.2590 g, 0.002211 mol), and diisopropylethylamine (0.40 mL, 0.0023 mol) in
31 mL of an equivolume tetrahydrofuran-dichloromethane solution was added 1.18 g (0.00227 mol) of solid PyBOP ([benzotriazolyloxy]triρyrrolidino phosphonium hexafluorophosphate, Advanced ChemTech) directly. The reaction mixture was stirred for 30 minutes after which time it was concentrated in vacuo. The brown oil was treated with 10% aqueous hydrochloric acid. The suspension was extracted with ether. The organic extraction was washed with 10%) sodium hydroxide followed by another 10% hydrochloric acid wash, was dried (MgSO4) and concentrated in vacuo to afford 1.0 g of a light-brown foam. 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. Scratching with pentane-hexanes and drying under high vacuum afforded 0.1541 g (23%) of an off-white foam; mp 61-75°C; iH NMR (400 MHz, DMSO): δ 11.34 (s, IH), 9.68 (s, IH), 9.18 (s, IH), 7.65 (d, IH, J=1.5 Hz), 7.58 (dd, IH, J=8.7, 6.8 Hz), 7.52 (dd, IH, J=8.4, 1.9 Hz), 7.15 (d, IH, J=8.4 Hz), 6.74 (dd, IH, J=l 1.8, 2.4 Hz), 6.62 (ddd, IH, J=8.4, 8.4, 2.7 Hz), 2.18 (s, 3H); 13C NMR (100 MHz, DMSO): δ 165.91, 164.36 (d, JC-F=247.1 Hz), 146.78,
139.18, 138.77, 135.43, 132.64, 130.60 (d, 1-5 Hz), 122.23, 112.52, 104.72 (d, J=22.1 Hz), 100.45 (d, JC-F=25.2 Hz), 86.77, 17.03;
19F NMR (376 MHz, DMSO): δ -107.20 to -107.27 (m);
IR (KBr) 3307 (broad, O-H stretch), 1636 (C=O stretch) cm"1; MS (Cl) M+1 = 387.
Analysis calculated for C14H12F1N2O2:
C, 43.54; H, 3.13; N, 7.25. Found: C, 43.62; H, 3.24; N, 6.98.
EXAMPLE 2a 5-Bromo-3,4-difluoro-N-hvdroxy-2-(4-iodo-2-methyl-phenylamino -benzamide
(a) Preparation of 5-Bromo-2 ,4-trifluorobenzoic acid
To a stirred solution comprised of l-bromo-2,3,4-trifluorobenzene (Aldrich, 99%; 5.30 g, 0.0249 mol) in 95 mL of anhydrous tetrahydrofuran cooled to -78°C was slowly added 12.5 mL of 2.0 M lithium diisopropylamide in heptane/tetrahydrofuran/ethylbenzene solution (Aldrich). The mixture was stirred for 1 hour and transferred by canula into 700 mL of a stirred saturated ethereal carbon dioxide solution cooled to -78°C. The cold bath was removed, and the reaction mixture was stirred for 18 hours at ambient temperature. Dilute (10%) aqueous hydrochloric acid (ca. 500 mL) was poured into the reaction mixture, and the mixture was subsequently concentrated on a rotary evaporator to a crude solid.
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 combined organic extracts were dried (MgSO4), concentrated in vacuo, and subjected to high vacuum until constant mass was achieved to afford 5.60 g (88% yield) of an off-white powder; mp 139-142.5°C; lH NMR (400 MHz, DMSO): δ 13.97 (broad s, IH, 8.00-7.96 (m, IH);
13C NMR (100 MHz, DMSO): δ 162.96, 129.34, 118.47, 104.54 (d, JC-F=22.9 Hz);
19F NMR (376 MHz, DMSO): δ -120.20 to -120.31 (m), -131.75 to -131.86 (m), -154.95 to -155.07 (m); IR (KBr) 1696 (C=O stretch^m"1 ;
MS (Cl) M+1 = 255. Analysis calculated for C74H2iBrF3θ2:
C, 32.97; H, 0.79; N, 0.00; Br, 31.34; F, 22.35. Found: C, 33.18; H, 0.64; N, 0.01; Br, 30.14; F, 22.75.
(b) Preparation of 5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)- benzoic acid
To a stirred solution comprised of 1.88 g (0.00791 mol) of 2-amino- 5-iodotoluene in 10 mL of tetrahydrofuran at -78°C was added 6 mL (0.012 mol) of a 2.0 M lithium diisopropylamide in tetrahydrofuran/heptane/ethylbenzene (Aldrich) solution. The resulting green suspension was stirred vigorously for 10 minutes, after which time a solution of 1.00 g (0.00392 mol) of 5-bromo- 2,3,4-trifluorobenzoic acid in 15 mL of tetrahydrofuran was added. The cold bath was subsequently removed, and the reaction mixture stirred 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 solid was rinsed with dichloromethane, and dried in the vacuum- oven (80°C) to afford 1.39 g (76%) of a yellow-green powder; mp 259.5-262°C;
!H NMR (400 MHz, DMSO): δ 9.03 (s, IH), 7.99 (dd, IH, J=7.5, 1.9 Hz), 7.57 (dd, IH, J=1.5 Hz), 7.42 (dd, IH, J=8.4, 1.9 Hz), 6.70 (dd, IH, J=8.4, 6.0 Hz), 2.24 (s, 3H);
19F NMR (376 MHz, DMSO): δ -123.40 to -123.47 (m); -139.00 to -139.14 (m); IR (KBr) 1667 (C=O stretch)cm" 1 ;
MS (Cl) M+1 = 469. Analysis calculated for Ci4H9BrF2lNO2:
C, 35.93; H, 1.94; N, 2.99; Br, 17.07; F, 8.12; 1, 27.11. Found: C, 36.15; H, 1.91; N, 2.70; Br, 16.40; F, 8.46; 1, 26.05. (c) Preparation of 5-Bromo-3.4-difluoro-N-hydroxy-2-(4-iodo-2-methyl- phenylamino)-benzamide
To a stirred solution comprised of 5-bromo-3,4-difluoro-2-(4-iodo- 2-methyl-phenylamino)-benzoic acid (0.51 g, 0.0011 mol), O-(tetrahydro-2H- pyran-2-yl)-hydroxylamine (0.15 g, 0.0013 mol), and diisopropylethylamine (0.25 mL, 0.0014 mol) in 20 mL of an equivolume tetrahydrofuran- dichloromethane solution was added 0.6794 g (0.001306 mol) of solid PyBOP (Advanced ChemTech) directly. The reaction mixture was stirred 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) and concentrated in vacuo to afford
0.62 g (100%) of an off-white foam. The foam was dissolved in ca. 15 mL of methanolic hydrogen chloride. After 5 minutes, the solution was concentrated in vacuo to an oil, and the oil was purified by flash silica chromatography. Elution with dichloromethane: dichloromethane-methanol (99:1) afforded 0.2233 g (42%) of a yellow powder. The powder was dissolved in diethyl ether and washed with dilute hydrochloric acid. The organic phase was dried (MgSO4) and concentrated in vacuo to afford 0.200 g of a foam. This product was triturated with pentane to afford 0.1525 g of a powder that was repurified by flash silica chromatography. Elution with dichloromethane afforded 0.0783 g ( 15%) of an analytically pure title compound, mp 80-90°C; lH NMR (400 MHz, DMSO): δ 11.53 (s, IH), 9.38 (s, IH), 8.82 (s, IH), 7.70 (dd, IH, J=7.0, 1.9 Hz), 7.53 (s, IH), 7.37 (dd, IH, J=8.4, 1.9 Hz), 6.55 (dd, IH, J=8.2, 6.5 Hz), 2.22 (s, 3H); 19F NMR (376 MHz, DMSO): δ -126.24 to -126.29 (m), -137.71 to -137.77 (m);
IR (KBr) 3346 (broad, O-H stretch), 1651 (C=O stretc cnr1;
MS (Cl) M+1 = 484.
Analysis calculated for Ci4Hκ)BrF2iN2θ2:
C, 34.81; H, 2.09; N, 5.80. Found: C, 34.53; H, 1.73; N, 5.52.
Examples 3 a to 12a in the table below were prepared by the general procedure of Examples la and 2a.
EXAMPLES 13a-77a Examples 13a to 77a were prepared utilizing combinatorial synthetic methodology by reacting appropriately substituted phenylamino benzoic acids
(e.g., as shown in Scheme 1) and hydroxylamines (e.g., (NHRβa )-O-R7a). A general method is given below: To a 0.8-mL autosampler vial in a metal block was added 40 μL of a
0.5 M solution of the acid in DMF and 40 μL of the hydroxylamine (2 M solution in Hunig's base and 1 M in amine in DMF). A 0.5 M solution of PyBrOP was freshly prepared, and 50 μL were added to the autosampler vial. The reaction was allowed to stand for 24 hours.
