WO2018081863A1 - Dérivés à substitution en position 6 de l'hexaméthylène amiloride en tant qu'inhibiteurs de upa et leurs utilisations - Google Patents

Dérivés à substitution en position 6 de l'hexaméthylène amiloride en tant qu'inhibiteurs de upa et leurs utilisations Download PDF

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WO2018081863A1
WO2018081863A1 PCT/AU2017/051208 AU2017051208W WO2018081863A1 WO 2018081863 A1 WO2018081863 A1 WO 2018081863A1 AU 2017051208 W AU2017051208 W AU 2017051208W WO 2018081863 A1 WO2018081863 A1 WO 2018081863A1
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compound
cancer
group
optionally substituted
alkyl
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PCT/AU2017/051208
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English (en)
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Michael Kelso
Marie Ranson
Benjamin Buckley
Ashraf Aboelela
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University Of Wollongong
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings

Definitions

  • the present invention broadly relates to 6-substituted derivatives of hexamethylene amiloride, the preparation thereof, and their use in the treatment of diseases such as cancer.
  • the plasminogen activation system comprises the serine protease urokinase plasminogen activator (uPA), its cognate cell surface receptor (uPAR) and two endogenous serpin inhibitors; plasminogen activator inhibitor 1 (PAI-1 ) and PAI-2.
  • uPA serine protease urokinase plasminogen activator
  • PAI-1 plasminogen activator inhibitor 1
  • PAI-2 plasminogen activator inhibitor 1
  • Plasmin activates a downstream cascade of extracellular proteases (e.g. matrix metalloproteinases) and latent growth factors and together these provide controlled pericellular proteolysis and directional remodeling of the local extracellular environment.
  • the PAS plays a central role in many cell migration and invasion processes and, not surprisingly, its upregulation is heavily implicated in tumour metastasis and invasive disease.
  • upregulated uPA is one of the strongest prognostic biomarkers of shortened disease-free survival and overall survival in breast cancer and one of the most accurate predictors of metastasis in lymph-node- negative breast tumours.
  • the present invention provides a compound of the general formula (I):
  • R is -T-Ft ! ;
  • Ri is selected from the group consisting of: an aryl group, a heteroaryl group, a carbocyclyl group and a heterocycyl group, each of which may be optionally substituted, and with the proviso that 3-amino-5-(azepan-1 -yl)-A/-carbamimidoyl-6-phenylpyrazine-2- carboxamide is excluded.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula (I) according to the first aspect together with a pharmaceutically acceptable carrier, diluent or excipient.
  • the present invention provides a method for the treatment of a disease or condition associated with uPA in a subject in need thereof, the method comprising administration to the subject of a therapeutically effective amount of a compound of formula (I) according to the first aspect, or a composition of the second aspect.
  • the disease or condition may be associated with upregulation or increased expression of uPA.
  • the disease or condition may be cancer.
  • the cancer may be locally advanced.
  • the cancer may be metastatic.
  • the cancer may be, for example, breast cancer, pancreatic cancer, upper Gl tract cancer, lower Gl tract cancer, colorectal cancer, prostate cancer, ovarian cancer, lung cancer, renal cancer, head and neck cancer, glioblastoma or melanoma.
  • the disease or condition may be an inflammatory disease or condition.
  • the inflammatory disease or condition may be, for example, chronic obstructive pulmonary disorder, coronary artery disease, rheumatoid arthritis, corneal inflammation, preeclampsia, colitis, cardiac fibrosis, pulmonary fibrosis, cirrhosis, asthma, psoriasis or periodontitis.
  • the disease or condition may be amyotrophic lateral sclerosis.
  • the disease or condition may be a wound.
  • the wound may be a chronic wound.
  • the wound is a dermal ulcer.
  • the present invention provides a method for preventing or slowing cancer spread in a subject in need thereof, the method comprising administration to the subject of a therapeutically effective amount of a compound of formula (I) according to the first aspect, or a composition of the second aspect.
  • the cancer may be associated with uPA.
  • the cancer may be associated with upregulation or increased expression of uPA.
  • the cancer may be localised.
  • the cancer may be locally advanced.
  • the cancer may be metastatic.
  • the present invention provides a method for inducing apoptosis in a cancer cell, the method comprising contacting the cancer cell with an effective amount of a compound of formula (I) according to the first aspect, or a composition of the second aspect.
  • the present invention provides a method for reducing incidences of, or risk of, cancer recurrence in a subject deemed to be at risk of cancer recurrence, the method comprising administration to the subject of an effective amount of a compound of formula (I) according to the first aspect, or a composition of the second aspect.
  • the subject deemed to be at risk of cancer recurrence may be a subject who is in cancer remission.
  • the present invention provides use of a compound of formula (I) according to the first aspect in the manufacture of a medicament for the treatment of a disease or condition associated with uPA.
  • the disease or condition may be associated with upregulation or increased expression of uPA.
  • the disease or condition may be cancer.
  • the cancer may be locally advanced.
  • the cancer may be metastatic.
  • the cancer may be, for example, breast cancer, pancreatic cancer, upper Gl tract cancer, lower Gl tract cancer, colorectal cancer, prostate cancer, ovarian cancer, lung cancer, renal cancer, head and neck cancer, glioblastoma or melanoma.
  • the disease or condition may be an inflammatory disease or condition.
  • the inflammatory disease or condition may be, for example, chronic obstructive pulmonary disorder, coronary artery disease, rheumatoid arthritis, corneal inflammation, preeclampsia, colitis, cardiac fibrosis, pulmonary fibrosis, cirrhosis, asthma, psoriasis or periodontitis.
  • the disease or condition may be amyotrophic lateral sclerosis.
  • the disease or condition may be a wound.
  • the wound may be a chronic wound.
  • the wound is a dermal ulcer.
  • the present invention provides use of a compound of formula (I) according to the first aspect in the manufacture of a medicament for preventing or slowing cancer spread.
  • the cancer may be associated with uPA.
  • the cancer may be associated with upregulation or increased expression of uPA.
  • the cancer may be localised.
  • the cancer may be locally advanced.
  • the cancer may be metastatic.
  • the present invention provides use of a compound of formula (I) according to the first aspect in the manufacture of a medicament for inducing apoptosis in a cancer cell.
  • the present invention provides use of a compound of formula (I) according to the first aspect in the manufacture of a medicament for reducing incidences of, or risk of, cancer recurrence in a subject deemed to be at risk of cancer recurrence.
  • the present invention provides a method for inhibiting uPA in a subject, the method comprising administration to the subject of a compound of formula (I) according to the first aspect, or a composition of the second aspect.
  • the present invention provides use of a compound of formula (I) according to the first aspect for inhibiting uPA.
  • the present invention provides a method for preparing a compound of the formula (I) as defined in the first aspect comprising the steps of: (i) reacting a compound of the following formula (IV):
  • R is as defined in the first aspect
  • the present invention provides a compound of the general formula (II):
  • R is as defined in the first aspect and R 2 is C C 20 alkyl, with the proviso that methyl 3-amino-5-(azepan-1 -yl)-6-phenylpyrazine-2-carboxylate is excluded
  • R 2 is C C 10 alkyl.
  • R 2 is C C 6 alkyl.
  • R 2 is C C 3 alkyl.
  • R 2 is methyl
  • an element means one element or more than one element.
  • alkyl is taken to mean straight chain or branched chain monovalent saturated hydrocarbon groups having the recited number of carbon atoms, such as methyl, ethyl, 1 -propyl, isopropyl, 1 -butyl, 2-butyl, isobutyl, tert- butyl, amyl, 1 ,2-dimethylpropyl, 1 ,1 -dimethylpropyl, pentyl, isopentyl, hexyl and the like.
  • halo and halogen are synonomous and refer to fluoro, chloro, bromo and iodo.
  • haloCrCe alkyl is taken to mean straight chain or branched chain monovalent saturated hydrocarbon groups having between 1 and 6 carbon atoms wherein one or more hydrogens are replaced by a halogen.
  • C 2 -C 6 alkenyl is taken to mean straight chain or branched chain monovalent hydrocarbon radicals having between 2 and 6 carbon atoms and at least one carbon-carbon double bond, such as vinyl, propenyl, 2- methyl-2-propenyl, butenyl and the like.
  • the group may contain a plurality of double bonds and the geometry about each double bond is independently cis or trans, E or Z.
