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US20140256767A1 - Direct inhibitors of keap1-nrf2 interaction as antioxidant inflammation modulators - Google Patents

Direct inhibitors of keap1-nrf2 interaction as antioxidant inflammation modulators Download PDF

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US20140256767A1
US20140256767A1 US14/355,419 US201214355419A US2014256767A1 US 20140256767 A1 US20140256767 A1 US 20140256767A1 US 201214355419 A US201214355419 A US 201214355419A US 2014256767 A1 US2014256767 A1 US 2014256767A1
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occurrence
haloalkyl
halogen
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Longqin Hu
Sadagopan Magesh
Lin Chen
Timothy Lewis
Ben Munoz
Lili Wang
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Rutgers State University of New Jersey
Broad Institute Inc
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Broad Institute Inc
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • 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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/44—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members
    • C07D207/444—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5
    • C07D207/448—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide
    • C07D207/452—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having three double bonds between ring members or between ring members and non-ring members having two doubly-bound oxygen atoms directly attached in positions 2 and 5 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. maleimide with hydrocarbon radicals, substituted by hetero atoms, directly attached to the ring nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D209/00—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D209/02—Heterocyclic compounds containing five-membered rings, condensed with other rings, with one nitrogen atom as the only ring hetero atom condensed with one carbocyclic ring
    • C07D209/44—Iso-indoles; Hydrogenated iso-indoles
    • C07D209/48—Iso-indoles; Hydrogenated iso-indoles with oxygen atoms in positions 1 and 3, e.g. phthalimide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D217/00—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems
    • C07D217/12—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with radicals, substituted by hetero atoms, attached to carbon atoms of the nitrogen-containing ring
    • C07D217/14—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with radicals, substituted by hetero atoms, attached to carbon atoms of the nitrogen-containing ring other than aralkyl radicals
    • C07D217/16—Heterocyclic compounds containing isoquinoline or hydrogenated isoquinoline ring systems with radicals, substituted by hetero atoms, attached to carbon atoms of the nitrogen-containing ring other than aralkyl radicals substituted by oxygen atoms
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D335/00—Heterocyclic compounds containing six-membered rings having one sulfur atom as the only ring hetero atom
    • C07D335/04—Heterocyclic compounds containing six-membered rings having one sulfur atom as the only ring hetero atom condensed with carbocyclic rings or ring systems
    • C07D335/06—Benzothiopyrans; Hydrogenated benzothiopyrans
    • 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
    • C07D401/02—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 two hetero rings
    • C07D401/06—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 two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • 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
    • C07D405/02—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 two hetero rings
    • C07D405/12—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 two hetero rings linked by a chain containing hetero atoms as chain links
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns

Definitions

  • the present invention relates to use of Keap1-Nrf2 protein-protein interaction as a target in discovery of drug candidates, and the discovery of small molecule direct inhibitors of Keap1-Nrf2 protein-protein interaction and potent activators of Nrf2 and ARE-mediated genes to modulate inflammatory processes.
  • GST glutathione S-transferases
  • UDP-glucuronyl transferase 1A1 UDP-glucuronyl transferase 1A1
  • NQO1 NAD(P)H:quinone oxidoreductase 1
  • SOD1 superoxide dismutase 1
  • HO-1 heme oxygenase 1
  • Keap1-Nrf2-ARE antioxidant response elements
  • Keap1-Nrf2-ARE signaling pathway targeting the Keap1-Nrf2-ARE signaling pathway is an attractive strategy to discover preventive and therapeutic agents as antioxidant inflammation modulators (AIMs) for diseases and conditions, including cancer, diabetes, Alzheimer's, and Parkinson's, that involve oxidative stress and inflammation.
  • AIMs antioxidant inflammation modulators
  • Nrf2-ARE inducing agents are already in human clinical trials as chemopreventive agents for cancer or as therapeutic agents for conditions involving inflammation.
  • sulforaphane an isothiocyanate found in cruciferous vegetables and a known ARE inducer
  • COPD chronic obstructive pulmonary disease
  • Bardoxolone methyl another potent inducer of the Nrf2 pathway, was tested in phase III clinical trials as a first-in-class AIM for the treatment of advanced chronic kidney disease (CKD) in patients with type 2 diabetes mellitus.
  • Nrf2/ARE inducers are believed to be irreversible modifying agents of cysteine sulfhydryl groups and these include many natural products (e.g., sulforaphane, curcumin, and epigallocatechin gallate from natural sources such as fruits, vegetables, and tea products) and synthetic compounds (e.g., oltipraz, anethole dithiolethione, bardoxolone methyl). All these compounds are either chemically reactive or can be converted to chemically reactive metabolites that readily oxidize or form covalent adduct with the sulfhydryl group of cysteines.
  • natural products e.g., sulforaphane, curcumin, and epigallocatechin gallate from natural sources such as fruits, vegetables, and tea products
  • synthetic compounds e.g., oltipraz, anethole dithiolethione, bardoxolone methyl. All these compounds are either chemically reactive or can be converted to chemically reactive metabolites that readily
  • the present invention is designed to meet the foregoing need, pertaining to use of Keap1-Nrf2 protein-protein interaction as a novel target in discovery of drug candidates, and the discovery of small molecule direct inhibitors of Keap1-Nrf2 protein-protein interaction as potent activators of Nrf2 and ARE-mediated genes to modulate inflammatory processes, in particular identification of lead compounds and development of useful drug candidates through modification and derivatization of these lead compounds to find suitable candidates for development into therapeutic agents.
  • inhibitors are useful as pharmacological probes for the elucidation of the role of the important Keap1-Nrf2-ARE pathway in inflammation, a central theme in a variety of diseases and conditions.
  • they can potentially be developed into chemopreventive and therapeutic agents for a variety of diseases and conditions including, but not limited to, cancer, diabetes, Alzheimer's, and Parkinson's.