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 μM 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 desired fractions were evaporated using a Zymark Turbovap. The product was dissolved in chloroform and transferred to a preweighed vial, evaporated, and weighed again to determine the yield. The structure was confirmed by mass spectroscopy.
EXAMPLES 3a-77a Example Compound Melting MS
No. Point (°C) (M-H+)
3a 2-(4-bromo-2-methyl-phenylamino)-4-fluoro-N- 56-75 dec 523 hydroxy-benzamide
4a 5-Chloro-N-hydroxy-2-(4-iodo-2-methyl- 65 dec phenylamino)-benzamide
5a 5-Chloro-N-hydroxy-2-(4-iodo-2-methyl- 62-67 phenylamino)-N-methyl-benzamide
6a 5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N- 105-108 (terahydropyran-2-yloxy)benzamide
7a 5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N- 64-68 methoxybenzamide
8a 4-Fluoro-N-hydroxy-2-(4-fluoro-2-methyl- 119-135 phenylamino)-benzamide
9a 4-Fluoro-N-hydroxy-2-(2 -methyl phenylamino)- 101-103 benzamide
10a 4-Fluoro-2-(4-fluor-2-methyl-phenylamino)-N- 142-146 (terahydropyran-2-yloxy)benzamide
11a 4-Fluoro-N-hydroxy-2-(4-cluoro-2-methyl- 133.5-135 phenylamino)-benzamide Example Compound Melting MS
No. Point (°C) (M-H+)
Ϊ2a 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N- 107-109.5 phenylmethoxy-benzamide
13a 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N- 399 methoxy-benzamide
14a 3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)- 417
N-methoxy-benzamide
15a 2-(4-Bromo-2-methyl-phenylamino)- 369
3 ,4-difluoro-N-methoxy-benzamide
16a 2-(4-Bromo-2-methyl-phenylamino)-N-ethoxy- 342*
3,4-difluoro-benzamide (M-EtO)
17a 5-Bromo-N-ethoxy-3,4-difluoro-2-(4-iodo- 509
2-methyl-phenylamino)-benzamide
18a 3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)- 445
N-isopropoxy-benzamide
19a 2-(4-Bromo-2-methyl-phenylamino)- 397
3,4-difluoro-N-isopropoxy-benzamide
20a 4-Fluoro-N-(furan-3-ylmethoxy)-2-(4-iodo- 465
2-methyl-phenylamino)-benzamide Example Compound Melting MS
No. Point (°C) (M-H+) 2Ϊa 3,4-Difluoro-N-(furan-3-ylmethoxy)-2-(4-iodo- 483
2-methyl-phenylamino)-benzamide
22a 2-(4-Bromo-2-methyl-pheny lamino)- 435
3,4-difluoro-N-(furan-3-ylmethoxy)-benzamide
23a 5-Bromo-3,4-difluoro-N-(furan-3-ylmethoxy)- 561
2-(4-iodo-2-methyl-phenylamino)-benzamide
24a 5-Bromo-N-(but-2-enyloxy)-3,4-difluoro- 536
2-(4-iodo-2-methyl-phenylamino)-benzamide
25a 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N- 423
(prop-2-ynyloxy)-benzamide
26a 3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)- 441
N-(prop-2-ynyloxy)-benzamide
27a 3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)- 455
N-( 1 -methyl-prop-2-ynyloxy)-benzamide
28a 2-(4-Bromo-2-methyl-phenylamino)- 407
3 ,4-difluoro-N-( 1 -methyl-prop-2-ynyloxy)- benzamide
29a N-(But-3-ynyloxy)-3,4-difluoro-2-(4-iodo- 455
2-methyl-phenylamino)-benzamide Example Compound Melting MS
No. Point (°C) (M-H+) 30a 2-(4-Bromo-2-methyl-phenylamino)-N-(but- 407
3-ynyloxy)-3,4-difluoro-benzamide
31a 5-Bromo-N-(but-3-ynyloxy)-3,4-difluoro- 533
2-(4-iodo-2-methyl-phenylamino)-benzamide
32a 3 ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)- 517
N-(3-phenyl-prop-2-ynyloxy)-benzamide
33a 3,4-Difluoro-2-(4-bromo-2-methyl- 469 phenylamino)-N-(3-phenyl-prop-2-ynyloxy)- benzamide
34a 3,4-Difluoro-N-[3-(3-fluoro-phenyl)-prop- 535
2-ynyloxy]-2-(4-iodo-2-methyl-phenylamino)- benzamide
35a 2-(4-Bromo-2-methyl-phenylamino)- 487
3 ,4-difluoro-N- [3 -(3 -fluoro-phenyl)-prop- 2-ynyloxy]-benzamide
36a 3,4-Difluoro-N-[3-(2-fluoro-phenyl)-prop- 535
2-ynyloxy]-2-(4-iodo-2-methyl-phenylamino)- benzamide
37a 5-Bromo-3,4-difluoro-N-[3-(2-fluoro-phenyl)- 613 prop-2-ynyloxy]-2-(4-iodo-2-methyl- phenylamino)-benzamide Example Compound Melting MS
No. Point (°C) (M-H+)
38a 3 ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)- 557* N-(3-methyl-5-phenyl-pent-2-en-4-ynyloxy)- *(M+H) benzamide
39a 2-(4-Bromo-2-methyl-phenylamino)- 510
3 ,4-difluoro-N-(3 -methyl-5 -phenyl-pent-2-en- 4-ynyloxy)-benzamide
40a N-Ethoxy-3 ,4-difluoro-2-(4-iodo-2-methyl- 431 phenylamino)-benzamide
41a 2-(4-Bromo-2-methyl-phenylamino)-N-ethoxy- 383 3 ,4-difluoro-benzamide
42a 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N- 427 propoxy-benzamide
43 a 3 ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)- 445 N-propoxy-benzamide
44a 2-(4-Bromo-2-methyl-phenylamino)- 397 3 ,4-difluoro-N-propoxy-benzamide
45a 5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl- 523 phenylamino)-N-propoxy-benzamide
46a 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N- 427 isopropoxy-benzamide Example Compound Melting MS
No. Point (°C) (M-H+) 47a 3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)- 445
N-isopropoxy-benzamide
48a 2-(4-Bromo-2-methyl-phenylamino)- 397
3,4-difluoro-N-isopropoxy-benzamide
49a 5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl- 523 phenylamino)-N-isopropoxy-benzamide
50a N-Cyclobutyloxy-3,4-difluoro-2-(4-iodo- 457
2-methyl-phenylamino)-benzamide
51a 2-(4-Bromo-2-methyl-phenylamino)-N- 409 cyclobutyloxy-3,4-difluoro-benzamide
52a N-Cyclopentyloxy-4-fluoro-2-(4-iodo-2-methyl- 453 phenylamino)-benzamide
53a N-Cyclopentyloxy-3,4-difluoro-2-(4-iodo- 471
2-methyl-phenylamino)-benzamide
54a 2-(4-Bromo-2-methyl-phenylamino)-N- 423 cyclopentyloxy-3,4-difluoro-benzamide
55a N-Cyclopropylmethoxy-4-fluoro-2-(4-iodo- 439
2-methyl-phenylamino)-benzamide
56a N-Cyclopropylmethoxy-3,4-difluoro-2-(4-iodo- 457
2-methyl-phenylamino)-benzamide Example Compound Melting MS
No. Point (°C) (M-H+)
57a 2-(4-Bromo-2-methyl-phenylamino)-N- 409 cyclopropylmethoxy-3 ,4-difluoro-benzamide
58a 5-Bromo-N-cyclopropylmethoxy-3,4-difluoro- 435
2-(4-iodo-2-methyl-phenylamino) 59a 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N- 505
(2-phenoxy-ethoxy)-benzamide
60a 3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)- 523
N-(2-phenoxy-ethoxy)-benzamide
61a 2-(4-Bromo-2-methyl-phenylamino)- 475
3,4-difluoro-N-(2-phenoxy-ethoxy)-benzamide
62a 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N- 481
(thiophen-2-ylmethoxy)-benzamide
63 a 3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)- 499
N-(thiophen-2-ylmethoxy)-benzamide
64a 2-(4-Bromo-2-methyl-phenylamino)- 451
3,4-difluoro-N-(thiophen-2-ylmethoxy)- benzamide
65a 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N- 439
(2-methyl-allyloxy)-benzamide Example Compound Melting MS
No. Point (°C) (M-H+) 66a 3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)- 457
N-(2-methyl-allyloxy)-benzamide
67a 2-(4-Bromo-2-methyl-phenylamino)- 410
3,4-difluoro-N-(2-methyl-allyloxy)-benzamide
68a N-(But-2-enyloxy)-4-fluoro-2-(4-iodo-2-methyl- 439 phenylamino)-benzamide
69a N-(But-2-enyloxy)-3,4-difluoro-2-(4-iodo- 457
2-methyl-phenylamino)-benzamide
70a 2-(4-Bromo-2-methyl-phenylamino)-N-(but- 410
2-enyloxy)-3 ,4-difluoro-benzamide
71a 3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)- 441
N-(prop-2-ynyloxy)-benzamide
72a N-(But-3-ynyloxy)-3,4-difluoro-2-(4-iodo- 455
2-methyl-phenylamino)-benzamide
73 a 2-(4-Bromo-2-methyl-phenylamino)-N- 449