  • C 2 -C 6 alkynyl is taken to mean straight chain or branched chain monovalent hydrocarbon radicals having between 2 and 6 carbon atoms and at least one carbon-carbon triple bond, such as ethynyl, propargyl and the like.
  • the group may contain a plurality of triple bonds.
  • aryl group is taken to mean monovalent aromatic hydrocarbon groups having between 6 and 20 ring carbon atoms, or between 6 and 14 ring carbon atoms, or between 6 and 10 ring carbon atoms.
  • the aryl group may have a single ring or multiple rings.
  • Aryl also includes bicyclic radicals comprising an aromatic ring fused to a saturated or partially unsaturated carbocyclic ring.
  • aryl groups include, but are not limited to, phenyl, naphthyl, phenanthrenyl, azulenyl, anthracenyl, 1 ,2-dihydronaphthyl, 1 ,2,3,4-tetrahydronaphthyl and the like.
  • heteroaryl group is taken to mean monovalent aromatic groups having between 5 and 20 ring atoms, or between 5 and 14 ring atoms, or between 5 and 10 ring atoms in which one or more of the ring atoms is O, N or S, the remaining ring atoms being carbon.
  • the heteroaryl group may have a single ring or multiple rings.
  • Heteroaryl also includes bicyclic radicals comprising a heteroaromatic ring fused to a saturated, partially unsaturated ring or aromatic carbocyclic ring.
  • heteroaryl groups include, but are not limited to, pyridyl, pyrazolyl, triazolyl, pyrimidinyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxadiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, benzothienyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, triazolyl, thiadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophen
  • carbocyclyl group is taken to mean monovalent, non-aromatic, saturated or partially unsaturated carbocyclic rings having between 3 and 12 carbon atoms, or between 5 and 10 carbon atoms.
  • the carbocyclyl ring may be monocyclic or bicyclic.
  • Bicyclic carbocycles may be arranged, for example, as a bicyclo [4,5], [5,5], [5,6] or [6,6], or as bridged systems, such as bicyclo[2.2.1 ]heptane.
  • carbocyclyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, 1 -cyclopent-1 -enyl, 1 -cyclopent-3-enyl, 1 -cyclohex- 2-enyl, cyclohexadienyl, cyclooctyl, cyclodecyl and the like.
  • heterocyclyl group is taken to mean monovalent, non-aromatic, saturated or partially unsaturated rings having between 3 and 12 ring atoms, or between 5 and 10 ring atoms, in which one or more of the ring atoms is O, N or S, the remaining ring atoms being carbon.
  • the heterocyclyl ring may be monocyclic or bicyclic.
  • heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, tetrahydropyrimidinyl, thioxanyl, piperazinyl, homopiperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1 ,3- dioxolanyl, t
  • tautomer refers to structural isomers of different energies which are interconvertible via a low energy barrier.
  • prodrug means a compound which is able to be converted in vivo by metabolic means (e.g. by hydrolysis, reduction or oxidation) to a compound of the formula (I).
  • cancer refers to a physiological condition characterised by unregulated cell growth.
  • the term "effective amount” includes a non-toxic but sufficient amount of an active compound to provide the stated effect.
  • “effective amount” means an amount of a compound of formula (I) that is required to reduce the incidence of, or risk of an individual experiencing cancer recurrence.
  • Those skilled in the art will appreciate that the exact amount of a compound required will vary based on a number of factors and thus it is not possible to specify an exact "effective amount”. However, for any given case an appropriate "effective amount” may be determined by one of ordinary skill in the art.
  • the term "therapeutically effective amount” includes a non-toxic but sufficient amount of an active compound to provide the desired therapeutic effect. Those skilled in the art will appreciate that the exact amount of a compound required will vary based on a number of factors and thus it is not possible to specify an exact “therapeutically effective amount”. However, for any given case an appropriate “therapeutically effective amount” may be determined by one of ordinary skill in the art.
  • treating and “treatment” refer to any and all uses, which remedy the stated disease or symptoms thereof, hinder, retard or otherwise reverse the progression of the disease or other undesirable symptoms in any way whatsoever.
  • treating and “treatment” are to be considered in their broadest context. For example, treatment does not necessarily imply that a subject is treated until total recovery.
  • locally advanced is synonomous with the term “locally invasive” and means a cancer that has spread from where it originated to nearby tissues or lymph nodes, but which has not metastasized.
  • the term "recurrence" as it relates to cancer is understood to mean the return of cancerous cells and/or a cancerous tumour after cancerous cells and/or a cancerous tumour have been successfully treated previously.
  • association with when used in the context of a disease or condition “associated with” uPA means that the disease or condition, or a symptom thereof, may result from, result in, be characterized by, or otherwise related to uPA.
  • association between the disease or condition and uPA activity may be direct or indirect and may be temporally separated.
  • the term "subject” includes human and also non- human animals.
  • the compounds of the present invention in addition to being useful in the treatment of diseases such as cancer in humans, the compounds of the present invention also find use in the treatment of diseases in non-human animals, for example mammals such as companion animals and farm animals.
  • Non-limiting examples of companion animals and farm animals include dogs, cats, horses, cows, sheep and pigs.
  • the subject is a human.
  • administering includes contacting, applying, delivering or providing a compound or composition of the invention to an organism by any appropriate means.
  • FIG. 1 Inhibition of low molecular weight (LMW) human uPA proteolytic activity by compounds 19 and 26 as determined by fluorometric enzyme inhibition assay.
  • LMW active human uPA (0.75 nM) was added to drug dilutions or vehicle control containing Urokinase III Fluorescent substrate (250 ⁇ ) in assay buffer (20 mM HEPES (pH 7.6), 100 mM NaCI, 0.5 mM EDTA, 0.01 % v/v Tween-20) in a final volume of 200 ⁇ and 1 % v/v DMSO. Reads were conducted every minute at 37°C for 45 min, ex. 355 nn, em. 460 nm.
  • FIG. 2 Dose-dependent effects of compounds of the formula (I) on cell viability and LDH release in U-937 Human histiocytic lymphoma cells.
  • Panels A) and B) show results from a CellTitre 96® Aqueous One Solution Cell Proliferation Assay experiment where colour intensity (490 nm) is indicative of cell metabolism.
  • Cells were harvested 48 h post-passage, plated at a density of 5000 cells/well and incubated at 37 °C, 5% C0 2 for 18h prior to drug treatment. Vehicle (DMSO) was present at concentration of 1 %. Cells were cultured for 48 h post-drug treatment, followed by 2 h incubation with MTS reagent and reading at 490 nm.
  • DMSO DMSO
  • Panels C) and D) show results from assays using the Roche Cytotoxicity Detection Kit (PLUS) .
  • Abs 490 corresponds to degree of cytotoxic cell death in response to drug treatment.
  • FIG. 3 Dose-dependent caspase 3/7 activation in HEK-293 cells treated with compound 19 or hexamethylene amiloride (HMA).
  • Cells were plated at a density of 2.0x10 4 cells/well in a black 96-well cell culture plate and incubated for 18 h at 37° C, 5% C0 2 .
  • Figure 4 Evaluation of the selectivity of compound 19 for a variety of trypsin-like serine proteases via a chromogenic enzyme activity assay. Enzymes were present at a concentration of 10 nM thrombin, trypsin, plasmin and t-PA, uPA. The substrate, S- 2288 was present at a concentration of 250 ⁇ . All components were present in assay buffer (10 mM HEPES, 150 mM NaCI, 0.01 % Tween-20, pH 7.4) containing a final volume of 1 % DMSO. Change in absorbance overtime at 405 nm was measured at 37 °C using a Molecular Devices SpectraMax Plus 384-weil plate reader. IC 50 values were determined by plotting the percentage of residual activity (V0) versus log drug concentration and fitted to a sigmoidal dose response curve using GraphPad Prism v.6.0.
  • Figure 6 Effects of Amiloride, HMA, 19 and 24 on diuresis and urinary K7Na + in male acetazolamide-treated Sprague Dawley rats. Excepting the vehicle control cohort, all animals received 25 mg/kg acetazolamide IP followed by 1 .5 mg/kg amiloride, HMA or 6-HMA analogues treatment IV, all delivered at 1 ml/kg. Following administration animals were housed in metabolic cages and urine collected for urinalysis over 6 h.