  • the present invention provides a method of identifying lead or candidate compounds useful for developing small-molecule therapeutic agents for treatment of inflammatory diseases or conditions associated with Keap1-Nrf2 protein-protein interaction, the method comprising screening a plurality of compounds against a Keap1-Nrf2 interaction target using an assay selected from FP, TR-FRET, and SPR-based solution competition assays.
  • the invention provides methods of preventing or treating an inflammatory disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of an agent that is a non-peptide small molecule direct inhibitor of Keap1-Nrf2 protein-protein interaction.
  • the invention provides a method of preventing or treating an inflammatory disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I):
  • X at each occurrence is independently nitrogen (N) or carbon (C or CH);
  • Y at each occurrence is independently nitrogen (N) or carbon (C or CH);
  • Z is nitrogen (N) or carbon (C or CH);
  • L 1 is —[C(R 10 ) 2 ] i —, wherein R 10 at each occurrence is independently hydrogen, halogen, C 1 -C 4 alkyl, or C 1 -C 4 haloalkyl, and i is 0, 1, 2, or 3;
  • L 2 is —[C(R 20 ) 2 ] j —, C 3 -C 8 cycloalkylene, C 6 -C 10 arylene, 5- to 10-membered heteroarylene, or 5- to 10-membered heterocyclylene, each optionally substituted by one to three substituents independently selected from halogen, hydroxyl, C 1 -C 4 alkyl, and C 1 -C 4 haloalkyl, —OR 9 , and —NR a R b , wherein R 20 at each occurrence is independently hydrogen, halogen, C 1 -C 4 alkyl, or C 1 -C 4 haloalkyl, and wherein j is an integer selected from 1 through 6;
  • R 1 is an amido or imido group, said amido or imido group comprising a group selected from unsubstituted or substituted C 6 -C 10 aryls and unsubstituted or substituted 5- to 10-membered heteroaryls;
  • R 2 is selected from
  • R 2a and R 2b are each independently —OR 9 or —NR a R b ;
  • R 3 and R 4 are each independently hydrogen, halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, C 3 -C 8 cycloalkyl, C 6 -C 10 aryl, 5- to 10-membered heterocyclyl, 5- to 10-membered heterocyclyl, —CN, nitro, —COOR 11 , or —NR a R b ;
  • R 9 at each occurrence is independently hydrogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, or benzyl;
  • any said cycloalkyl or heterocyclyl is optionally substituted by one or more substituents independently selected from halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, oxo, —COOR 11 , and —NR a R b ;
  • each said aryl or heteroaryl is optionally substituted by one or more substituents independently selected from halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, —COOR 11 , and —NR a R b ;
  • R a and R b at each occurrence are independently hydrogen or C 1 -C 6 alkyl
  • n at each occurrence is independently 0 or an integer selected from 1 to 4.
  • the invention provides a method of preventing or treating an inflammatory disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (II):
  • X at each occurrence is independently nitrogen (N) or carbon (C or CH);
  • Y at each occurrence is independently nitrogen (N) or carbon (C or CH);
  • R 1 is selected from unsubstituted or substituted C 6 -C 10 aryls and unsubstituted or substituted 5- to 10-membered heteroaryls;
  • R 3 is hydrogen, halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —COOR 11 , or —NR a R b ;
  • any said cycloalkyl, or heterocyclyl is optionally substituted by one or more substituents independently selected from halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, oxo, —COOR 11 , and —NR a R b ;
  • each said aryl or heteroaryl is optionally substituted by one or more substituents independently selected from halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, —COOR 11 , and —NR a R b ;
  • R a and R b are each independently hydrogen or C 1 -C 6 alkyl
  • R 15 at each occurrence is independently hydrogen, halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, —COOR 11 , —CONR a R b , and —NR a R b ;
  • n at each occurrence is independently 0 or an integer selected from 1 to 4.
  • R 11 at each occurrence is independently hydrogen, C 1 -C 6 alkyl, or benzyl.
  • the present invention provides compounds of formula (Ia):
  • L 1 is —[C(R 10 ) 2 ] i —, wherein R 10 at each occurrence is independently hydrogen, halogen, C 1 -C 4 alkyl, and i is 1, 2, or 3;
  • L 2 is —[C(R 20 ) 2 ] j —, C 3 -C 8 cycloalkylene, C 6 -C 10 arylene, 5- to 10-membered heteroarylene, 5- to 10-membered heterocyclylene, wherein R 20 at each occurrence is independently hydrogen, halogen, or C 1 -C 4 alkyl, and j is an integer selected from 1 through 6;
  • R 1 is selected from
  • n at each occurrence is independently 0 or an integer from 1 to 4, and R 25 at each occurrence is independently hydrogen, halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, —COOR 11 , —CONR a R b , substituted or unsubstituted C 6 -C 10 aryl, and —NR a R b ;
  • R 2 is —OR 9 or —NR a R b ;
  • R 3 through R 8 are each independently hydrogen, halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, C 3 -C 8 cycloalkyl, C 6 -C 10 aryl, 5- to 10-membered heteroaryl, 5- to 10-membered heterocyclyl, —CN, nitro, —COOR 11 , or —NR a R b ;
  • R 9 at each occurrence is independently hydrogen or C 1 -C 6 alkyl
  • any said cycloalkyl, or heterocyclyl is optionally substituted by one or more substituents independently selected from halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, oxo, —COOR 11 , and —NR a R b ;
  • each said aryl or heteroaryl is optionally substituted by one or more substituents independently selected from halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, —COOR 11 , and —NR a R b ;
  • R a and R b are each independently hydrogen or C 1 -C 6 alkyl
  • R 11 at each occurrence is independently hydrogen or C 1 -C 6 alkyl
  • the invention provides a composition comprising a compound of formula (I) according to any of embodiments described herein, and a pharmaceutically acceptable carrier.