(4,4-dimethyl-pent-2-ynyloxy)-3,4-difluoro- benzamide
74a N-(But-2-enyloxy)-3,4-difluoro-2-(4-iodo- 457
2-methyl-phenylamino)-benzamide Example Compound Melting MS
No. Point (°C) (M-H+)
75a 2-(4-Bromo-2-methyl-phenylamino)-N-(but- 410 2-enyloxy)-3 ,4-difluoro-benzamide
76a N-(3-tert-butyl-propyn-2-yl)oxy-4-fluoro- 479
2-(4-iodo-2-methyl-phenylamino)-benzamide
77a 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N- 577 phenylmethoxy-benzamide
PHYSICAL DATA FOR SELECTED COMPOUNDS
PD 0171984
mp 80-90 °C
PD 0184161
mp 174-175 °C
PD 0203311
mp 141-144 °C
PD 0297189
mp l67-169 °C
1H-NMR (400 MHz; DMSO) δ 11.70 (s, IH), 8.59 (s, IH), 7.55 (s, IH), 7.43 (d, IH, J=6.5 Hz), 7.27 (d,lH, J=8.7 Hz), 6.46 (m, IH), 3.42 (d, 2H, J=7.0 Hz), 0.84 (m, 1 H), 0.27 (m, 2H), 0.00 (m, 2H) PD 0297190 mp 125.5-133 °C
1H-NMR (400 MHz; DMSO) δ 11.48 (s, IH), 8.32 (s, IH), 7.34 (d, IH, J=7.5 Hz), 7.28 (d, 2H, J=8.2 Hz), 6.48 (d, 2H, J=7.7 Hz), 3.32 (d, 2H, J=6.8 Hz), 0.81 (m, IH), 0.28 (m, 2H), 0.00 (m, 2H)
PD 0296771
mp 266.7-268.9 °C
1H-NMR (400 MHz; DMSO) δ 13.85 (broad s, IH), 8.99 (s, IH), 7.87 (dd, IH, J=7.9, 2.1 Hz), 7.55 (d,2H, J=8.6 Hz), 6.82 (dd, 2H, J=8.7, 2.8 Hz)
PD 0296770
mp 293.2-296.3 °C
Η-NMR (400 MHz; DMSO) δ 14.05 (broad s, IH), 9.21 (s, IH), 7.93 (dd, IH, J=7.8, 2.2 Hz), 7.82 (d,lH, J=1.9 Hz), 7.54 (dd, IH, J=8.6, 1.9 Hz), 6.82 (dd, IH, J=8.6, 6.7 Hz)
PD 0296767
mp 249-251 °C
1H-NMR (400 MHz; DMSO) δ 13.99 (broad s, IH), 9.01 (s, IH), 7.90 (dd, IH, J=7.9, 2.3 Hz), 7.58 (d,lH, J=1.6 Hz), 7.42 (dd, IH, J=8.4, 1.9 Hz), 6.69 (dd, IH, J=8.4, 6.0 Hz), 2.24 (s, 3H)
PD 298127
mp 127-135 °C
5-chloro-N-cyclopropyl methoxy-3,4-difluoro-2-[4-iodo-2 -methyl phenylamino]benzamide
1H NMR (440 MHz; DMSO) δ 11.64 (s, IH), 8.28 (s, IH), 7.38 (dd, IH, J=7.6, 1.7 Hz), 7.31 (d, 1 H, J=1.2 Hz), 7.15 (dd, IH, J=8.5, 1.7 Hz), 3.35 (d, 2H, J=7.3 Hz), 2.01 (s, 3H), 0.83 (m, IH), 0.28 (m,2H), 0.01 (m, 2H) BIOLOGICAL ASSAYS
Example 1
Inhibition of IL-2 Production Induced by Concanavalin A (Con A) Several of the 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). In this assay, the cells (HPBMC) were prepared by first centrifuging tubes of heparinized blood
(obtained from normal healthy volunteers) at 1400 rpm for 10 minutes at room temperature. The interphase containing mostly leukocytes was removed and added to a 50 mL centrifuge tube, and diluted with phosphate buffered saline (PBS) to a volume of 40 mL. The diluted PBS solution was added to a 50 mL centrifuge tube containing 7 mL of Histopague (Sigma, Sp. Gr. 1.077). The mixture was centrifuged at 2200 rpm for 20 minutes at room temperature. The middle layer, comprised mostly of peripheral blood mononuclear cells (PBMC), was removed and added to a clean 50 mL centrifuge tube. These cells were diluted with PBS to a volume of 30 mL, and centrifuged at 1000 rpm for 10 minutes at room temperature. The supernatant was removed, and the remaining cells were washed twice with 30 mL portions of PBS. The PBMC were resuspended in medium (Roswell Park Memorial Institute No. 1640 (RPMI-1640), from Gibco BRL, Gaithersburg, MD), and 10%) fetal bovine serum (FBS) culture medium. The cells were adjusted to 2.5 x 10^ cells/mL. The compounds to be tested were prepared by dissolving them in dimethylsulfoxide (DMSO) to a concentration of 30 micromolar. Additional dilutions were made in RPMI-1640, and then in RPMI-1640 containing 1% DMSO so that the final in- well concentration of DMSO was 0.25% in all wells. Concanavalin A (Con 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 carried out by adding 50 μL of the diluted test compounds to appropriate wells of a plate. To the wells were added 100 μL of the 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. For the HPBMC assay, 50 μL of the Con A solution (80 μg/mL Con A in RPMI-1640) were added to the appropriate wells. For the HWB assay, 50 μL of a Con A solution (800 μg/mL Con A in RPMI-1640) were added to the appropriate wells. Control wells contained medium plus 50 μL 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 μL) 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.
The results of the foregoing assay are shown in Figures 1 and 9. A preferred compound to be used in accordance with this invention is 2-(2-chloro-
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. The Figure shows that PD 184352 causes a dramatic dose dependent inhibition of IL-2 production, and has an IC50 of 71 nM.
Figure 9 shows the inhibition of IL-2 production in cells caused by several of the phenyl amine MEK inhibitors of Formulas I and II, compared to known immunosuppressive agents dexamethasone, a steroid, which is 9-fluoro- 1 lβ,17,21-trihydroxy-16α-methylpregna-l,4-diene-3,20-dione, and rolipram, a phosphodiesterase-4 inhibitor which is 4-[3-(cyclopentyloxy)-4-methoxyphenyl]-
2-pyrrolidinone. The data establish that the phenyl amine MEK inhibitors are in general very potent in their ability to inhibit IL-2 production.
Example 2
Inhibition of IL-2 Production Induced by Anti-CD3 & Anti-CD28 Stimulation of T cells through direct activation of the T cell receptor is felt to be more representative of physiologic T cell activation than when cells are activated by mitogens, such as Con A. The 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.
The most prominent and best-characterized T cell co-receptor is CD28. Monoclonal antibodies to CD3 and CD28 and be used together to induce release of IL-2.
Anti-CD3 was purchased from BioSource Int. (catalog #AHS2812). A working solution was prepared containing 10 μg/mL of anti-CD3 in PBS. A
100 μL aliquot was added to appropriate wells and incubated for 3 hours at 37°C, and then unbound anti-CD3 was washed off with PBS. Anti-CD28 was purchased from BioSource Int. (catalog #AH0312) and was added as a solution (0.5 μg/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% CO2 in air, supernatant was collected and assayed for IL-2 as described in Example 1. The results of the foregoing assay are shown in Figure 2. 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 IC50 of 47 nM.
Example 3
Inhibition of Interferon-γ Production
The foregoing procedure was followed to evaluate the ability of the phenyl amine MEK inhibitors of Formulas I and II to inhibit the release of interferon gamma (IFN-gamma) from human peripheral blood mononuclear cells (HPBMC) and human whole blood (HWB). The cell samples and compound samples were prepared by the general procedure described above. The assays of the incubated well plates were carried out using an IFN-gamma ELISA kit (No. DIF00 from R & D Systems). The results of the assays are shown in Figures 3 and 10. Figure 3 shows that Con A causes a large production of IFN-gamma, and that such production is totally inhibited by PD 184352 at some concentrations. The Figure shows that the IC50 for PD 184352 against IFN-gamma is 148 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 μM or higher. The ability of the 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.