  • Figure 8 Representative images showing migration of SKOV-3 human ovarian carcinoma cells into collagen I matrix contracted by human-skin derived telomerase- immortalized fibroblasts. Cells were allowed to invade for 21 days prior to formalin fixing. Cytokeratin stained plugs were imaged using brightfield microscopy with 10 images collected across three plugs for each treatment group. Compound or vehicle containing media was refreshed every 72 h. Images for each treatment group were obtained from different replicate plugs that received identical drug or vehicle treatments. All drugs were present at 2 ⁇ .
  • Figure 9 Inhibition of SKOV-3 ovarian adenocarcinoma cell invasion by compounds of formula (I).
  • Figure 12 X-ray co-crystal structures of compounds 19 and 24 bound to the active site of human uPA.
  • Ligand, Ser146 and the amino acid side chains within 6 A of the inhibitors are presented as cylinders. H-bonds are represented as dashed lines.
  • the present invention provides a compound of the general formula (I)
  • R is -T-Ft ! ;
  • Ri is selected from the group consisting of: an aryl group, a heteroaryl group, a carbocyclyl group and a heterocycyl group, each of which may be optionally substituted, and with the proviso that 3-amino-5-(azepan-1 -yl)-A/-carbamimidoyl-6-phenylpyrazine-2- carboxamide is excluded.
  • T is absent.
  • the aryl group may comprise between 6 and 14 ring carbon atoms
  • the heteroaryl group comprises between 5 and 14 ring atoms in which one or more of the ring atoms is O, N or S and the remaining ring atoms are carbon
  • the carbocyclyl group comprises between 3 and 12 ring carbon atoms
  • the heterocycyl group comprises between 3 and 12 ring atoms in which one or more of the ring atoms is O, N or S and the remaining ring atoms are carbon
  • each of the groups may be optionally substituted.
  • the aryl group may comprise between 6 and 10 ring carbon atoms
  • the heteroaryl group comprises between 5 and 10 ring atoms in which one or more of the ring atoms is O, N or S and the remaining ring atoms are carbon
  • the carbocyclyl group comprises between 5 and 10 ring carbon atoms
  • the heterocycyl group comprises between 5 and 10 ring atoms in which one or more of the ring atoms is O, N or S and the remaining ring atoms are carbon
  • each of the groups may be optionally substituted.
  • the aryl group may be phenyl or naphthyl, each of which may be optionally substituted.
  • the aryl group may be optionally substituted with one or more substituents independently selected from: d-C 6 alkyl, SH, SCi-C 6 alkyl, halo, OH, haloCi-C 6 alkyl, OCi-C 6 alkyl, methylenedioxy, ethylenedioxy, CH 2 -morpholinyl, (CH 2 ) 2 - morpholinyl, (CH 2 ) 3 -morpholinyl, NH 2 , NHC C 6 alkyl, NH(CH 2 )OH, NH(CH 2 ) 2 OH, N(C C 6 alkyl) 2 , -NHS0 2 C C 6 alkyl, -S0 2 NHC C 6 alkyl, C(0)NH 2 , NHC(0)C C 6 alkyl and C(0)NHC C 6 alkyl.
  • substituents independently selected from: d-C 6 alkyl, SH, SCi-C 6 alkyl, halo, OH, hal
  • the aryl group may be optionally substituted with 1 , 2 or 3 substituents independently selected from: SC C 6 alkyl, haloC C 6 alkyl, OC C 6 alkyl, halo, methylenedioxy, C C 6 alkyl, CH 2 -morpholinyl, (CH 2 ) 2 -morpholinyl, (CH 2 ) 3 - morpholinyl, NHC C 6 alkyl, NH 2 , NH(CH 2 )OH and NH(CH 2 ) 2 OH.
  • the aryl group may be optionally substituted with 1 , 2 or 3 substituents independently selected from: SMe, halomethyl, OMe, halo, methylenedioxy, (CH 2 ) 2 -morpholinyl, methyl, NHC C 3 alkyl, NH 2 and NH(CH 2 ) 2 OH.
  • the aryl group may be optionally substituted with 1 , 2 or 3 substituents independently selected from: SMe, CF 3 , OMe, halo, methylenedioxy and methyl.
  • aryl group may be optionally substituted with 1 or 2 substituents independently selected from: SMe, CF 3 , OMe, methylenedioxy and methyl.
  • the carbocyclyl group may be cyclopentyl or cyclohexyl, each of which may be optionally substituted.
  • the carbocyclyl group may be optionally substituted with 1 , 2 or 3 substituents independently selected from: carbonyl, Sd-C 6 alkyl, haloCi-C 6 alkyl, OC C 6 alkyl, halo, methylenedioxy, Ci-C 6 alkyl, CH 2 -morpholinyl, (CH 2 ) 2 -morpholinyl, (CH 2 ) 3 - morpholinyl, NHC C 6 alkyl, NH 2 , NH(CH 2 )OH and NH(CH 2 ) 2 OH.
  • the carbocyclyl group may be optionally substituted with 1 , 2 or 3 substituents independently selected from: carbonyl, SMe, halomethyl, OMe, halo, methylenedioxy, (CH 2 ) 2 -morpholinyl, methyl, NHCi-C 3 alkyl, NH 2 and NH(CH 2 ) 2 OH.
  • the carbocyclyl group may be optionally substituted with 1 , 2 or 3 substituents independently selected from: carbonyl, SMe, CF 3 , OMe, halo, methylenedioxy and methyl.
  • the carbocyclyl group may be optionally substituted with 1 or 2 substituents independently selected from: carbonyl, SMe, CF 3 , OMe, methylenedioxy and methyl.
  • the heteroaryl group may comprise between 5 and 10 ring atoms in which between 1 and 4 of the ring atoms is O, N or S and the remaining ring atoms are carbon.
  • the heteroaryl group may comprise between 5 and 10 ring atoms in which 1 or 2 of the ring atoms is O, N or S and the remaining ring atoms are carbon.
  • the heteroaryl group may be selected from the group consisting of:
  • the heteroaryl group may be optionally substituted with 1 , 2 or 3 substituents independently selected from: Sd-C 6 alkyl, haloCi-C 6 alkyl, OCi-C 6 alkyl, halo, methylenedioxy, C C 6 alkyl, CH 2 -morpholinyl, (CH 2 ) 2 -morpholinyl, (CH 2 ) 3 - morpholinyl, NHC C 6 alkyl, NH 2 , NH(CH 2 )OH and NH(CH 2 ) 2 OH.
  • substituents independently selected from: Sd-C 6 alkyl, haloCi-C 6 alkyl, OCi-C 6 alkyl, halo, methylenedioxy, C C 6 alkyl, CH 2 -morpholinyl, (CH 2 ) 2 -morpholinyl, (CH 2 ) 3 - morpholinyl, NHC C 6 alkyl, NH 2 , NH(CH 2
  • heteroaryl group may be optionally substituted with 1 , 2 or 3 substituents independently selected from: SMe, halomethyl, OMe, halo, methylenedioxy, (CH 2 ) 2 -morpholinyl, methyl, NHC 1 -C 3 alkyl, NH 2 and NH(CH 2 ) 2 OH.
  • heteroaryl group may be optionally substituted with 1 , 2 or 3 substituents independently selected from: halomethyl, OMe, halo, (CH 2 ) 2 - morpholinyl, methyl, NHC 1 -C 3 alkyl, NH 2 and NH(CH 2 ) 2 OH.
  • heteroaryl group may be optionally substituted with 1 or 2 substituents independently selected from: halomethyl, OMe, halo, (CH 2 ) 2 -morpholinyl, methyl, NHC 1 -C 3 alkyl, NH 2 and NH(CH 2 ) 2 OH.
  • the heterocycyl group may comprise 6 ring atoms in which one or more of the ring atoms is N and the remaining ring atoms are carbon, and wherein the heterocycyl group is optionally substituted with 1 , 2 or 3 substituents independently selected from: carbonyl, SMe, halomethyl, OMe, halo, methylenedioxy, (CH 2 ) 2 - morpholinyl, methyl, NHCi-C 3 alkyl, NH 2 and NH(CH 2 ) 2 OH.
  • the heterocycyl group may comprise 6 ring atoms in which 1 or 2 of the ring atoms is N and the remaining ring atoms are carbon, and wherein the heterocycyl group is optionally substituted with 1 or 2 substituents independently selected from : carbonyl, OMe, halo and methyl.
  • Ri is an aryl group, a heteroaryl group or a heterocyclyl group, each of which may be optionally substituted.