  • the invention provides a method of treating a disease or disorder associated with Keap1-Nrf2 interaction, comprising administering to a subject in need thereof a therapeutically effective amount of the compound of formula (I), (II), or (Ia), or composition thereof, according to any of the embodiments described herein.
  • the present invention thus provides use of a compound of formula (I), (II), or (Ia), or composition thereof, according to any of the embodiments described herein in the manufacture of a medicament for treatment of an inflammatory disease or disorder associated with Keap1-Nrf2 interaction.
  • the disease or disorder can include any of those associated with Keap1-Nrf2 interaction and/or caused by oxidative stress and inflammation, including but not limited to cancers, diabetes, Alzheimer's and Parkinson's.
  • FIG. 1 illustrates a simplified scheme depicting the activation of Nrf2 transcription factor.
  • Nrf2 is sequestered in the cytosol by the protein inhibitor, Keap1, and is transcriptionally inactive.
  • Reactive oxygen species or electrophiles either cause the dissociation of Keap1 or inhibition of ubiquitination and degradation of Keap1-Nrf2 complex, both of which would allow more Nrf2 to translocate to the nucleus and form a transcriptionally active complex with Maf, leading to the induced expression of Nrf2 itself and oxidative stress response enzymes that deactivate reactive oxygen species and electrophiles.
  • FIG. 2 illustrates HTS screening flowchart of MLPCN (the Molecular Libraries Probe Centers Network) compounds.
  • FIG. 3 illustrates (A) the structures of two hits (LH601 and LH602) identified through HTS, (B) dose-response curves of the two hits in the confirmative FP assay, (C) sensorgrams of two hits in the SPR assay, (D) the curve-fitting of the sensorgram data shown in C to derive the K d for the two hits, (E) comparison of K d of different stereoisomers of LH601, and (F) X-ray crystal structures of LH601A/B as a pair and LH601D as a single enantiomer.
  • FIG. 4 illustrates (A) a comparison of binding of FITC-9mer-Nrf2-NH 2 to the Keap1 Kelch domain and to the full length Keap1 protein, and (B) similar % inhibition of binding of FITC-9mer-Nrf2-NH 2 to the full length Keap1 protein for the two leads LH601A and LH602 as compared to their inhibition of binding of FITC-9mer-Nrf2-NH 2 to the Keap1 Kelch domain.
  • FIG. 5 illustrates representative replacements that could be made for each of the three parts in LH601A with improved potency, better cellular permeability, and good pharmaceutical properties.
  • FIG. 6 illustrates representative replacements that could be made for each of the three parts in Hit 2 (LH602) with improved potency, better cellular permeability, and good pharmaceutical properties.
  • FIGS. 7 (A)-(D) illustrate a hit list in Keap1-retest in dose.
  • FIG. 8 illustrates a preliminary SAR of LH601.
  • A Some structure modifications made on the three highlighted regions and a linker. Active modifications inside the blue rectangular box refer to those structural changes that retain most, if not enhance, binding affinity (K d ⁇ 10 ⁇ M) while inactivating modifications outside the blue rectangular box result in dramatic loss of binding affinity (K d >50 ⁇ M).
  • B Inhibition of Keap1-Nrf2 binding for some LH601 analogs synthesized. % inhibition was measured using our SPR assay and represents the inhibition of Keap1 binding (40 nM) to 16mer Nrf2 peptide immobilized on SA chip at inhibitor concentrations of 50 ⁇ M (dark blue bars) or 5 ⁇ M (red bars).
  • the data are grouped by regions modified; double red lines ( ) above compound ID (LH601A/B) indicate enantiomers while double green lines ( ) indicate diastereomers.
  • the red stars ( ⁇ ) indicate promising single modifications from each region, when combined, could lead to more potent inhibitors of Keap1-Nrf2 interaction.
  • the numbers (1-4) in brackets after compound ID indicate the number of stereoisomers present in the sample.
  • FIG. 9 illustrates ARE inducing activity of the two leads (LH601 and LH602) in CellSensorTM ⁇ -lactamase reporter assay.
  • the present invention provides a novel approach to the discovery and identification of drug candidates for treatment of inflammatory diseases or disorders. It provides new methods for fighting oxidative stress and inflammation mediated diseases, including but not limited to cancers, diabetes, Alzheimer's, and Parkinson's.
  • the method protects cells and tissues against various oxidative, inflammatory, and carcinogenic compounds or metabolites derived from exogenous or endogenous sources.
  • the invention uses Keap1-Nrf2 protein-protein interaction as a novel target for the discovery of direct inhibitors of Keap1-Nrf2 protein-protein interaction to obtain potent activators of Nrf2 and ARE-mediated genes to modulate inflammatory processes.
  • Oxidative stress and inflammation are byproducts of normal cellular processes. Oxidative stress can damage cellular proteins, membranes and genes, and can lead to inflammation. Oxidative stress and inflammation are not diseases themselves, but they are implicated in many diseases and conditions including cancer, Alzheimer's, Parkinson's, asthma, and diabetes. Controlling oxidative stress can reduce inflammation and potentially prevent and treat the many oxidative stress-related diseases and conditions.
  • the Keap1-Nrf2-ARE system has been identified as the central regulatory pathway in response to oxidative stress.
  • Keap1 binding to Nrf2 prevents Nrf2 from entering the nucleus and leads to ubiquitination and subsequent degradation of Nrf2 under normal conditions; it acts as a chemical sensor that controls the steady state level of Nrf2 based on cellular redox conditions.
  • Keap1-Nrf2 protein-protein interaction is, thus, an attractive target to regulate the cellular Nrf2 level and ARE gene expression, for the control of oxidative stress and inflammation and for the management of many diseases and conditions.