Example 4
Human Mixed Lymphocyte Reaction
Several of the MEK inhibitors which are to be used in the method of this invention have been evaluated in an in vitro test in which lymphocytes (or 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). This test measures the degree of histoincompatibility. The assay is a mixed lymphocyte (or leukocyte) reaction, and is referred to as the "MLR". In this assay, inhibition of tritiated thymidine (3H-TDR) incorporation is measured. Tritiated thymidine was supplied from Amersham (Catalog No. TRK.758, 250 μCi). The commercial product was diluted in RPMI-1640 in a 50 mL conical centrifuge tube to provide a working stock solution of 5-10 μCi/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 of the assay plate was labeled with the 3H-TDR working stock solution (total of 0.1 - 0.5 μCi 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 (3H-TDR) uptake. The Figure shows that untreated MLR values are in excess of 4500 CPM, whereas the test compound causes a dose dependent inhibition of 3H-TDR uptake, with almost total inhibition occurring at 10 μM. 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.
The data presented in Figures 4 and 11 further establish that the selective MEK inhibitors of Formulas I and II are useful for preventing the rejection of transplanted organs, tissues, cells, and limbs in mammals.
Example 5
Inhibition of T-Cell Proliferation induced by Con A
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 of the 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 3H-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 of the Con A induced proliferation at the lowest dose tested (0.12 μM), and causes almost total inhibition at the highest dose tested (10.0 μM). The IC50 for the compound was determined to be 340 nM.
Figure 12 shows that all of the phenyl amine MEK inhibitors that were tested caused a dramatic and dose dependent inhibition of T-cell proliferation.
Example 6
Inhibition of T-Cell Proliferation induced by Phytohemagglutinin (PHA)
The T-cell inhibition study was carried out using the agent PHA to induce the proliferation. Figure 6 shows the effects of PD 184352. In this study, the test compound failed to cause inhibition at the low dose (0.12 μM), but caused a measurable inhibition at all other doses, with almost total inhibition at the high dose (10 μM). The IC50 was determined to be 1.9 μM in this assay. The data further establish the ability of the phenyl amine MEK inhibitor to inhibit T-cell proliferation, and thereby to be useful in the prophylaxis of transplant rejections in mammals.
Example 7 Toxicity Assay
As noted above, 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 of the 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 μM, and caused only slight color change even at concentrations as high as 100 μM. 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 μM. These data establish that the phenyl amine MEK inhibitors are essentially devoid of any toxic effects in this assay. Figure 13 shows the toxicity of several of the phenyl amine MEK inhibitors when evaluated in the MTT assay. The data establish that all of the 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 rej ection in mammals .
Figure 8 shows the relative activities of several of the phenyl amine MEK inhibitors of Formulas I and II, compared with the activities of rolipram and dexamethasone, in a number of the 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.
Much of the foregoing data is summarized below in Pharmacological Table 1. 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 of the measured parameter (the IC50), except in the case of the toxicity data, which is presented as
TC50 (concentration required to produce toxicity in 50 percent of the cells). In the
Table, "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 l,4-diamino-2,3-dicyano-l,4-bis[2-aminophenylthio]butadiene, an immunosuppressive compound described in US Patent No. 2,779,780. PHARMACOLOGICAL TABLE 1
MEK Inhibitors: In Vitro Effects on Human Leukocytes (All data are mean (*) IC5QS or % inhibition at the concentration given, except toxicity data
( MTT) which are TC50S)
APK Human IFN- U937 MTT JH-TDR JH-TDR 3H-TDR
IC50 IL-2 gamma TC50 PHA MLR Con A (nM) (μM) (μM) (μM) or (μM) (μM) (μM)
(% dead)
171984-0000 3.0 0.019 *0.034 50.6 *2.4 0.35 *0.19
177098-0000 14.0 0.006 ♦0.076 *29.9 *NJ *NJ *3.9
177168-0000 18.0 0.052 *0.17 *13.9 *2.5 0.69 *0.52
180841-0000 4.4 *0.11 *0.21 *5.8 *ND *NJ *ND
184161-0000 1.6 *0.19 *0.15 *12.6 *1.8 0.53 *0.61
184352-0000 1.3 0.068 *0.14 >100 (6%) *4.5 0.64 *0.52
184386-0000 1.4 0.039 *0.040 61 *4.0 0.41 ♦0.31
185625-0000 5.1 0.071 •0.12 •12.4 *NJ *0.39 *4.0
185848-0000 1.0 0.018 *0.024 38.1 *NJ *0.51 *NJ
188563-0000 1.3 0.013 ♦0.15 40 *NJ •0.17 *NJ
198306-0000 8.0 0.037 *0.15 13.1 *1.40 *1.8 *1.9
20331 1-0000 - 0.032 *0.10 *32.2 *ND *0.076 *ND
ND = not determined. NJ = no judgment: Studies indeterminant or incomplete.
The foregoing extensive biological evaluations clearly establish the selective MEK inhibitors described above, especially the phenyl amines of Formulas I and II, are well-suited to the prophylaxis of transplant rejections in mammals, preferably humans. Like other immunosuppressive agents, the MEK inhibitors can be used in combination with other such agents for even better results. For example, 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 of the 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 of the 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.
If any grafts are rejected at any time during the study, the animal is sacrificed by dry ice (CO2) asphyxiation as soon as graft rejection is determined.
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 of the 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 of the 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.
D. Other Embodiments
From the above disclosure and examples, and from the claims below, the essential features of the invention are readily apparent. The scope of the invention also encompasses various modifications and adaptations within the knowledge of a person of ordinary skill. Examples include a disclosed compound modified by addition or removal of a protecting group, or an ester, pharmaceutical salt, hydrate, acid, or amide of a disclosed compound. Publications cited herein are hereby incorporated by reference in their entirety.
What is claimed is:

Claims

A method for preventing and controlling the rejection, in a patient, of a transplanted organ, cell, tissue, or limb, said method comprising administering to the patient who has undergone a transplant, or who is scheduled to undergo a transplant, an effective immunosuppressive amount of a MEK inhibitor.
A method according to Claim 1 wherein the MEK inhibitor administered is 2-(2-amino-3 -methoxyphenyl)-4-oxo-4H- [ 1 ]benzopyran.
The method according to Claim 1, wherein said MEK inhibitor is a selective MEK 1 or MEK 2 inhibitor.
4. The method according to Claim 1 wherein the MEK inhibitor is a compound of Formula I
wherein:
R\ is hydrogen, hydroxy, Ci-Cg alkyl, Ci -Cg alkoxy, halo, trifluoromethyl, or CN; R2 is hydrogen;
R3, R4, and R5 independently are hydrogen, hydroxy, halo, trifluoromethyl, Cj-Cg alkyl, Cj-Cg alkoxy, nitro, CN, or
-(O or NH)m-(CH2)n-R9, where R9 is hydrogen, hydroxy, COOH, or NRioRπ; n is 0-4; m is 0 or 1 ;
RjO and R\ \ independently are hydrogen or Cj-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, or N-Cj-Cg alkyl; Z is COOR7, tetrazolyl, CONRgRγ, CONHNRioRl l, or CH2OR7; R6 and R7 independently are hydrogen, C1 -Cg alkyl, C2-Cg alkenyl, C2-Cg alkynyl, (CO)-Cj-Cg alkyl, aryl, heteroaryl, C3-C1 Q cycloalkyl, or 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; and wherein any of the foregoing alkyl, alkenyl, aryl, heterocyclic, and alkynyl groups can be unsubstituted or substituted by halo, hydroxy, Cj-C6 alkoxy, amino, nitro, Cι-C4 alkylamino, di(d- C4)alkylamino, C3-C cycloalkyl, phenyl,phenoxy, C3-C5 heteroaryl, or C3- C5 heteroaryloxy; or a pharmaceutically acceptable salt, ester, amide, or prodrug thereof.