  • the aryl group, heteroaryl group and heterocyclyl group may be as defined in any of the foregoing embodiments and combinations thereof.
  • R is a heteroaryl group or a heterocyclyl group, each of which may be optionally substituted.
  • the heteroaryl group and the heterocyclyl group may be as defined in any of the foregoing embodiments and combinations thereof.
  • R is a heteroaryl group, a heterocyclyl group or an aryl group, each of which may be optionally substituted.
  • the heteroaryl group, the heterocyclyl group and the aryl group may be as defined in any of the foregoing embodiments and combinations thereof.
  • Ri is a heteroaryl group.
  • the heteroaryl group may be as defined in any of the foregoing embodiments and combinations thereof.
  • each of which may be optionally substituted with one or more substitutents selected from the group consisting of: SC C 6 alkyl, haloC C 6 alkyl, OC C 6 alkyl, halo, methylenedioxy, C C 6 alkyl, CH 2 -morpholinyl, (CH 2 ) 2 -morpholinyl, (CH 2 ) 3 -morpholinyl, NHCi-C 6 alkyl, NH 2 , NH(CH 2 )OH and NH(CH 2 ) 2 OH.
  • substitutents selected from the group consisting of: SC C 6 alkyl, haloC C 6 alkyl, OC C 6 alkyl, halo, methylenedioxy, C C 6 alkyl, CH 2 -morpholinyl, (CH 2 ) 2 -morpholinyl, (CH 2 ) 3 -morpholinyl, NHCi-C 6 alkyl, NH 2 , NH(CH 2 )OH and NH
  • each of which may be optionally substituted with one or more substitutents selected from the group consisting of: SMe, halomethyl, OMe, halo, methylenedioxy, C C 6 alkyl, CH 2 -morpholinyl, (CH 2 ) 2 -morpholinyl, NHCi-C 6 alkyl, NH 2 , NH(CH 2 )OH and NH(CH 2 ) 2 OH.
  • substitutents selected from the group consisting of: SMe, halomethyl, OMe, halo, methylenedioxy, C C 6 alkyl, CH 2 -morpholinyl, (CH 2 ) 2 -morpholinyl, NHCi-C 6 alkyl, NH 2 , NH(CH 2 )OH and NH(CH 2 ) 2 OH.
  • Exemplary compounds of the formula (I) include:
  • the present invention further extends to compounds of formula (II), which are intermediates in the synthesis of the compounds of formula (I). Accordingly, in another aspect the present invention provides compounds of the formula (II):
  • R 2 is Ci-C 20 alkyl, with the proviso that methyl 3-amino-5-(azepan-1 -yl)-6-phenylpyrazine-2-carboxylate is excluded.
  • R 2 is C C 10 alkyl.
  • R 2 is C C 6 alkyl.
  • R 2 is C C 3 alkyl.
  • R 2 is methyl or ethyl.
  • R 2 is methyl.
  • Exemplary compounds of the formula (II) include: ⁇
  • Solvates are complexes formed by association of molecules of a solvent with a compound of the formula (I) or (II).
  • solvents that are capable of forming solvates include, but are not limited to water, isopropanol, ethanol, methanol, DMSO, ethyl acetate and ethanolamine.
  • the compounds of formula (I) and (II) may be in the form of pharmaceutically acceptable salts.
  • Such salts are well known to those skilled in the art. S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66:1 - 19.
  • Pharmaceutically acceptable salts can be prepared in situ during the final isolation and purification of compounds of the formula (I) or (II), or separately by reacting the free base compound with a suitable organic acid.
  • Suitable pharmaceutically acceptable acid addition salts of the compounds of the present invention may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric, and phosphoric acid.
  • Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucoronic, fumaric, maleic, pyruvic, alkyl sulfonic, arylsulfonic, aspartic, glutamic, benzoic, anthranilic, mesylic, salicylic, p-hydroxybenzoic, phenylacetic, mandelic, ambonic, pamoic, pantothenic, sulfanilic, cyclohexylaminosulfonic, stearic, algenic, ⁇ -hydroxybutyric, galactaric, and galacturonic acids.
  • Suitable pharmaceutically acceptable base addition salts of the compounds of the present invention include metallic salts made from lithium, sodium, potassium, magnesium, calcium, aluminium, and zinc, and organic salts made from organic bases such as choline, diethanolamine and morpholine.
  • the compounds of formula (I) may also exist in different tautomeric forms, and all such forms are within the scope of the invention.
  • the compounds of formula (I) also extend to include all derivatives with physiologically cleavable leaving groups that can be cleaved in vivo to provide the compounds of the formula (I).
  • the compounds of formula (I) also extend to include prodrugs thereof.
  • An exemplary prodrug of a compound formula (I) is the following compound:
  • This compound includes an amino-1 ,2,4-oxadiazolyl group at the 2-position of the pyrazine ring which can be converted in vivo to a guanidino group, thereby liberating compound 19.
  • compounds of the formula (I) are potent inhibitors of uPA. By virtue of this inhibitory activity, compounds of the formula (I) are useful in the treatment of diseases and conditions associated with uPA. Typically, the diseases or conditions are associated with upregulation or increased expression of uPA.
  • the disease or condition is cancer.
  • the cancer may be, for example, breast cancer, lung cancer (NSCLC and SCLC), prostate cancer, pancreatic cancer, upper Gl tract cancer, lower Gl tract cancer, ovarian cancer, renal cancer, glioblastoma, cervical cancer, testicular cancer, lip cancer, tongue cancer, urinary tract cancer, laryngeal cancer, esophageal cancer, bone cancer, uterine cancer, brain cancer (including, for example, glioma and glioblastoma), skin cancer, thyroid cancer, bladder cancer, colorectal cancer, gastric cancer, liver cancer, head and neck cancer, cancer of the central nervous system, melanoma, lymphoma, follicular carcinoma, seminoma, neuroblastoma or mesothelioma.
  • the cancer is breast cancer, pancreatic cancer, upper Gl tract cancer, lower Gl tract cancer, colorectal cancer, prostate cancer, ovarian cancer, lung cancer, renal cancer, head and neck cancer, glioblastoma or melanoma.
  • the compounds of formula (I) may be used in conjunction with, or alternatively in the absence of, other chemotherapeutic agents.
  • u PA is a determinant of cell invasiveness and metastatic potential and is overexpressed in a variety of aggressive malignancies.
  • the compounds of formula (I) inhibit uPA through the reversible competitive inhibition of the uPA active site.
  • the 6-substitution increases the affinity of binding with uPA through the formation of additional favourable non-specific interactions between the inhibitor and the enzyme relative to HMA (as determined by X-ray crystallography herein).
  • the compounds of formula (I) show a high therapeutic window between their uPA inhibitory potencies (IC 50 10's-100's nM) and their cytotoxic potencies (IC 50 10's-100 ⁇ ) and therefore find particular use in the treatment of metastatic cancers.
  • the compounds of formula (I) inhibited the migration and invasion of highly metastatic breast cancer cells in two dimensional in vitro and three dimensional ex vivo assays, demonstrating their ability as antimetastatic agents.
  • Compounds of the formula (I) may therefore also find use in preventing or slowing cancer spread, and in particular, slowing the spread of metastatic cancer. Compounds of the formula (I) may also find use in reducing incidences of, or risk of, cancer recurrence in a subject deemed to be at risk of cancer recurrence.
  • the disease or condition may be an inflammatory disease or condition, such as for example chronic obstructive pulmonary disorder, coronary artery disease, rheumatoid arthritis, corneal inflammation, preeclampsia, colitis, cardiac fibrosis, pulmonary fibrosis, cirrhosis, asthma, psoriasis or periodontitis.
  • an inflammatory disease or condition such as for example chronic obstructive pulmonary disorder, coronary artery disease, rheumatoid arthritis, corneal inflammation, preeclampsia, colitis, cardiac fibrosis, pulmonary fibrosis, cirrhosis, asthma, psoriasis or periodontitis.
  • the disease or condition may be amyotrophic lateral sclerosis.
  • the disease or condition may be a wound, and more particularly a chronic wound.
  • the wound is a dermal ulcer.
  • Another embodiment of the invention involves a method for inhibiting uPA in a subject comprising administration to the subject of a compound of formula (I).