  • Keap1-Nrf2 interaction Most research efforts in chemoprevention and antioxidant inflammation modulation over the past decade have focused on natural products that are isolated from fruit, vegetables, and tea products, but are indirect inhibitors of Keap1-Nrf2 interaction. These compounds include sulforaphane, phenethyl isothiocyanate, curcumin, resveratrol and epigallocatechin gallate (EGCG). Structurally, these compounds are either chemically reactive (an isothiocyanate or a Michael acceptor) or can be metabolized to chemically reactive species that covalently modify Keap1 and activate Nrf2-mediated ARE genes.
  • the present invention uses state-of-the-art methodologies in drug design high throughput screening for lead discovery, X-ray crystallography (co-crystal structures), structure-based rational drug design, and computer-assisted lead discovery.
  • the direct inhibitors of Keap1-Nrf2 interaction identified will be useful as pharmacological probes for the elucidation of Keap1-Nrf2-ARE signaling pathway and as potential preventive and therapeutic agents for a variety of diseases and conditions.
  • the present invention provides the discovery of novel Nrf2 activators/ARE inducers through high throughput screening of the MLPCN small molecule library.
  • the screening provided novel leads for potent direct inhibitors of the protein-protein interaction between Keap1 and Nrf2, including developing and submitting the HTS FP assay to the MLPCN, screening leads in terms of analog design and synthesis, investigating their in vitro and in vivo biological activities, including chemopreventive and anti-inflammatory properties using a variety of molecular biological techniques and in vivo pharmacology studies, crystallizing and analyzing the cocrystal structure of Keap1 Ketch domain with potent direct inhibitors obtained.
  • the method of identifying novel direct inhibitors of the Keap1-Nrf2 interaction also includes synthesizing analogs of the identified lead compounds, for example, the eight lead compounds identified from an existing library of compounds in a Keap1-retest in dose-response curves and screening their inhibitory activity against the Keap1-Nrf2 interaction:
  • the present invention provides methods of preventing or treating an inflammatory disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of an agent that is a non-peptide small molecule direct inhibitor of Keap1-Nrf2 protein-protein interaction.
  • the present invention provides a method of preventing or treating an inflammatory disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (I):
  • X at each occurrence is independently nitrogen (N) or carbon (C or CH);
  • Y at each occurrence is independently nitrogen (N) or carbon (C or CH);
  • Z is nitrogen (N) or carbon (C or CH);
  • L 1 is —[C(R 10 ) 2 ] i —, wherein R 10 at each occurrence is independently hydrogen, halogen, C 1 -C 4 alkyl, or C 1 -C 4 haloalkyl, and i is 0, 1, 2, or 3;
  • L 2 is —[C(R 20 ) 2 ] j —, C 3 -C 8 cycloalkylene, arylene, 5- to 10-membered heteroarylene, or 5- to 10-membered heterocyclylene, each optionally substituted by one to three substituents independently selected from halogen, hydroxyl, C 1 -C 4 alkyl, and C 1 -C 4 haloalkyl, —OR 9 , and —NR a R b , wherein R 20 at each occurrence is independently hydrogen, halogen, C 1 -C 4 alkyl, or C 1 -C 4 haloalkyl, and wherein j is an integer selected from 1 through 6;
  • R 1 is an amido or imido group, said amido or imido group comprising a group selected from unsubstituted or substituted C 6 -C 10 aryls and unsubstituted or substituted 5- to 10-membered heteroaryls;
  • R 2 is selected from
  • R 2a and R 2b are each independently —OR 9 or —NR a R b ;
  • R 3 and R 4 are each independently hydrogen, halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, C 3 -C 8 cycloalkyl, C 6 -C 10 aryl, 5- to 10-membered heterocyclyl, 5- to 10-membered heterocyclyl, —CN, nitro, —COOR 11 , or —NR a R b ;
  • R 9 at each occurrence is independently hydrogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, or benzyl;
  • any said cycloalkyl or heterocyclyl is optionally substituted by one or more substituents independently selected from halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, oxo, —COOR 11 , and —NR a R b ;
  • each said aryl or heteroaryl is optionally substituted by one or more substituents independently selected from halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, —COOR 11 , and —NR a R b ;
  • R a and R b at each occurrence are independently hydrogen or C 1 -C 6 alkyl
  • R 11 at each occurrence is independently hydrogen, C 1 -C 6 alkyl, or benzyl;
  • R 15 at each occurrence is independently hydrogen, halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, —COOR 11 , —CONR a R b , and —NR a R b ; and
  • n at each occurrence is independently 0 or an integer selected from 1 to 4.
  • R 2a is OH
  • the amido or imido of R 1 group has a formula of
  • R 1a and R 1b are each independently hydrogen or a group selected from:
  • R 1a and R 1b together form a group selected from:
  • n at each occurrence is independently 0 or an integer from 1 to 4, and R 25 at each occurrence is independently hydrogen, halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 halo alkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, —COOR 11 , —CONR a R b , substituted or unsubstituted phenyl, and —NR a R b .
  • R 1a and R 1b together form a group selected from:
  • R 1a and R 1b together form a group selected from:
  • n at each occurrence is independently 0, 1, or 2; and R 25 at each occurrence is independently halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, —COOR 11 , phenyl, and —NR a R b , wherein R 11 , R a , and R b are each independently hydrogen or C 1 -C 6 alkyl.