The method according to Claim 4 wherein the MEK inhibitor is a compound selected from:
[4-Chloro-2-( 1 H-tetrazol-5-yl)-phenyl-(4-iodo-2-methyl-phenyl)- amine;
(4-iodo-2-methyl-phenyl)-[2-(lH-tetrazol-5-yl)-phenyl]amine; [4-nitro-2-( 1 H-tetrazol-5-yl)-phenyl-(4-iodo-2-methyl-phenyl)- amine;
4-Fluoro-2-(4-iodo-2-methylphenylarnino)benzoic acid; 3,4,5-Trifluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid; 3 ,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid; 5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid; 5 -Chloro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid;
Sodium 5-Chloro-2-(4-iodo-2-methyl-phenylamino)-benzoate;
5-Bromo-2-(4-iodo-2-methyl-phenylamino)-benzoic acid;
2-(4-Iodo-2-methyl-phenylamino)-5-nitro-benzoic acid; 4-Chloro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid;
2-(4-Iodo-2-methyl-phenylamino)-benzoic acid;
5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-benzoic acid;
5-Iodo-2-(4-iodo-2-methyl-phenylamino)-benzoic acid;
2,3,5-Trifluoro-4-(4-iodo-2-methyl-phenylamino)-benzoic acid; 2-(4-Iodo-phenylamino)-5-methoxy-benzoic acid;
5-Methyl-2-(4-iodo-2-methyl-phenylamino)-benzoic acid;
2-(4-Iodo-2-methyl-phenylamino)-4-nitro-benzoic acid;
2-(4-Bromo-2-methyl-phenylamino)-4-fluoro-benzoic acid;
2-(2-Bromo-4-iodo-phenylamino)-5-nitro-benzoic acid; 2-(4-Bromo-2-methyl-phenylamino)-3 ,4-difluoro-benzoic acid;
5-Chloro-N-(2-hydroxyethyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide;
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-benzamide;
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-methyl-benzamide; N-Ethyl-4-fluoro-2-(4-iodo-2-methyl-phenylamino)-benzamide;
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N,N-dimethyl- benzamide;
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-( 1 H-tetrazol-5-yl)- benzamide; 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-benzamide;
5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N,N-dimethyl- benzamide;
[5-Chloro-2-(4-iodo-2-methyl-phenylamino)-benzoylamino]-acetic acid; 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-propyl-benzamide;
5-Bromo-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide; N,N-Diethyl-4-fluoro-2-(4-iodo-2-methyl-phenylamino)- benzamide;
4-Fluoro-N- { 3 - [4-(2-hydroxy-ethyl)-piperazin- 1 -yl] -propyl } - 2-(4-iodo-2-methyl-phenylamino)-benzamide; N,N-Diethyl-2-(4-iodo-2-methyl-phenylamino)-5-nitro-benzamide;
N-Butyl-4-fluoro-2-(4-iodo-2-methyl-phenylamino)-benzamide;
5-Chloro-N,N-diethyl-2-(4-iodo-2-methyl-phenylamino)- benzamide;
5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N,N-dimethyl- benzamide;
5-Bromo-3,4-difluoro-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl- phenylamino)-benzamide;
N-(2,3-Dihydroxy-propyl)-3,4-difluoro-2-(4-iodo-2-methyl- phenylamino)-benzamide; 5-Bromo-3 ,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N-
(2 -piperidin- 1 -yl-ethyl)-benzamide;
3,4-Difluoro-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl- phenylamino)-benzamide;
N-(2,3-Dihydroxy-propyl)-4-fluoro-2-(4-iodo-2-methyl- phenylamino)-benzamide;
3,4-Difluoro-N-(3-hydroxy-propyl)-2-(4-iodo-2-methyl- phenylamino)-benzamide;
5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N- (2-pyrrolidin- 1 -yl-ethyl)-benzamide; 5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N-
(2-pyridin-4-yl-ethyl)-benzamide;
4-Fluoro-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide;
5-Bromo-N-(3-dimethylamino-propyl)-3,4-difluoro-2-(4-iodo- 2-methyl-phenylamino)-benzamide;
5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N- (2-morpholin-4-yl-ethyl)-benzamide; 3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-morpholin- 4-yl-ethyl)-benzamide;
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-pyrrolidin- 1 -yl-ethyl)-benzamide; 3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-pyridin-4-yl- ethyl)-benzamide;
N-(3 -Dimethylamino-propyl)-3 ,4-difluoro-2-(4-iodo-2-methyl- phenylamino)-benzamide ;
N-Benzyl-4-fluoro-2-(4-iodo-2-methyl-phenylamino)-benzamide; 2-(4-Bromo-2-methyl-phenylamino)-3 ,4-difluoro-N-(2-hydroxy- ethyl)-benzamide;
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-morpholin-4-yl- ethyl)-benzamide;
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(3 -piperidin- 1 -yl- propyl)-benzamide;
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(3-piperidin- 1 -yl-propyl)-benzamide;
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-thiophen-2-yl- ethyl)-benzamide; 4-Fluoro-2-(4-iodo-2 -methyl -phenylamino)-N-(2-pyrrolidin- 1 -yl- ethyl)-benzamide;
2-(4-Bromo-2-methyl-phenylamino)-3,4-difluoro-N-(2-moφholin- 4-yl-ethyl)-benzamide ;
5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N- pyridin-4-ylmethyl-benzamide;
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-pyridin- 4-ylmethyl-benzamide ;
2-(4-Bromo-2-methyl-phenylamino)-N-(3-dimethylamino- propyl)-3 ,4-difluoro-benzamide; 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-pyridin-4-ylmethyl- benzamide;
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-pyridin-4-yl- ethyl)-benzamide; 2-(4-Bromo-2-methyl-phenylamino)-3,4-difluoro-N-(2-pyridin- 4-yl-ethyl)-benzamide;
2-(4-Bromo-2-methyl-phenylamino)-3,4-difluoro-N-(3-hydroxy- propyl)-benzamide ; 2-(4-Bromo-2-methyl-phenylamino)-3 ,4-difluoro-N-(2-pyrrolidin-
1 -yl-ethyl)-benzamide;
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-phenethyl- benzamide;
2-(4-Bromo-2-methyl-phenylamino)-3,4-difluoro-N-(2-thiophen- 2-yl-ethyl)-benzamide;
2-(4-Bromo-2-methyl-phenylamino)-3,4-difluoro-N-pyridin- 4-ylmethyl-benzamide;
2-(4-Bromo-2-methyl-phenylamino)-3,4-difluoro-N-phenethyl- benzamide; 2-(4-Bromo-2 -methyl -phenylamino)-3 ,4-difluoro-N-(2-piperidin-
1 -yl-ethyl)-benzamide;
5 -Chloro-N- { 3 - [4-(2-hydroxy-ethyl)-piperazin- 1 -yl] -propyl } - 2-(4-iodo-2-methyl- phenylamino)- benzamide;
5 -Fluoro-N- { 3 - [4-(2-hydroxy-ethyl)-piperazin- 1 -yl] -propyl } - 2-(4-iodo-2-methyl- phenylamino)- benzamide;
2-(4-Iodo-2-methyl-phenylamino)-5-nitro-N-pyridin-4-yl methyl- benzamide;
5 -Bromo-N- { 3 - [4-(2-hydroxy-ethyl)-piperazin- 1 -yl] -propyl } - 2-(4-iodo-2-methyl- phenylamino)- benzamide; 5-Chloro-N-(2-diethylamino-ethyl)-2-(4-iodo-2-methyl- phenylamino)- benzamide;
5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N-(2-piperidin-l-yl- ethyl)-benzamide;
(3-Hydroxy-pyrrolidin- 1 -yl)-[2-(4-iodo-2-methyl-phenylamino)- 5-nitro-phenyl]-methanone;
5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N-(2 -pyrrolidin- 1 -yl- ethyl)-benzamide; 5-Bromo-N-(2-diethylamino-ethyl)-2-(4-iodo-2-methyl- phenylamino)- benzamide;
N-{2-[Bis-(2-hydroxy-ethyl)-amino]-ethyl}-5-chloro-2-(4-iodo- 2-methyl- phenylamino)- benzamide; N-{2-[Bis-(2-hydroxy-ethyl)-amino]-ethyl}-5-bromo-2-(4-iodo-
2-methyl- phenylamino)- benzamide;
N- { 3 - [4-(2-Hydroxy-ethyl)-piperazin- 1 -yl] -propyl } -2-(4-iodo- 2-methyl-phenylamino)- benzamide;
5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-pyridin-4-ylmethyl- benzamide;
5-Bromo-2-(4-iodo-2-ethyl-phenylamino)-N-(2-pyrrolidin-l-yl- ethyl)-benzamide;
5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-(2-piperidin- 1 -yl- ethyl)-benzamide ; 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-pyrrolidin- 1 -yl- ethyl)-benzamide;