  • the compounds of formula (I) also show a high degree of selectivity (30-1000's fold) for uPA over related trypsin-like serine protease (TLSP) off-targets and are therefore not expected to suffer from side-effects relating to inhibition of these off- targets in vivo.
  • TLSP trypsin-like serine protease
  • compounds and pharmaceutical compositions of the present invention may be administered via any route that delivers an effective amount of the compounds to the tissue or site to be treated.
  • the compounds and compositions may be administered by the parenteral (for example intravenous, intraspinal, subcutaneous or intramuscular), oral, inhalation, or topical route. Administration may be systemic, regional or local.
  • the particular route of administration to be used in any given circumstance will depend on a number of factors, including the nature of the disease or condition to be treated, the severity and extent of the disease or condition, the required dosage of the particular compound to be delivered and the potential side-effects of the compound.
  • suitable compositions may be prepared according to methods that are known to those of ordinary skill in the art and may include pharmaceutically acceptable carriers, diluents and/or excipients.
  • the carriers, diluents and excipients must be "acceptable” in terms of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof.
  • Examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable-based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysiloxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; Cremaphor; cyclodextrins; lower alcohols, for example ethanol or /-propanol; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1 ,3-butylene glycol or glycerin; fatty
  • compositions of the invention may be in a form suitable for administration by injection, in the form of a formulation suitable for oral ingestion (such as capsules, tablets, caplets and elixirs for example), in the form of an ointment, cream or lotion suitable for topical administration, in a form suitable for delivery as an eye drop, in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation, in a form suitable for parenteral administration, that is, subcutaneous, intramuscular or intravenous injection.
  • a formulation suitable for oral ingestion such as capsules, tablets, caplets and elixirs for example
  • an ointment cream or lotion suitable for topical administration
  • an eye drop in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation
  • parenteral administration that is, subcutaneous, intramuscular or intravenous injection.
  • non-toxic parenterally acceptable diluents or carriers can include cyclodextrins (for example Captisol®) Cremaphor, Ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1 ,2-propylene glycol.
  • cyclodextrins for example Captisol®
  • the compounds may also be added to PEG and non-PEGylated liposomes or micelles with specific targeting tags attached to PEG moieties, such as the RGD peptide or glutathione, for aiding passage across the blood brain barrier.
  • suitable carriers, diluents, excipients and adjuvants for oral use include cyclodextrins, Cremaphor, peanut oil, liquid paraffin, sodium carboxymethylcellulose, methylcellulose, sodium alginate, gum acacia, gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatine and lecithin.
  • these oral formulations may contain suitable flavouring and colourings agents.
  • the capsules When used in capsule form the capsules may be coated with compounds such as glyceryl monostearate or glyceryl distearate that delay disintegration.
  • Adjuvants typically include emollients, emulsifiers, thickening agents, preservatives, bactericides and buffering agents.
  • Solid forms for oral administration may contain binders acceptable in human and veterinary pharmaceutical practice, sweeteners, disintegrating agents, diluents, flavourings, coating agents, preservatives, lubricants and/or time delay agents.
  • Suitable binders include gum acacia, gelatine, corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol.
  • Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharin.
  • Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar.
  • Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate.
  • Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring.
  • Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten.
  • Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite.
  • Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc.
  • Suitable time- delay agents include glyceryl monostearate or glyceryl distearate.
  • Liquid forms for oral administration may contain, in addition to the above agents, a liquid carrier.
  • suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides or mixtures thereof.
  • Suspensions for oral administration may further comprise dispersing agents and/or suspending agents. Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, sodium alginate or acetyl alcohol.
  • Suitable dispersing agents include lecithin, polyoxyethylene esters of fatty acids, such as stearic acid, polyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate, polyoxyethylene sorbitan mono- or di-oleate, -stearate or -laurate and the like.
  • Emulsions for oral administration may further comprise one or more emulsifying agents.
  • Suitable emulsifying agents include dispersing agents as exemplified above or natural gums such as guar gum, gum acacia or gum tragacanth.
  • a further suitable emulsifying agent for use in oral or parenteral formulations, which may also function as a solubilizer, is Kolliphor® HS 15.
  • parenterally administrable compositions are apparent to those skilled in the art, and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, PA, hereby incorporated by reference herein.
  • Topical formulations may comprise an active ingredient together with one or more acceptable carriers, and optionally any other therapeutic ingredients.
  • Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site where treatment is required, such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose.
  • Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions. These may be prepared by dissolving the active ingredient in an aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and optionally including a surface active agent. The resulting solution may then be clarified by filtration, transferred to a suitable container and sterilised. Sterilisation may be achieved by autoclaving or maintaining at 90 °C to 100 °C for half an hour, or by filtration, followed by transfer to a container by an aseptic technique.
  • bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01 %) and chlorhexidine acetate (0.01 %).
  • Suitable solvents for the preparation of an oily solution include glycerol, diluted alcohol and propylene glycol.
  • Lotions according to the present invention include those suitable for application to the skin or eye.
  • An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those described above in relation to the preparation of drops.
  • Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisiteriser such as glycerol, or oil such as olive oil.
  • Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with a greasy or non-greasy basis.
  • the basis may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives, or a fatty acid such as stearic or oleic acid together with an alcohol, such as propylene glycol or macrogols.
  • composition may incorporate any suitable surfactant such as an anionic, cationic or non-ionic surfactant, such as sorbitan esters or polyoxyethylene derivatives thereof.
  • suitable surfactant such as an anionic, cationic or non-ionic surfactant, such as sorbitan esters or polyoxyethylene derivatives thereof.
  • Suspending agents such as natural gums, cellulose derivatives or inoraganic materials such as silicaceous silicas, and other ingredients such as lanolin, may also be included.
  • compositions are administered in the form of suppositories suitable for rectal administration of the compounds.
  • suppositories suitable for rectal administration of the compounds.
  • These compositions are prepared by mixing the compound with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the zeolite or zeolite-like material.
  • suitable non-irritating excipient include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol.
  • compositions may also be administered or delivered to target cells in the form of liposomes.
  • Liposomes are generally derived from phospholipids or other lipid substances and are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium.
  • liposomes used in administering or delivering a composition to target cells are synthetic cholesterol (Sigma), the phospholipid 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); Avanti Polar Lipids), the PEG lipid 3-N-[(-methoxy poly(ethylene glycol)2000)carbamoyl]-1 ,2- dimyrestyloxy-propylamine (PEG-cDMA), and the cationic lipid 1 ,2-di-o-octadecenyl-3- (N,N-dimethyl)aminopropane (DODMA) or 1 ,2-dilinoleyloxy-3-(N,N- dimethyl)aminopropane (DLinDMA) in the molar ratios 55:20:10:15 or 48:20:2:30, respectively, PEG-cDMA, DODMA and DLinDMA.
  • DSPC phospholipid 1 ,2-distearoyl-
  • any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes can be used.
  • the compositions in liposome form may contain stablisers, preservatives, excipients and the like.
  • the preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art, and in relation to this, specific reference is made to: Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq., the contents of which is incorporated herein by reference.
  • compositions may also be administered in the form of microparticles or nanoparticles.
  • Biodegradable microparticles formed from polyactide (PLA), polylactide- co-glycolide (PLGA), and epsilon-caprolactone ( ⁇ -caprlactone) have been extensively used as drug carriers to increase plasma half life and thereby prolong efficacy (R. Kumar, M., 2000, J. Pharm. Pharmaceut. Sci. 3(2) 234-258).
  • Microparticles have been formulated for the delivery of a range of drug candidates including vaccines, antibiotics, and DNA. Moreover, these formulations have been developed for various delivery routes including parenteral subcutaneous injection, intravenous injection and inhalation.
  • compositions may incorporate a controlled release matrix that is composed of sucrose acetate isobutyrate (SAIB) and an organic solvent or organic solvents mixture.
  • SAIB sucrose acetate isobutyrate
  • Polymer additives may be added to the vehicle as a release modifier to further increase the viscosity and slow down the release rate.
  • SAIB is a well known food additive. It is a very hydrophobic, fully esterified sucrose derivative, at a nominal ratio of six isobutyrate to two acetate groups. As a mixed ester, SAIB does not crystallise but exists as a clear viscous liquid. Mixing SAIB with a pharmaceutically acceptable organic solvent, such as ethanol or benzyl alcohol decreases the viscosity of the mixture sufficiently to allow for injection.
  • An active pharmaceutical ingredient may be added to the SAIB delivery vehicle to form SAIB solution or suspension formulations.