  • the agent is a compound of formula (Ia):
  • L 1 is —[C(R 10 ) 2 ] i —, wherein R 10 at each occurrence is independently hydrogen, halogen, C 1 -C 4 alkyl, or C 1 -C 4 haloalkyl, and i is 0, 1, 2, or 3;
  • L 2 is —[C(R 20 ) 2 ] j —, C 3 -C 8 cycloalkylene, C 6 -C 10 arylene, 5- to 10-membered heteroarylene, 5- to 10-membered heterocyclylene, wherein R 20 at each occurrence is independently hydrogen, halogen, or C 1 -C 4 alkyl, or C 1 -C 4 haloalkyl, and wherein j is an integer selected from 1 through 6;
  • R 1 is an amido or imido group, said amido or imido group comprising a group selected from C 6 -C 14 aryls and 5- to 10-membered heteroaryls;
  • R 2 is —OR 9 or —NR a R b ;
  • R 3 through R 8 are each independently hydrogen, halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, C 3 -C 8 cycloalkyl, C 6 -C 10 aryl, 5- to 10-membered heterocyclyl, 5- to 10-membered heterocyclyl, —CN, nitro, —COOR 11 , or —NR a R b ;
  • R 9 at each occurrence is independently hydrogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, or benzyl;
  • any said cycloalkyl, or heterocyclyl is optionally substituted by one or more substituents independently selected from halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, oxo, —COOR 11 , and —NR a R b ;
  • each said aryl or heteroaryl is optionally substituted by one or more substituents independently selected from halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, —COOR 11 , and —NR a R b ;
  • R a and R b are each independently hydrogen or C 1 -C 6 alkyl
  • R 11 at each occurrence is independently hydrogen or C 1 -C 6 alkyl.
  • the compound of formula (Ia) is defined as follows:
  • L 1 is —(CH 2 ) i —, wherein i is 1 or 2;
  • L 2 is —(CH 2 ) j — (j is 1, 2, or 3) or C 3 -C 8 cycloalkylene;
  • R 1 is selected from
  • n at each occurrence is independently 0 or an integer from 1 to 4, and R 25 at each occurrence is independently hydrogen, halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, —COOR 11 , —CONR a R b , substituted or unsubstituted phenyl, and —NR a R b ;
  • R 2 is —OR 9 or —NR a R b ;
  • R 3 through R 8 are each independently hydrogen, halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, C 6 -C 10 aryl, nitro, —CN, or —NR a R b ;
  • R 9 at each occurrence is independently hydrogen, C 1 -C 6 alkyl, or benzyl
  • R a and R b are each independently hydrogen or C 1 -C 6 alkyl.
  • the compound of formula (Ia) is defined as follows:
  • L 1 is —CH 2 — or —(CH 2 ) 2 —;
  • n at each occurrence is independently 0, 1, or 2; and R 25 at each occurrence is independently halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, —COOR 11 , phenyl, and —NR a R b ;
  • R 2 is —OR 9 ;
  • R 3 and R 4 are each hydrogen
  • R 5 through R 8 are each independently hydrogen, halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, phenyl, or —NR a R b ;
  • R 9 is hydrogen
  • R a and R b are each independently hydrogen or C 1 -C 6 alkyl.
  • the compound of formula (Ia) is defined as follows:
  • L 1 is —CH 2 — or —(CH 2 ) 2 —;
  • L 2 is —(CH 2 ) 2 —, —(CH 2 ) 3 —,
  • R 1 is selected from:
  • R 2 is —OH, —OCH 3 , —OCH 2 Ph, or —NH 2 ;
  • R 3 and R 4 are each hydrogen
  • R 5 through R 8 are each independently hydrogen, F, Cl, Br, OMe, —NO 2 , or —NH 2 .
  • the compound of formula (Ia) is defined as follows:
  • L 1 is —CH 2 —
  • R 1 is selected from:
  • R 2 is —OH
  • R 3 and R 4 are each hydrogen
  • R 5 through R 8 are each independently hydrogen, F, OMe, or —NH 2 .
  • the compound of formula (Ia) is selected from:
  • the compound of formula (Ia) is selected from the group consisting of:
  • the present invention provides a method of preventing or treating an inflammatory disease or disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula (II):
  • X at each occurrence is independently nitrogen (N) or carbon (C or CH);
  • Y at each occurrence is independently nitrogen (N) or carbon (C or CH);
  • R 1 is selected from unsubstitued or substituted C 6 -C 10 aryls and unsubstituted or substituted 5- to 10-membered heteroaryls;
  • R 3 is hydrogen, halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —COOR 11 , or —NR a R b ;
  • any said cycloalkyl, or heterocyclyl is optionally substituted by one or more substituents independently selected from halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, oxo, —COOR 11 , and —NR a R b ;
  • each said aryl or heteroaryl is optionally substituted by one or more substituents independently selected from halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, —COOR 11 , and —NR a R b ;
  • R a and R b are each independently hydrogen or C 1 -C 6 alkyl
  • R 15 at each occurrence is independently hydrogen, halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, —COOR 11 , —CONR a R b , and —NR a R b ;
  • n at each occurrence is independently 0 or an integer selected from 1 to 4.
  • R 11 at each occurrence is independently hydrogen, C 1 -C 6 alkyl, or benzyl.
  • the compound of formula (II) is defined as follows:
  • said bioisosteric group is selected from
  • R 4 is hydrogen, halogen, hydroxyl, C 1 -C 4 alkyl, C 1 -C 4 haloalkyl, or C 1 -C 4 alkoxy
  • R 5 is hydrogen, C 1 -C 4 alkyl, or C 1 -C 4 haloalkyl
  • R 11 is hydrogen or C 1 -C 4 alkyl.
  • R 1 group of formula (II) is selected from
  • n at each occurrence is independently 0 or an integer selected from 1 to 4; and R 25 at each occurrence is independently hydrogen, halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, —COOR 11 , —CONR a R b , substituted or unsubstituted phenyl, and —NR a R b .
  • formula (II) has a formula selected from
  • R 3 is hydrogen, halogen, hydroxyl, or NH 2 ; and R 4 is hydrogen or NH 2 .
  • the compound has a formula (IIa)
  • R 3 is hydrogen, halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —COOR 11 , or —NR a R b ;
  • n at each occurrence is independently 0 or an integer selected from 1 to 4;
  • R 15 at each occurrence is independently hydrogen, halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, —COOR 11 , —CONR a R b , and —NR a R b ;
  • R 25 at each occurrence is independently hydrogen, halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, —COOR 11 , —CONR a R b , substituted or unsubstituted phenyl, and —NR a R b ;
  • R 11 at each occurrence is independently hydrogen, C 1 -C 6 alkyl, or benzyl
  • R a and R b are each independently hydrogen or C 1 -C 6 alkyl.