5-Chloro-N-(3-dimethylamino-propyl)-2-(4-iodo-2-methyl- phenylamino)-benzamide;
N- { 2- [Bis-(2-hydroxy-ethyl)-amino] -ethyl } -5 -fluoro-2-(4-iodo- 2-methyl- phenylamino)- benzamide;
5-Chloro-N-(3-hydroxy-propyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide;
5-Chloro-N-(3-diethylamino-2-hydroxy-propyl)-2-(4-iodo- 2-methyl-phenylamino)- benzamide; 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-piperidin- 1 -yl- ethyl)-benzamide ;
5-Bromo-N-(3-hydroxy-propyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide;
5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-(3-piperidin- 1 -yl- propyl)-benzamide;
N-{2-[Bis-(2-hydroxy-ethyl)-amino]-ethyl}-2-(4-iodo-2-methyl- phenylamino)-5 -nitro- benzamide; 5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N-(2-morpholin-4-yl- ethyl)-benzamide;
5-Chloro-N-(3-diethylamino-propyl)-2-(4-iodo-2-methyl- phenylamino)-benzamide; 5-Chloro-N-(2-diisopropylamino-ethyl)-2-(4-iodo-2-methyl- phenylamino)-benzamide;
5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N-(3-piperidin- 1 -yl- propyl)-benzamide;
2-(4-Iodo-2-methyl-phenylamino)-5-nitro-N-(2-piperidin- 1 -yl- ethyl)-benzamide;
5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-(2-piperazin- 1 -yl- ethyl)-benzamide;
N-(2-Diethylamino-ethyl)-5-fluoro-2-(4-iodo-2-methyl- phenylamino)-benzamide; 5-Bromo-N-(3-dimethylamino-propyl)-2-(4-iodo-2-methyl- phenylamino)-benzamide;
N-(3-Hydroxy-propyl)-2-(4-iodo-2-methyl-phenylamino)-5-nitro- benzamide;
5-Fluoro-N-(3-hydroxy-propyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide;
N-(3-Diethylamino-propyl)-5-fluoro-2-(4-iodo-2-methyl- phenylamino)-benzamide;
N-(3-Diethylamino-propyl)-2-(4-iodo-2-methyl-phenylamino)- 5-nitro-benzamide; 5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-(2-morpholin-4-yl- ethyl)-benzamide;
2-(4-Iodo-2-methyl-phenylamino)-5-nitro-N-(3-piperidin- 1 -yl- propyl)-benzamide;
[5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-phenyl] -(3 -hydroxy- pyrrolidin- 1 -yl)-methanone ;
5-Bromo-N-(2-diisopropylamino-ethyl)-2-(4-iodo-2-methyl- phenylamino)-benzamide; 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-morpholin-4-yl- ethyl)-benzamide ;
5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(3-piperidin-l-yl- propyl)-benzamide; [5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-phenyl] -
[4-(2-hydroxy-ethyl)-piperazin- 1 -yl]-methanone;
N-(3-Diethylamino-2-hydroxy-propyl)-5-fluoro-2-(4-iodo- 2-methyl-phenylamino)- benzamide;
N-Cyclopropyl-5-fluoro-2-(4-iodo-2-methyl-phenylamino)- benzamide;
5-Chloro-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide;
5-Fluoro-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide; N-Benzyloxy-5-fluoro-2-(4-iodo-2-methyl-phenylamino)- benzamide;
N-Benzyloxy-5-bromo-2-(4-iodo-2-methyl-phenylamino)- benzamide;
2-(4-Iodo-2-methyl-phenylamino)-5-nitro-N-(4-sulfamoyl-benzyl)- benzamide;
5-Bromo-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide;
N-(2-Hydroxy-ethyl)-5-iodo-2-(4-iodo-2-methyl-phenylamino)- benzamide; N-(2-Hydroxy-ethyl)-2-(4-iodo-2-ethyl-phenylamino)-5-nitro- benzamide;
2-(4-Iodo-2-methyl-phenylamino)-N-methyl-5-nitro-N-phenyl- benzamide;
5-Chloro-N-cyclopropyl-2-(4-iodo-2-methyl-phenylamino)- benzamide;
5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-methyl-N-phenyl- benzamide;
N-Allyl-5-fluoro-2-(4-iodo-2-methyl-phenylamino)-benzamide; N-Benzyloxy-5-iodo-2-(4-iodo-2-methyl-phenylamino)- benzamide;
5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(4-sulfamoyl- benzyl)-benzamide; N-Allyl-5-chloro-2-(4-iodo-2-methyl-phenylamino)-benzamide;
N-Cyclopropyl-2-(4-iodo-2-methyl-phenylamino)-5-nitro- benzamide;
5-Bromo-N-cyclopropyl-2-(4-iodo-2-methyl-phenylamino)- benzamide; 5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N-methyl-N-phenyl- benzamide;
5-Iodo-2-(4-iodo-2-methyl-phenylamino)-N-(4-sulfamoyl-benzyl)- benzamide;
5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-(4-sulfamoyl- benzyl)-benzamide;
N-AUyl-2-(4-iodo-2-methyl-phenylamino)-5-nitro-benzamide;
2-(4-Iodo-2-methyl-phenylamino)-5-nitro-N-(4-sulfamoyl-benzyl)- benzamide;
N-AUyl-5-bromo-2-(4-iodo-2-methyl-phenylamino)-benzamide; 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(3-methyl-benzyl)- benzamide;
N-Cyclopropyl-5-iodo-2-(4-iodo-2-methyl-phenylamino)- benzamide;
5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-methyl-N-phenyl- benzamide;
N-Benzyloxy-2-(4-iodo-2-methyl-phenylamino)-5-nitro- benzamide;
N-Cyclohexyl-5-iodo-2-(4-iodo-2-methyl-phenylamino)- benzamide; N-Allyl-5-iodo-2-(4-iodo-2-methyl-phenylamino)-benzamide;
5 -Iodo-2-(4-iodo-2-methy l-phenylamino)-N-(3 -methy 1-benzyl)- benzamide; 2-(4-Iodo-2-methyl-phenylamino)-N-(3-methyl-benzyl)-5-nitro- benzamide;
5-Iodo-2-(4-iodo-2-methyl-phenylamino)-N-methyl-N-phenyl- benzamide; N-Cyclohexyl-5-fluoro-2-(4-iodo-2-methyl-phenylamino)- benzamide;
5-Chloro-N-cyclohexyl-2-(4-iodo-2-methyl-phenylamino)- benzamide;
5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-(3-methyl-benzyl)- benzamide;
5-Bromo-N-cyclohexyl-2-(4-iodo-2-methyl-phenylamino)- benzamide;
5 -Chloro-2-(4-iodo-2-methyl-phenylamino)-N-(3 -methyl-benzy 1)- benzamide; N-Cyclohexyl-2-(4-iodo-2-methyl-phenylamino)-5-nitro- benzamide;
N-Benzyloxy-5-bromo-2-(4-iodo-2-methyl-phenylamino)- benzamide;
N-Benzyloxy-5-fluoro-2-(4-iodo-2-methyl-phenylamino)- benzamide;
5-Chloro-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide;
5-Bromo-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide; 2-(4-Iodo-2-methyl-phenylamino)-N-methyl-5-nitro-N-phenyl- benzamide;
5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N-methyl-N-phenyl- benzamide;
N-(2-Hydroxy-ethyl)-5-iodo-2-(4-iodo-2-methyl-phenylamino)- benzamide;
5-Chloro-N-cyclopropyl-2-(4-iodo-2-methyl-phenylamino)- benzamide;
N-Allyl-5-chloro-2-(4-iodo-2-methyl-phenylamino)-benzamide; 5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-methyl-N-phenyl- benzamide;
N-(2-Hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)-5-nitro- benzamide; 5 -Fluoro-N-(2-hydroxy-ethyl)-2-(4-iodo-2-methyl-phenylamino)- benzamide;
5-Bromo-N-cyclopropyl-2-(4-iodo-2-methyl-phenylamino)- benzamide;
N-Cyclopropyl-5-fluoro-2-(4-iodo-2-methyl-phenylamino)- benzamide;
5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(4-sulfamoyl- benzyl)-benzamide ;
N-Cyclopropyl-2-(4-iodo-2-methyl-phenylamino)-5-nitro- benzamide; N-Allyl-5-fluoro-2-(4-iodo-2-methyl-phenylamino)-benzamide;
N-Benzyloxy-5-iodo-2-(4-iodo-2-methyl-phenylamino)- benzamide;
N-Allyl-5-bromo-2-(4-iodo-2-methyl-phenylamino)-benzamide;
5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-(4-sulfamoyl- benzyl)-benzamide;
5-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-methyl-N-phenyl- benzamide;
N-Allyl-2-(4-iodo-2-methyl-phenylamino)-5-nitro-benzamide;
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-benzyl alcohol; [5-Chloro-2-(4-iodo-2-methyl-phenylamino)-phenyl]-methanol;
[2-(4-Iodo-2-methyl-phenylamino)-5-nitro-phenyl]-methanol;
[5-Bromo-2-(4-iodo-2-methyl-phenylamino)-phenyl]-methanol; and
N-Allyl-2-(4-iodo-2-methyl-phenylamino)-5-nitro-benzamide.