  • the solvent differs from the matrix allowing the SAIB-drug or SAIB-drug-polymer mixtures to set up as an in situ forming depot.
  • compositions may be administered to subjects either therapeutically or preventively.
  • compositions are administered to a patient already suffering from a disease or condition in an amount sufficient to cure or at least partially arrest the disease or condition and its complications.
  • the composition should provide a quantity of the compound or agent sufficient to effectively treat the subject.
  • the therapeutically effective amount for any particular subject will depend upon a variety of factors including: the disease or condition being treated and the severity thereof; the activity of the compound administered; the composition in which the compound is present; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of sequestration of the compound; the duration of the treatment; drugs used in combination or coincidental with the compound, together with other related factors well known in medicine.
  • One skilled in the art would be able, by routine experimentation, to determine an effective, non-toxic amount of a compound that would be required to treat or prevent a particular disease or condition.
  • an effective dosage is expected to be in the range of about 0.0001 mg to about 1000 mg per kg body weight per 24 hours; typically, about 0.001 mg to about 750 mg per kg body weight per 24 hours; about 0.01 mg to about 500 mg per kg body weight per 24 hours; about 0.1 mg to about 500 mg per kg body weight per 24 hours; about 0.1 mg to about 250 mg per kg body weight per 24 hours; about 1 .0 mg to about 250 mg per kg body weight per 24 hours.
  • an effective dose range is expected to be in the range about 1 .0 mg to about 200 mg per kg body weight per 24 hours; about 1 .0 mg to about 100 mg per kg body weight per 24 hours; about 1 .0 mg to about 50 mg per kg body weight per 24 hours; about 1 .0 mg to about 25 mg per kg body weight per 24 hours; about 5.0 mg to about 50 mg per kg body weight per 24 hours; about 5.0 mg to about 20 mg per kg body weight per 24 hours; about 5.0 mg to about 15 mg per kg body weight per 24 hours.
  • an effective dosage may be up to about 500 mg/m 2 .
  • an effective dosage is expected to be in the range of about 25 to about 500 mg/m 2 , preferably about 25 to about 350 mg/m 2 , more preferably about 25 to about 300 mg/m 2 , still more preferably about 25 to about 250 mg/m 2 , even more preferably about 50 to about 250 mg/m 2 , and still even more preferably about 75 to about 150 mg/m 2 .
  • the treatment would be for the duration of the disease or condition.
  • the compounds of formula (I) may be used alone or alternatively in combination with photodynamic therapy and/or radiotherapy and/or surgery and/or other therapeutic agents, for example chemotherapeutic agents and immunostimulatory agents, as part of a combination therapy.
  • the compounds of formula (I) may sensitise cancer cells to other chemotherapeutic agents and/or radiotherapy.
  • combination therapy and "adjunct therapy” are intended to embrace administration of multiple therapeutic agents in a sequential manner in a regimen that will provide beneficial effects and is intended to embrace administration of these agents in either a single formulation or in separate formulations.
  • Combination therapy may involve the active agents being administered together, sequentially, or spaced apart as appropriate in each case.
  • Combinations of active agents, including compounds of the invention, may be synergistic.
  • the co-administration of compounds of the formula (I) with other therapeutic agent(s) may be effected by a compound of the formula (I) being in the same unit dose form as the other therapeutic agent(s), or the compound of the formula (I) and the other therapeutic agent(s) may be present in individual and discrete unit dosage forms that are administered sequentially, at the same, or at a similar time.
  • Sequential administration may be in any order as required, and may require an ongoing physiological effect of the first or initial agent to be current when the second or later agent is administered, especially where a cumulative or synergistic effect is desired.
  • one or more compounds of formula (I) may be included in combination therapy with surgery and/or radiotherapy and/or one or more chemotherapeutic agents.
  • chemotherapeutic agents that are currently in use, in clinical evaluation and in pre-clinical development, which could be selected for treatment of cancers in combination with compounds of the formula (I). Such agents fall into several major categories, namely, antibiotic-type agents, alkylating agents, anti-metabolite agents, hormonal agents, immunological agents, interferon-type agents and a category of miscellaneous agents.
  • Suitable agents which may be used in combination therapies include those listed, for example, in the Merck Index, An Encyclopaedia of Chemicals, Drugs and Biologicals, 15th Ed., 2014 the entire contents of which are incorporated herein by reference.
  • compounds of the formula (I) When used in the treatment of cancer, compounds of the formula (I) may be administered with vinblastine.
  • the present invention is further described below by reference to the following non- limiting examples.
  • Methyl 3-amino-5-(azepan-1 -yl)-6-chloropyrazine-2-carboxylate (Ilia) was combined with K 2 C0 3 (1 0 mol eq) , 1 .5 mole equivalents of the appropriately substituted (hetero)aryl boronic acid and Pd(PPh 3 ) 4 (5 mole %) in a round-bottom flask under argon. Depending on the reaction scale, either 15 mL or 30 mL of 4:1 dry toluene:MeOH was then added and the mixture heated at reflux (30 min - 48 h), while monitoring by TLC.
  • uPA inhibitory potency was determined for compounds 1 to 29 using a 96-well plate in vitro enzymatic assay.
  • a commercially available fluorogenic substrate (Cat # 672159 - Urokinase substrate III (Fluorogenic) (Merck Millipore, Merck KgaA, Darmstadt, Germany)) was diluted in HEPES assay buffer (20 mM HEPES, 100 mM NaCI, 0.5 mM EDTA, 0.01 % (v/v) Tween-20, pH 7.6) to give a final concentration of 250 ⁇ used with active low molecular weight uPA (Cat # U4010 - Urokinase from Human Kidney Cells, Sigma-Aldrich, St.
  • Assay plates were read at 355 nm (excitation), 460 nm (emission) using a BMG Labtech POLARStar Omega Fluorescence Plate Reader (BMG Labtech, Ortenberg, Germany), with cycle time 60 s, 45-60 cycles and 3s orbital shaking before each read. Absorbance values were recorded for time points (AF/min) taken from the linear region of plots of absorbance versus time and IC 50 values were calculated from sigmoidal dose response curves of absorbance versus log nhibitor] using GraphPad PRISM v6.0 (Graphpad Software, La Jolla, California, USA). The results are shown in Table 1 and Figure 1 .
  • Inhibitory constants (K,) against uPA were determined for select compounds using a 96- well plate in vitro enzyme assay.
  • Urokinase substrate III (Fluorogenic) (Cat #672159 - Merck Millipore, Merck KgaA, Darmstadt, Germany) was serially diluted in HEPES assay buffer (20 mM HEPES, 100 mM NaCI, 0.5 mM EDTA, 0.01 % (v/v) Tween-20, pH 7.6) to give final concentrations between 2.0 ⁇ and 250 ⁇ . 200x inhibitor stocks in DMSO were diluted 1 in 100 into HEPES assay buffer to yield a 2x working stock in 1 % DMSO.
  • the diluted substrate was added to a Greiner CELLSTAR black 96 well microtitre plate (Cat. # M9936, Greiner Bio-One GmbH, Kremsmunster, Austria), followed by addition of 2x working stock drug dilutions or equivalent volumes of assay buffer for controls such that all wells contained 200 ⁇ solution, 0.5% DMSO v/v.
  • HMA is hexamethylene amiloride.
  • MDA-MB-231 breast adenocarcinoma cell viability and LDH release was measured following treatment with 19 or 26 ( Figure 2).
  • Cell viability was determined using the CellTitre 96 ® Aqueous One Solution Cell Proliferation Assay (Cat # G3581 , Promega Corporation, Fitchburg, Wisconsin, USA). Sub-confluent cells ( « 70-80% confluent) were harvested and counted and dissociated into a single cell suspension as described above.
  • Lactate Dehydrogenase is a cytosolic enzyme whose release can be used to measure plasma membrane damage caused by drug treatment and thus can be used to determine the cytotoxic activity of a compound.
  • MDA-MB-231 cells were assayed for LDH release following treatment with compounds 19 or 26 ( Figures 2C and D).
  • Assay and compound dilution plates were prepared as described for the MTS assay described in Example 4. After 48h treatment time assay plates were removed from the incubator and 5 ⁇ of Lysis solution (Bottle 3) of the Roche Cytotoxicity Detection Kit (PLUS) (LDH) (Roche Diagnostics Australia Pty. Ltd., NSW, Australia), was added to cell lysis controls.