  • the compound is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl
  • the disease or disorder is selected from cancer, diabetes, Alzheimer's, and Parkinson's, or a disease or disorder caused by oxidative stress and inflammation.
  • the present invention provides compounds of formula (Ia):
  • L 1 is —[C(R 10 ) 2 ] i —, wherein R 10 at each occurrence is independently hydrogen, halogen, C 1 -C 4 alkyl, and i is 1, 2, or 3;
  • L 2 is —[C(R 20 ) 2 ] j —, C 3 -C 8 cycloalkylene, C 6 -C 10 arylene, 5- to 10-membered heteroarylene, 5- to 10-membered heterocyclylene, wherein R 20 at each occurrence is independently hydrogen, halogen, or C 1 -C 4 alkyl, and j is an integer selected from 1 through 6;
  • R 1 is selected from
  • n at each occurrence is independently 0 or an integer from 1 to 4, and R 25 at each occurrence is independently hydrogen, halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, —COOR 11 , —CONR a R b , substituted or unsubstituted C 6 -C 10 aryl, and —NR a R b ;
  • R 2 is —OR 9 or —NR a R b ;
  • R 3 through R 8 are each independently hydrogen, halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, C 3 -C 8 cycloalkyl, C 6 -C 10 aryl, 5- to 10-membered heteroaryl, 5- to 10-membered heterocyclyl, —CN, nitro, —COOR 11 , or —NR a R b ;
  • R 9 at each occurrence is independently hydrogen or C 1 -C 6 alkyl
  • any said cycloalkyl, or heterocyclyl is optionally substituted by one or more substituents independently selected from halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, oxo, —COOR 11 , and —NR a R b ;
  • each said aryl or heteroaryl is optionally substituted by one or more substituents independently selected from halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, —COOR 11 , and —NR a R b ;
  • R a and R b are each independently hydrogen or C 1 -C 6 alkyl
  • R 11 at each occurrence is independently hydrogen or C 1 -C 6 alkyl
  • L 1 is —(CH 2 ) i —, wherein i is 1 or 2;
  • L 2 is —(CH 2 ) j — (j is 1, 2, or 3) or C 3 -C 8 cycloalkylene.
  • the compound of formula (Ia) is defined as follows:
  • L 1 is —(CH 2 ) i —, wherein i is 1 or 2;
  • L 2 is —(CH 2 ) j — (j is 1, 2, or 3) or C 3 -C 8 cycloalkylene;
  • R 1 is selected from
  • n at each occurrence is independently 0 or an integer from 1 to 4, and R 25 at each occurrence is independently hydrogen, halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, —COOR 11 , —CONR a R b , substituted or unsubstituted phenyl, and —NR a R b ;
  • R 2 is —OR 9 or —NH 2 ;
  • R 3 through R 8 are each independently hydrogen, halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, C 6 -C 10 aryl, nitro, —CN, or —NR a R b ;
  • R 9 at each occurrence is independently hydrogen, C 1 -C 6 alkyl, or benzyl
  • R a and R b are each independently hydrogen or C 1 -C 6 alkyl.
  • the compound of formula (Ia) is defined as follows:
  • L 1 is —CH 2 — or —(CH 2 ) 2 —;
  • R 1 is selected from
  • n at each occurrence is independently 0, 1, or 2; and R 25 at each occurrence is independently halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, —CN, nitro, —COOR 11 , phenyl, and —NR a R b ;
  • R 2 is —OR 9 ;
  • R 3 and R 4 are each hydrogen
  • R 5 through R 8 are each independently hydrogen, halogen, hydroxyl, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkoxy, phenyl, or —NR a R b ;
  • R 9 is hydrogen
  • R a and R b are each independently hydrogen or C 1 -C 6 alkyl.
  • the compound of formula (Ia) is defined as follows:
  • L 1 is —CH 2 — or —(CH 2 ) 2 —;
  • L 2 is —(CH 2 ) 2 —, —(CH 2 ) 3 —,
  • R 1 is selected from:
  • n is 0 or 1; and R 25 is selected from H, F, Cl, Br,-Ph, —NO 2 , —NH 2 , C 1 -C 4 alkyl, and —CO 2 H;
  • R 2 is —OH, —OCH 3 , —OCH 2 Ph, or —NH 2 ;
  • R 3 and R 4 are each hydrogen
  • R 5 through R 8 are each independently hydrogen, F, Cl, Br, OMe, —NO 2 , or —NH 2 .
  • the compound of formula (Ia) is defined as follows:
  • L 1 is —CH 2 —
  • R 1 is selected from:
  • R 2 is —OH
  • R 3 and R 4 are each hydrogen
  • R 5 through R 8 are each independently hydrogen, F, —OMe, or —NH 2 .
  • the compound of formula (Ia) is selected from the group consisting of:
  • the present invention provides a substantially enantiomerically pure compound selected from the group consisting of:
  • the substantially enantiomerically pure compound is selected from:
  • the present invention provides a compound according to any embodiments described above for use in the treatment of an inflammatory disease or disorder associated with Keap1-Nrf2 interaction, including but not limited to cancers, diabetes, Alzheimer's, Parkinson's, or a disease caused by oxidative stress and inflammation
  • the present invention provides a composition comprising a compound according to any of the embodiments described above, and a pharmaceutically acceptable carrier.
  • the present invention provides a method of treating a disease or disorder associated with Keap1-Nrf2 interaction, comprising administering to a subject in need thereof a therapeutically effective amount of the compound according to any of the embodiments described.
  • the present invention provides a method of treating a disease or disorder associated with Keap1-Nrf2 interaction, comprising administering to a subject in need thereof a composition comprising a therapeutically effective amount of a compound according to any of the embodiments described above.