6. The method of claim 4, wherein the MEK inhibitor is a compound of Formula (I) wherein (a) R\ is hydrogen, methyl, methoxy, fluoro, chloro, or bromo; (b) R2 is hydrogen; (c) R3, i, and R5 independently are hydrogen, fluoro, chloro, bromo, iodo, methyl, methoxy, or nitro; (d) R10 and Ri ι independently are hydrogen or methyl; (e) Z is COOR , tetrazolyl, CONReR?, CONHNRioRn, or CH2OR7; Re and R7 independently are hydrogen, C 1-4 alkyl, heteroaryl, or C 3-5 cycloalkyl optionally containing one or two heteroatoms selected from O, S, or NH; or Re and R together with the nitrogen to which they are attached complete a 5-6 member cyclic ring optionally containing 1 or 2 additional heteroatoms selected from O, NH or N-alkyl; and wherein any of the foregoing alkyl or aryl groups can be unsubstituted or substituted by halo, hydroxy, methoxy, ethoxy, or heteroaryloxy; (f) Z is COOR ; (g) R7 is H, pentafluorophenyl, or tetrazolyl; (h) R3, t, and R5 are independently H, fluoro, or chloro; (i) Rt is fluoro; (j) two of R3, Rt, and R5 are fluoro; or (k) combinations of the above.
The method of claim 6, wherein the MEK inhibitor is a compound of Formula (I) wherein: Z is COOR ; R is H, pentafluorophenyl, or tetrazolyl; R3 and R5 are independently H, fluoro, or chloro; and R4 is fluoro.
A method according to claim 1, where the MEK inhibitor is a compound of Formula II
wherein:
R\a is hydrogen, hydroxy, C^-Cg alkyl, Cj-Cg alkoxy, halo, trifluoromethyl, or CN; R2a is hydrogen; R3a, R4a, and R5a independently are hydrogen, hydroxy, halo, trifluoromethyl, Cj-Cg alkyl, Cj-Cg alkoxy, nitro, CN, or
(O or NH)m-(CH2)n-R9a> where R9a is hydrogen, hydroxy, CO2H or NRiOaRl la- n is 0-4; m is O or l;
RlOa an(i Rl la independently are 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-Ci-Cg alkyl;
Rga is hydrogen, Cj-Cg alkyl, (CO)-Cj-Cg alkyl, aryl, aralkyl, or
C3-C10 cycloalkyl; R7a is hydrogen, Cj-Cg alkyl, C2-C alkenyl, C2-Cg alkynyl,
C3-C10 (cycloalkyl or cycloalkyl optionally containing a heteroatom selected from O, S, or NR9a); and wherein any of the foregoing alkyl, alkenyl, aryl, heteroaryl, heterocyclic, and alkynyl groups can be unsubstituted or substituted by halo, hydroxy, Cι-C6 alkoxy, amino, nitro, Cι-C4 alkylamino, C4)alkylamino, C3-C cycloalkyl, phenyl, phenoxy, C3-C5 heteroaryl or heterocyclic radical, or C3-C5 heteroaryloxy or heterocyclic radical-oxy; or
R a and R7a 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 NRjOaRl la> or a pharmaceutically acceptable salt, ester, amide or prodrug thereof.
9. The method of Claim 8, comprising a MEK inhibitor having a structure of Formula (II) wherein: (a) Rla is H, methyl, fluoro, or chloro; (b) R2a is H; R3a, ia, and R5aare each H, Cl, nitro, or F; (c) R^ is H; (d) R7a is methyl, ethyl, 2-propenyl, propyl, butyl, pentyl, hexyl, cyclopropylmethyl, cyclobutyl methyl, cyclopropylmethyl, or cyclopropylethyl; and (e) the 4' position is I, rather than Br.
10. The method of claim 9, comprising a MEK inhibitor having a structure of Formula (II) wherein: Ria is F at the 4 position, para to the CO-N-R6a-OR a group and meta to the bridging nitrogen; at least one of R3a and R5a is F or
Cl; and Rιa is methyl or chloro.
11. The method of Claim 8, comprising a MEK inhibitor having a formula selected from:
4-Fluoro-N-hydroxy-2-(4-iodo-2-methyl-phenylamino)-benzamide; 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(methoxy)- benzamide;
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(prop-2-ynyloxy)- benzamide;
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-phenoxyethoxy)- benzamide;
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(2-thienylmethoxy)- benzamide;
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(prop-2-enyloxy)- benzamide; 4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-
(cyclopropylmethoxy)-benzamide;
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(cyclopentoxy)- benzamide;
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N- (3-furylmethoxy)-benzamide;
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino)-N-ethoxy- benzamide; 3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino -N-(but-2-enyloxy)- benzamide;
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino ■N-(cyclopropyl- methoxy)-benzamide;
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino N-( 1 -methylprop- 2-ynyloxy)-benzamide;
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino N-(3 -phenylprop- 2-ynyloxy)-benzamide;
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino N-(3-methyl- 5-phenylpent-2-en-4-ynyloxy).-benzamide;
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino -N-(prop- 2-ynyloxy)-benzamide;
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino ■N-(propoxy)- benzamide;
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino ■N-(cyclobutyloxy)- benzamide;
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino >-N- (2-thienylmethoxy)-benzamide;
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino •N-(2-methyl-prop- 2-enyloxy)-benzamide;
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino' •N- (2-phenoxyethoxy)-benzamide;
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino ■N-(but-2-enyloxy)- benzamide;
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino -N-(but-3 -yny loxy)- benzamide;
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino •N- (cyclopentyloxy)-benzamide;
3,4-Difluoro-2-(4-iodo-2-methyl-phenylamino •N- (3 -(2-fluorophenyl)-prop-2-ynyloxy)-benzamide ;
5 -Bromo-3 ,4-difluoro-N-hydroxy-2-(4-iodo-2-methyl phenylamino)-benzamide; 5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N- (n-propoxy)-benzamide;
5 -Bromo-3 ,4-difluoro-N-(furan-3 -y lmethoxy)-2-(4-iodo-2-methyl- phenylamino)-benzamide; 5-Bromo-N-(but-2-enyloxy)-3,4-difluoro-2-(4-iodo-2-methyl- phenylamino)-benzamide
5-Bromo-N-butoxy-3,4-difluoro-2-(4-iodo-2-methyl- phenylamino)-benzamide;
5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N- (3-methyl-but-2-enyloxy)-benzamide;
5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N- (3-methyl-pent-2-en-4-ynyloxy)-benzamide;
5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-benzyl)-N- [5-(3-methoxy-phenyl)-3-methyl-pent-2-en-4-ynyloxy]-benzamide; 5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N-(prop-
2-ynyloxy)-benzamide;
5 -Bromo-3 ,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N- [3 -(3 -methoxy-phenyl)-prop-2-ynyloxy] -benzamide;
5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N- (thiopen-2-ylmethoxy)-benzamide;
5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N- (pyridin-3 -ylmethoxy)-benzamide;
5-Bromo-3-4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N- (3-(2-fluorophenyl)-prop-2-ynyloxy)-benzamide; 5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N-
(ethoxy)-benzamide;
5 -Bromo-3 ,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N- (cyclopropylmethoxy)-benzamide;
5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N- (isopropoxy)-benzamide;
5-Bromo-3,4-difluoro-2-(4-iodo-2-methyl-phenylamino)-N-but- 3 -ynyloxy)-benzamide; 5-Chloro-N-hydroxy-2-(4-iodo-2-methyl-phenylamino)- benzamide;
5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N-(tetrahydro-pyran- 2-yloxy)-benzamide; 5-Chloro-2-(4-iodo-2-methyl-phenylamino)-N-methoxy- benzamide;
4-Bromo-2-(4-iodo-2-methyl-phenylamino)-N-phenylmethoxy- benzamide;
4-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-phenylmethoxy- benzamide;