  • Lysis solution Bottle 3
  • PLUS Roche Cytotoxicity Detection Kit
  • the plate was gently mixed by hand in a circular motion atop a bench (similar to the manner in which one might wax a car) for 30 s and incubated for a further 15 min. During this period 2.5 ⁇ Catalyst solution (Bottle 1 ) was added to 1 12.5 ⁇ Dye solution (Bottle 2) to form Reaction mixture for each well to be assayed. The plate was removed from the incubator and 100 ⁇ Reaction mixture added to each well. The plate was then wrapped in aluminium foil and incubated for 30 min. After 30 min 50 ⁇ Stop solution was added to each well and read at 490 nm using a SpectraMax Pius 384-weil plate reader (Molecular Devices LLC, Sunnyvale, California, USA).
  • pro-apoptotic caspase 3 and 7 activity was quantitated in HEK- 293 cells post-treatment using the Apo-ONE ® Homogeneous Caspase-3/7 Assay ( Figure 3, Cat # G7790, Promega Corporation, Fitchburg, Wisconsin, USA).
  • Sub- confluent HEK-293 cells were harvested and seeded into a black Greiner CELLSTAR ® 96 well plate (Cat # 655079, Greiner Bio-One GmBH, Kremsmijnster, Austria) at a density of 5000 cells/well in a volume of 40 ⁇ RPMI 1640 media with incubation for 18h.
  • IC 50 values were determined by plotting percentage of residual activity (V0) versus log drug concentration and fitted to a sigmoidal dose response curve using GraphPad Prism v.6.0 (GraphPad Software, La Jolla, CA, USA).
  • GraphPad Prism v.6.0 GraphPad Software, La Jolla, CA, USA.
  • S-2366 pyroGlu-Pro- Arg-pNA
  • S-2288 H-D- lle-Pro-Arg-pNA, Factor Xia
  • S-2444 pyroGlu-Gly-Arg-pNA, uPA H99Y
  • Table 2 Evaluation of compound selectivity against a variety of trypsin-like serine proteases via a chromogenic enzyme activity assay
  • Example 7 Inhibition of Epithelial Sodium Channel (ENaC) activity by amiloride, HMA and compounds 5, 19, 23 and 24
  • ENaC epithelial sodium channels
  • the major and most common side-effect associated with amiloride treatment are cardiac arrhythmias caused by amiloride- induced hyperkalemia. For this reason, ENaC inhibition is not desirable for amiloride- derived anticancer therapeutics.
  • certain compounds were screened against HEK-293 cells transiently expressing the ⁇ , ⁇ and ⁇ subunits of Human ENaC using a fluorescent membrane potential dye kit and an automated fluorescence plate reader ( Figure 5, FLIPR TETRA , Molecular Devices, Sunnyvale, California, USA).
  • HEK-293 Cells were be cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (D-MEM/F-12) supplemented with 10% fetal bovine serum, 100 U/mL penicillin G sodium, 100 ⁇ g/mL streptomycin sulfate and appropriate selection antibiotics.
  • Semiconfluent cells were transfected with cDNA for ENaC ⁇ , ⁇ and ⁇ subunits (genes SCNN1 A, SCNN1 B and SCNN1 G) prior to experimentation.
  • Cells were plated at a density of 20,000 cells/well in a 384-well black wall, flat clear bottom microtiter plates (Type: BD Biocoat Poly-D-Lysine Multiwell Cell Culture Plate) and incubated at 37 e C overnight. Following manufacturer instructions, cells were loaded with 20 ⁇ of membrane potential dye solution for 30 min at 37 °C from the FLIPR Membrane Potential Red Dye Fluorescence Assay Kit (Molecular Devices, Sunnyvale, California, USA). This assay allows the fluorometric detection of changes in membrane potential following modulation of transmembrane ion channel/exchanger activity, namely antagonism of stimulated ENaC activity in this case.
  • Example 8 Effects of Amiloride, HMA, 19 and 24 on diuresis and urinary K + /Na + levels in Sprague Dawley Rats
  • the wells were washed with 1 xPBS and seeded with MDA-MB-231 cells at a density of 250,000 cells/ml in a volume of 100 ⁇ of DM EM/10% FCS followed by incubation overnight at 37 °C, 5% C0 2 . After * 18h, culture media was removed via multichannel pipette and replaced with 100 ⁇ of fresh sreum-free DMEM. On a separate 96-weli plate drugs were diluted to 5 ⁇ serum-free DMEM (Final concentration of vehicle 0.2% DMSO v/v).
  • Isolated collagen I was adjusted to a concentration of 2.0 mg/ml for use. Purity of extracted collagen I was verified by SDS- PAGE. Human skin-derived telomerase -immortalized fibroblasts were maintained in culture for 4-14 days post-confluency without changing of media prior to harvesting to ensure quiescence. On day of collagen I seeding a collagen I matrix solution was prepared by adding 3 ml 10x MEM solution to 25 ml collagen I solution along with 3 ml 0.22 M NaOH and subsequent dropwise addition of NaOH until a pH of « 7.4 was achieved as determined by colour change of internal Phenol Red indicator.
  • SKOV-3 ovarian adenocarcinoma cells were seeded with a volume of 100 ⁇ of cell suspension at a density of 1 x10 s cells/mi.
  • Seeded plugs were transferred to sterile steel mesh grids in 60 mm ceil culture dishes (3 plugs per grid, 1 grid/treatment group per 60 mm dish) and w 10 ml media added such that an air liquid interface was established with the bottom of the plug (as growth media diffuses from the bottom of the plug towards the top a chemotactic gradient is established, inducing epithelial ceils seeded atop to invade downwards through the collagen matrix towards the bulk growth media).
  • culture media was removed from each dish and 3 ⁇ 4 10 ml of the appropriate drug treatment (ail drugs present at 2 ⁇ ) /vehicle control was added to establish an air-liquid interface as described above. Media was refreshed with drug or vehicle containing media solutions every 2 days.
  • Dilutions of drug or vehicle in media were prepared from DMSO stocks every 7 days and stored at 4 °C until used. After 21 days plugs were fixed in 4% neutral buffered formalin for 24-48h. Fixed samples were processed using a Peioris Dual Retort tissue processor (Leica, Germany). Histological staining was conducted on 4 ⁇ sections deparfinized in xylene and rehydrated via graded washing in ethanol (70% v/v ethanol/water-100% ethanol). Cytokeratin staining was performed using an Autostainer XL (Leica, Germany).
  • invasive index is defined as: (# epithelial ceils invaded into collagen plug/ # epithelial cells forming contiguous monolayer along top of the piug)x100. Scores for each image were collated and statistical analysis performed using Microsoft Excel for Mac 201 1 v.14.1 .0. Graphs were generated using GraphPad PRISM v6.0 (GraphPad Software, San Diego, California, USA). Representative photomicrographs from distinct replicate plugs for each treatment group are shown in Figure 8.
  • Compounds 19 and 24 were selected for experiments in mice aimed at determining their dose-tolerability and acute toxicity profiles (Figure 10). Cohorts of four female age- matched C57AlbPLG1 mice (transgenic mice engineered to stably express the human gene for plasminogen) were treated with 2.5 mg/kg, 5 mg/kg and 10 mg/kg of HMA, 19 or 24 via intraperitoneal bolus injection (100 ⁇ injection volume, 20% DMSO v/v in saline). Mice were weighed and monitored twice daily for signs of moribundity. The toxicity endpoint for the study was a greater than 10% decrease in initial body.
  • Example 12 Maximum tolerated single dose of HMA, 19 and 24 in male Balb/c mice via IV bolus injection
  • mice 9-10 week- old male Balb/c mice were administered a single IV bolus injection of amiloride, HMA, 19 or 24 and monitored for any signs of acute toxicity or distress over 72 h.
  • HMA was well tolerated at 40 mg/kg with no signs of ill effects over 72 h. Both 19 and 24 caused death of animals immediately after dosing at 40 mg/kg. 19 at 20 mg/kg caused mild lethargy 2 h following dosing with recovery to normal activity by 24 h. Similar to the 40 mg/kg dose, 24 at 20 mg/kg caused immediate death following dosing. 24 at 10 mg/kg was well tolerated with no signs of ill effects over 72 h. Necropsy and histopathological analysis revealed no distinct cause of death with no evidence of major organ toxicity.