  • the present invention provides use of a compound according to any of the embodiments described above in the manufacture of a medicament for treatment of an inflammatory disease or disorder associated with Keap1-Nrf2 interaction.
  • the disease or disorder can include any diseases or disorders associated with Keap1-Nrf2 interaction and/or caused by oxidative stress and inflammation, including, but not limited to cancer, diabetes, Alzheimer's, Parkinson's.
  • the present invention provides a method of identifying a lead or candidate compound useful for developing small-molecule therapeutic agents for treatment of inflammatory diseases or conditions associated with Keap1-Nrf2 protein-protein interaction, comprising screening a plurality of compounds against a Keap1-Nrf2 interaction target using an assay selected from FP, TR-FRET, and SPR-based solution competition assays.
  • the method of identifying a lead or candidate compound contains the steps of:
  • the compound is screened at a pre-determined concentration of 5 ⁇ M and 50 ⁇ M, and the percent inhibition target is at least 50%.
  • the method further includes steps of:
  • step (e) evaluating selected compounds from step (d) in cell-based ARE ⁇ -lactamase reporter assay using CellSensor HepG2 cell line;
  • step (f) selecting compounds from step (e) exhibiting an ARE-inducing activity in the CellSensor® ARE-b1a Hep G2 cell line with an EC 50 of less than 20 ⁇ M using 150 ⁇ M tBHQ as 100%.
  • the present invention provides eight hit compounds that can be used as lead drug candidates for developing into therapeutic agents.
  • the present invention provides a compound selected from the group consisting of:
  • Keap1-Nrf2 for use in the treatment of an inflammatory disease or disorder associated with Keap1-Nrf2 interaction, (b) for use as a direct inhibitor to Keap1-Nrf2 protein-protein interaction, or (c) for use as lead compounds to obtain useful therapeutic agents for Keap1-Nrf2 related diseases or disorders, including but not limited to cancer, diabetes, Alzheimer's, Parkinson's, or a disease or disorder caused by oxidative stress and inflammation.
  • the compounds of the present invention may be optically active and may be isolated in either their optically active, i.e., enantiomerically enriched, or racemic forms.
  • Cis and trans geometric isomers of the compounds of the present invention may be isolated as a mixture of isomers or as separated isomeric forms and are intended as a disclosed variation. In the present application, all chiral, diastereomeric, racemic forms and all geometric isomeric forms of a structure are intended to be disclosed.
  • alkyl is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups containing 1 to 10 carbons, more preferably 1 to 6 carbons, in the main (longest) chain.
  • the term encompasses, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, or the like.
  • cycloalkyl as used herein alone or as a part of another group, includes saturated cyclic hydrocarbon radical having 3 to 8 carbons forming the ring.
  • aryl refers to monocyclic or bicyclic aromatic radical containing 6 to 10 carbons in the ring portion (such as phenyl, benzyl, or naphthyl, including 1-naphthyl and 2-naphthyl).
  • Halo or halogen refers to fluoro, chloro, bromo, and iodo; and “haloalkyl” is intended to include both branched and straight-chain saturated aliphatic hydrocarbon radicals containing one or more halogen substituents, such as for example CF 3 or the like.
  • alkoxyl as used herein alone or as a part of another group, includes an alkyl group linked to a molecular moiety through an oxygen atom, for example, RO—, where R is alkyl.
  • heteroaryl is intended to mean a stable 5- to 10-membered monocyclic or bicyclic, heterocyclic aromatic group which consists of carbon atoms and from 1 to 4 heteroatoms independently selected from the group consisting of N, O and S, and is aromatic in nature, including but not limited to pyridinyl, furyl, pyrimidyl, indolyl, etc.
  • heterocyclyl is intended to mean a 5- to 10-membered monocyclic or bicyclic, heterocyclic non-aromatic group which consists of carbon atoms and from 1 to 4 heteroatoms independently selected from the group consisting of N, O and S, including but not limited to piperidinyl, piperazinyl, pyrrolidinyl, tetrhydrofuranyl, etc.
  • substituted “cycloalkyl,” “aryl,” “heterocyclyl,” or “heteroaryl” includes the corresponding groups substituted with from one to five substituents independently selected from halogen, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkyl, hydroxy, hydroxy-alkyl, C 1 -C 6 acyl, cyano, nitro, and amino group;
  • cycloalkylene represents the corresponding divalent “cycloalkyl,” “aryl”, “heteroaryl”, and “heterocyclyl” groups, respectively, connected to two other molecular moieties, and may be used exchangeably with the corresponding “cycloalkyl,” “aryl”, “heteroaryl”, and “heterocyclyl” groups, respectively.
  • cyano refers to a —CN group and the two terms are used interchangeably.
  • nitro refers to an —NO 2 group and the two terms are used interchangeably.
  • hydroxy or “hydroxyl”, as used herein, refers to an —OH group and the two terms are used interchangeably.
  • moiety refers to a portion or portions of a molecule.
  • bioisosteric groups or “bioisosteres”, or the like, as used herein, refers to substituents or functional groups with similar physical or chemical properties which produce broadly similar biological properties to a chemical compound, as generally understood in medicinal chemistry or pharmaceutical sciences.
  • substantially enantiomerically pure refers to a chiral compound having an enantiomeric excess (ee) of at least 95%, preferably at least 97%, and more preferably at least 99%, and most preferably at least 99.5%.
  • phrases “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in human beings and animals commensurate with a reasonable therapeutic benefit/risk ratio.
  • “pharmaceutically acceptable salts” refer to derivatives of the disclosed compounds wherein the parent compound is modified by making counterpart acid or base salts thereof.
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • Pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • the term may refer to counter ions of any moiety that is designated in this disclosure in an ionic form.
  • basic addition salts can be prepared during the final isolation and purification of the compounds by reacting a carboxylic acid group with a suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation or with ammonia or an organic primary, secondary, or tertiary amine.