5-Fluoro-N-hydroxy-2-(4-iodo-2-methyl-phenylamino)-benzamide;
5-Iodo-2-(4-iodo-2-methyl-phenylamino)-N-phenylmethoxy- benzamide;
5-Fluoro-2-(4-iodo-2-methyl-phenylamino)-N-(tetrahydropyran- 2-yloxy)-benzamide;
3,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N- (3 -phenylprop-2-ynyloxy)-benzamide ;
3,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N- (3-furylmethoxy)-benzamide; 3 ,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N-
(2-thienylmethoxy)-benzamide;
3,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N-(but- 3 -ynyloxy)-benzamide;
3,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N-(2-methyl- prop-2-enyloxy)-benzamide;
3,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N-(but- 2-enyloxy)-benzamide;
3,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N-(methoxy)- benzamide; 3,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N-(ethoxy)- benzamide;
3,4-Difluoro-2-(4-bromo-2-methyl-phenylamino)-N- (cyclobutoxy)-benzamide; 3,4-Difluoro-2-(4-bromo-2-methyl-phenylamino) ■N-(isopropoxy)- benzamide;
3,4-Difluoro-2-(4-bromo-2-methyl-phenylamino) ■N- (2-phenoxyethoxy)-benzamide;
3,4-Difluoro-2-(4-bromo-2-methyl-phenylamino) ■N-(cyclopropyl- methoxy)-benzamide;
3,4-Difluoro-2-(4-bromo-2-methyl-phenylamino) •N-(n-propoxy)- benzamide;
3,4-Difluoro-2-(4-bromo-2-methyl-phenylamino) -N-(l-methyl- prop-2-ynyloxy)-benzamide;
3,4-Difluoro-2-(4-bromo-2-methyl-phenylamino) ■N- (3-(3-fluorophenyl)-prop-2-ynyloxy)-benzamide;
3,4-Difluoro-2-(4-bromo-2-methyl-phenylamino) -N- (4,4-dimethylpent-2-ynyloxy)-benzamide;
3,4-Difluoro-2-(4-bromo-2-methyl-phenylamino) •N- (cyclopentoxy)-benzamide ;
3,4,5-Trifluoro-N-hydroxy-2-(4-iodo-2-methyl-phenylamino)- benzamide;
5 -Chloro-3 ,4-difluoro-N-hydroxy-2-(4-iodo-2-methyl- phenylamino)-benzamide;
5-Bromo-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-N- hydroxy-benzamide;
N-Hydroxy-2-(4-iodo-2-methyl-phenylamino)-4-nitro-benzamide;
3,4,5-Trifluoro-2-(2-fluoro-4-iodo-phenylamino)-N-hydroxy- benzamide;
5-Chloro-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-N- hydroxy-benzamide;
5-Bromo-2-(2-chloro-4-iodo-phenylamino)-3,4-difluoro-N- hydroxy-benzamide;
2-(2-Fluoro-4-iodo-phenylamino)-N-hydroxy-4-nitro-benzamide;
2-(2-Chloro-4-iodo-phenylamino)-3,4,5-trifluoro-N-hydroxy- benzamide; 5-Chloro-2-(2-chloro-4-iodo-phenylamino)-3,4-difluoro-N- hy droxy-benzamide ;
5-Bromo-2-(2-bromo-4-iodo-phenylamino)-3,4-difluoro-N- hydroxy-benzamide; 2-(2-Chloro-4-iodo-phenylamino)-N-hydroxy-4-methyl- benzamide;
2-(2-Bromo-4-iodo-phenylamino)-3,4,5-trifluoro-N-hydroxy- benzamide;
2-(2-Bromo-4-iodo-phenylamino)-5-chloro-3,4-difluoro-N- hydroxy-benzamide;
2-(2-Bromo-4-iodo-phenylamino)-N-hydroxy-4-nitro-benzamide;
4-Fluoro-2-(2-fluoro-4-iodo-phenylamino)-N-hydroxy-benzamide;
3,4-Difluoro-2-(2-fluoro-4-iodo-phenylamino)-N-hydroxy- benzamide; 2-(2-Chloro-4-iodo-phenylamino)-4-fluoro-N-hydroxy-benzamide;
2-(2-Chloro-4-iodo-phenylamino)-3,4-difluoro-N-hydroxy- benzamide;
2-(2-Bromo-4-iodo-phenylamino)-4-fluoro-N-hydroxy-benzamide;
2-(2-Bromo-4-iodo-phenylamino)-3,4-difluoro-N-hydroxy- benzamide;
N-Cyclopropylmethoxy-3,4,5-trifluoro-2-(4-iodo-2-methyl- phenylamino)-benzamide;
5-Chloro-N-cyclopropylmethoxy-3,4-difluoro-2-(4-iodo-2-methyl- phenylamino)-benzamide; 5-Bromo-N-cyclopropylmethoxy-3,4-difluoro-2-(2-fluoro-4-iodo- phenylamino)-benzamide;
N-Cyclopropylmethoxy-2-(4-iodo-2-methyl-phenylamino)-4-nitro- benzamide;
N-Cyclopropylmethoxy-3,4,5-trifluoro-2-(2-fluoro-4-iodo- phenylamino)-benzamide;
5 -Chloro-N-cyclopropylmethoxy-3 ,4-difluoro-2-(2-fluoro-4-iodo- phenylamino)-benzamide; 5-Bromo-2-(2-chloro-4-iodo-phenylamino)-N- cyclopropylmethoxy-3,4-difluoro-benzamide;
N-Cyclopropylmethoxy-2-(2-fluoro-4-iodo-phenylamino)-4-nitro- benzamide; 2-(2-Chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-
3,4,5-trifluoro-benzamide;
5-Chloro-2-(2-chloro-4-iodo-phenylamino)-N- cyclopropylmethoxy-3,4-difluoro-benzamide;
5-Bromo-2-(2-bromo-4-iodo-phenylamino)-N-ethoxy-3,4-difluoro- benzamide;
2-(2-Chloro-4-iodo-phenylamino)-N-ethoxy-4-nitro-benzamide;
2-(2-Bromo-4-iodo-phenylamino)-N-cyclopropylmethoxy- 3,4,5-trifluoro-benzamide;
2-(2-Bromo-4-iodo-phenylamino)-5-chloro-N- cyclopropylmethoxy-3 ,4-difluoro-benzamide
2-(2-Bromo-4-iodo-phenylamino)-N-cyclopropylmethoxy-4-nitro- benzamide;
N-Cyclopropylmethoxy-4-fluoro-2-(2-fluoro-4-iodo-phenylamino)- benzamide; N-Cyclopropylmethoxy-3,4-difluoro-2-(2-fluoro-4-iodo- phenylamino)-benzamide;
2-(2-Chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy- 4-fluoro-benzamide;
2-(2-Chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy- 3,4-difluoro-benzamide;
2-(2-Bromo-4-iodo-phenylamino)-N-cyclopropylmethoxy- 4-fluoro-benzamide; and
2-(2-Bromo-4-iodo-phenylamino)-N-cyclopropylmethoxy- 3 ,4-difluoro-benzamide.
12. The method of claim 1 , comprising a MEK inhibitor having a structure selected from: 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-methylphenylamino)- benzoic acid (PD 296767); and 5-chloro-N-cyclopropylmethoxy -3,4- difluoro-2-(4-iodo-2-methylphenylamino)-benzamide (PD 298127).
13. A method for preventing and controlling the rejection in a patient of a transplanted organ, cell, tissue or limb, said method comprising the step of administering to the patient who has undergone a transplant, or who is scheduled to undergo a transplant, an effective immunosuppressive amount of a compound selected from:
2-(2-Chloro-4-iodophenylamino)-N-cyclopropylmethoxy- 3,4-difluorobenzamide (PD184352);
2-(2-Methyl-4-iodophenylamino)-N-hydroxy-4-fluorobenzamide (PD 170611);
2-(2-Methyl-4-iodophenylamino)-N-hydroxy-3,4-difluoro- 5-bromobenzamide (PD171984); 2-(2-Methyl-4-iodophenylamino)-N-cyclopropylmethoxy-
3,4-difluoro-5-bromobenzamide (PDl 77168);
2-(2-Methyl-4-iodophenylamino)-N-cyclobutylmethoxy- 3,4-difluoro-5-bromobenzamide (PD 180841);
2-(2-Chloro-4-iodophenylamino)-N-cyclopropylmethoxy- 3,4-difluoro-5-bromobenzamide (PD 184161);
2-(2-Chloro-4-iodophenylamino)-N-hydroxy-3,4-difluoro- 5-bromobenzamide (PD 184386);
2-(2-Chloro-4-iodophenylamino)-N-cyclobutylmethoxy- 3,4-difluorobenzamide (PD 185625); 2-(2-Chloro-4-iodophenylamino)-N-hydroxy-4-fluorobenzamide
(PD 185848); 2-(2-Methyl-4-iodophenylamino)-N-hydroxy- 3,4-difluorobenzamide(PD 188563);
2-(2-Methyl-4-iodophenylamino)-N-cyclopropylmethoxy- 3,4,5-trifluorobenzamide (PD 198306); and
2-(2-Chloro-4-iodophenylamino)-N-cyclopropylmethoxy- 4-fluorobenzamide (PD 203311).
14. A method for the prophylaxis or maintenance of transplant rejection in a mammal comprising administering to a patient in need of prophylaxis or maintenance an effective amount of 2-(2-chloro-4-iodophenylamino)-N- cyclopropylmethoxy-3,4-difluorobenzamide.
15. A method for the prophylaxis or maintenance of transplant rejection in a mammal comprising administering to a patient in need of prophylaxis or maintenance an effective amount of 2-(2-methyl-4-iodophenylamino)-N- cyclopropylmethoxy-3 ,4,5-trifluorobenzamide.
EP99966203A 1998-12-15 1999-12-14 Use of a mek inhibitor for preventing transplant rejection Withdrawn EP1140046A1 (en)

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ZA200103765B (en) 2002-05-09
CA2346684A1 (en) 2000-06-22
WO2000035435A1 (en) 2000-06-22
WO2000035435A9 (en) 2001-03-22
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HUP0104607A3 (en) 2002-12-28
KR20010101203A (en) 2001-11-14

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