  • HMA full pharmacokinetic profiles were determined for Amiloride, HMA, compounds 19 and 24 in male Sprague Dawley rats over a 24h period (Table 4). All animals were fasted overnight prior to infusion. Three animals per treatment cohort received a nominal dose of 3 mg/kg via I.V. infusion (10 min infusion, indwelling jugular vein catheter, 1 ml infusion volume). Arterial blood was sampled and blood plasma extracted pre-dose, 5 min and 10 min during infusion and 5 min, 20 min, 1 h, 2.5h, 5h, 7h, 10h, 16h and 24h post-infusion.
  • Amiloride behaved in a relatively polar manner when intrinsic hydrophobicity was determined by comparative analytical HPLC (Amiloride gLog D 7 4 0.8). Compound 19 was considerably more hydrophobic, whereas compound 24 showed decreased hydrophobicity relative to HMA, increases attributable to the inclusion of the hexamethylene group at the 5-position and heteroaryl substituents at the 6-position respectively (Table 4, 19 gLog D 7 4 5.1 , 24 gLog D 7 4 3.4, HMA gLog D 7 4 3.8).
  • HMA, compounds 19 and 24 showed large increases in plasma clearance rates relative to amiloride, attributable to their prevalence for non-renal metabolism (>7.5-fold increase relative to Amiloride). Amiloride was primarily eliminated via renal excretion with 72.6% of the administered dose recovered unchanged in the urine.
  • Example 14 Pharmacokinetic analysis of amiloride, HMA, 19 and 24 in male Balb/c mice following I. V. administration
  • Pharmacokinetic profiles were determined for Amiloride, HMA, 19 and 24 in male Balb/c mice over a 24h period (Table 5). All animals were fasted overnight prior to infusion. Three animals per treatment cohort received a nominal dose of 3 mg/kg via I.V. infusion (10 min infusion, indwelling jugular vein catheter, 1 ml infusion volume). Arterial blood was sampled and blood plasma extracted pre-dose, 5 min and 10 min during infusion and 5 min, 20 min, 1 h, 2.5h, 5h, 7h, 10h, 16h and 24h post-infusion. All compounds were formulated in 50 mM acetate buffer/10 % (v/v) DMSO/5% Kolliphor® HS-15.
  • uPA overexpression is a well-characterized determinant of cell invasiveness and metastatic potential in a variety of aggressive malignancies. Enhancement of invasive capacity is conferred by increased plasminogen activation and consequent downstream protease activation which results in broad scale remodelling of the surrounding stroma necessary for metastatic spread.
  • amiloride and 24 were evaluated in a late-stage mouse model of experimental metastasis. Luciferase-tagged HT-1080 human fibrosarcoma cells (BioWare Brite HT-1080-Red-FLuc) were used due to their high endogenous expression of uPA/uPAR and high metastatic potential.
  • mice 7-8 week old male and female NOD.Cg- Prkdc ⁇ scid>IL2rg ⁇ tm 1 Wjl>SzJAusb mice were sourced from Australian BioResources, Moss Vale, NSW, Australia and allowed to acclimatize for 4 days prior to beginning experimentation. Males and females were randomized into 6 treatment groups of 6 mice each, equal numbers male and female. Animals were dosed daily from day -1 to day 20 with 7.5 mg/kg amiloride, 19, 24 or vehicle (50 mM acetate buffer (pH 5.5), 7.5% DMSO v/v, 0.75% Tween-80) via IP injections not exceeding 1 00 uL total volume.
  • vehicle 50 mM acetate buffer (pH 5.5), 7.5% DMSO v/v, 0.75% Tween-80
  • Lungs were homogenized on ice using a PRO Scientific Bio-Gen PRO200 Homogenizer in cell lysis buffer (1 50 mM NaCI, 1 0 mM Tris Base, 1 % Triton X100 v/v, pH 8.0) and centrifuged twice at 4 °C, 1 ,000 rpm, 15 min to remove cell debris. Homogenates were prepared to approximately 50 mg/ml protein based on individual wet lung weights. Luciferase activity was quantified according to manufacturers instructions using the Molecular Diagnostics SpectraMax Glo Steady-Luc Report Assay Kit.
  • Example 17 Screening of amiloride, HMA, 19 and 24 against a panel of human cardiac ion channels
  • Cardiotoxic risk arises through the blockade of a variety of pro-torsadogenic cardiac ion channels (e.g. hERG) which when inhibitied to a high enough extent can result in lethal cardiac episode (e.g. torsades des pointes).
  • hERG pro-torsadogenic cardiac ion channels
  • torsades des pointes pro-torsadogenic cardiac ion channels
  • Understanding hERG inhibitory potential has become a common triage point in the development of novel therapeutics with potent hERG inhibition needing to be addressed prior to further pre-clinical or clinical evaluation.
  • HMA in contrast showed potent inhibition of hERG (IC 50 3.3 ⁇ ) and NaV1 .2 (IC 50 8.3 ⁇ ) and moderate inhibition of CaV1 .2 (IC 50 30 ⁇ ).
  • 19 showed highly potent activity against all channels, with IC 50 values extrapolated to be below the lowest concentration tested ( ⁇ 3 ⁇ ).
  • 24 in comparison showed decreased activity relative to all other compounds with only moderate activity against NaV1 .5 observed (12 ⁇ ).
  • Table 6 Inhibitory potencies of amiloride, HMA, 19 and 24 against a panel of human cardiac ion channels.

Abstract

La présente invention concerne, d'une manière générale, des dérivés à substitution en position 6 de l'hexaméthylène amiloride, leur préparation et leur utilisation dans le traitement de maladies, tel que le cancer.
PCT/AU2017/051208 2016-11-04 2017-11-02 Dérivés à substitution en position 6 de l'hexaméthylène amiloride en tant qu'inhibiteurs de upa et leurs utilisations WO2018081863A1 (fr)

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CN110075303A (zh) * 2019-05-08 2019-08-02 长治医学院 Na+-H+交换泵抑制剂联合顺铂促卵巢癌耐药细胞凋亡的方法
US10793561B2 (en) 2017-07-18 2020-10-06 Nuvation Bio Inc. 1,8-naphthyridinone compounds and uses thereof
US11028058B2 (en) 2017-07-18 2021-06-08 Nuvation Bio Inc. Heterocyclic compounds as adenosine antagonists
US11198699B2 (en) 2019-04-02 2021-12-14 Aligos Therapeutics, Inc. Compounds targeting PRMT5
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US11306071B2 (en) 2019-01-18 2022-04-19 Nuvation Bio Inc. Heterocyclic compounds as adenosine antagonists
WO2022120150A1 (fr) * 2020-12-03 2022-06-09 Duke University Dérivés d'amiloride et procédés d'utilisation de ceux-ci pour le traitement d'infections virales
CN115181092A (zh) * 2018-08-17 2022-10-14 迪哲(江苏)医药股份有限公司 吡嗪化合物和其用途
WO2023180964A1 (fr) * 2022-03-24 2023-09-28 Novartis Ag Dérivés d'amide de pyrazine

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10793561B2 (en) 2017-07-18 2020-10-06 Nuvation Bio Inc. 1,8-naphthyridinone compounds and uses thereof
US11028058B2 (en) 2017-07-18 2021-06-08 Nuvation Bio Inc. Heterocyclic compounds as adenosine antagonists
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CN115181092B (zh) * 2018-08-17 2024-05-03 迪哲(江苏)医药股份有限公司 吡嗪化合物和其用途
US11254670B2 (en) 2019-01-18 2022-02-22 Nuvation Bio Inc. 1,8-naphthyridinone compounds and uses thereof
US11306071B2 (en) 2019-01-18 2022-04-19 Nuvation Bio Inc. Heterocyclic compounds as adenosine antagonists
US11198699B2 (en) 2019-04-02 2021-12-14 Aligos Therapeutics, Inc. Compounds targeting PRMT5
CN110075303A (zh) * 2019-05-08 2019-08-02 长治医学院 Na+-H+交换泵抑制剂联合顺铂促卵巢癌耐药细胞凋亡的方法
WO2022120150A1 (fr) * 2020-12-03 2022-06-09 Duke University Dérivés d'amiloride et procédés d'utilisation de ceux-ci pour le traitement d'infections virales
WO2023180964A1 (fr) * 2022-03-24 2023-09-28 Novartis Ag Dérivés d'amide de pyrazine

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