  • a suitable base such as the hydroxide, carbonate, or bicarbonate of a metal cation or with ammonia or an organic primary, secondary, or tertiary amine.
  • the cations of pharmaceutically acceptable salts include lithium, sodium, potassium, calcium, magnesium, and alum-Mum, as well as nontoxic quaternary amine cations such as ammonium, tetramethyl-ammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, diethylamine, ethylamine, tributylamine, pyridine, N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine, dicyclohexylamine, ethylenediamine, ethanolamine, diethanolamine, triethanolamine, piperidine, piper-azine, 1H-imidazole, choline, lysine, arginine, benethamine, benzathine, betaine, or the like.
  • nontoxic quaternary amine cations such as ammonium, tetramethyl-ammonium, tetraethylam
  • prodrug refers to compounds that are transformed in vivo to yield the parent compound of the above formulae, for example, by hydrolysis in blood. Common examples include, but are not limited to, ester and amide forms of a compound having an active form bearing a carboxylic acid moiety.
  • solvate means a physical association of a compound of this invention with one or more, preferably one to three, solvent molecules, whether organic or inorganic.
  • exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Methods of solvation are generally known in the art.
  • terapéuticaally effective amount refers to the total amount of each active component that is sufficient to show a meaningful patient benefit, e.g., a sustained reduction of symptoms.
  • the compounds of this invention can be administered for any of the uses described herein by any suitable means, for example, orally, such as tablets, capsules (each of which includes sustained release or timed release formulations), pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions; sublingually; bucally; parenterally, such as by subcutaneous, intravenous, intramuscular, or intrasternal injection, or infusion techniques (e.g., as sterile injectable aqueous or non-aqueous solutions or suspensions); nasally, including administration to the nasal membranes, such as by inhalation spray; topically, such as in the form of a cream or ointment; or rectally such as in the form of suppositories. They can be administered alone, but generally will be administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.
  • composition means a composition comprising a compound of the invention in combination with at least one additional pharmaceutically acceptable carrier.
  • a “pharmaceutically acceptable carrier” refers to media generally accepted in the art for the delivery of biologically active agents to animals, in particular, mammals, including, i.e., adjuvant, excipient or vehicle, such as diluents, preserving agents, fillers, flow regulating agents, disintegrating agents, wetting agents, emulsifying agents, suspending agents, sweetening agents, flavoring agents, perfuming agents, antibacterial agents, antifungal agents, lubricating agents and dispensing agents, depending on the nature of the mode of administration and dosage forms.
  • Pharmaceutically acceptable carriers are formulated according to a number of factors well within the purview of those of ordinary skill in the art. These include, without limitation: the type and nature of the active agent being formulated; the subject to which the agent-containing composition is to be administered; the intended route of administration of the composition; and the therapeutic indication being targeted. Pharmaceutically acceptable carriers include both aqueous and non-aqueous liquid media, as well as a variety of solid and semi-solid dosage forms.
  • the dosage regimen for the compounds of the present invention will vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition and weight of the recipient; the nature and extent of the symptoms; etc.
  • the daily oral dosage of each active ingredient when used for the indicated effects, will range between about 0.01 to about 5000 mg per day, preferably between about 0.1 to about 1000 mg per day, and most preferably between about 0.1 to about 250 mg per day.
  • This section describes, among others, the approaches to (a) advance two of the eight hits to leads for development through chemical synthesis of the hit compounds and activity confirmation in biochemical and cell-based assays, (b) investigate the stereochemical requirement of binding to Keap1 Ketch domain and assign the stereochemistry of active stereoisomers using spectroscopic and crystallographic methods and chemical synthesis, (c) achieve the stereoselective synthesis of the active stereoisomer, and (d) biochemical, in vitro, and in vivo assays.
  • FIG. 3A shows the structures of the two potent hits and FIG. 3B shows the FP assay dose-response curves demonstrating their 3 ⁇ M IC 50 .
  • the Keap1-binding activity of the two potent hits was confirmed in our SPR assay with K d between 1.6 and 1.9 ⁇ M as shown in FIGS. 3C and 3D .
  • Human Keap1 Kelch domain protein was cloned into expression vector pET15b and transformed into E.coli BL21 (DE3) strain. The protein was purified through Ni-NTA affinity column. Purified protein was stored in the buffer containing 20 mM HEPES (pH7.4), 5 mM DTT at ⁇ 80° C.
  • FP assay dose-response retest was performed using the same FP assay protocol above for HTS with the exception that the compound concentration started at 25 ⁇ M with 2-fold dilution for 8 points (25-0.2 ⁇ M) in duplicate.
  • the fluorescence-based thermal shift assay was developed using the standard protocol to confirm the ligand-binding affinity of hit compounds by assessing the shift of the unfolding temperature (DT) obtained in the presence of varying concentrations of the inhibitor.
  • Streptavidin 200 ⁇ g/mL was immobilized on Fc1 and Fc2 following the general procedure to obtain surface immobilization level of 7000 RU.
  • Biotinylated 16mer Nrf2 peptide was diluted into the running buffer to obtain 10 nM solution, which was injected through Fc2 at a flow rate of 10 ⁇ L/min to saturate the sensor chip surface.
  • An immobilization level of 300 RU for 16mer Nrf2 peptide was achieved in Fc2 and the free streptavidin at Fc1 of the chip was used as the blank.
  • Keap1 Kelch domain was diluted serially 2-fold in the running buffer to obtain as standards 6 concentrations of Keap 1 Ketch domain ranging from 125 nM to 4 nM.
  • concentrations of Keap 1 Ketch domain ranging from 125 nM to 4 nM.
  • Each solution of Keap1 Ketch domain was injected through Fc1 and Fc2 with a 1-min association time and a 3-min dissociation time at 25° C. and a flow rate of 50 ⁇ L/min.
  • the sensor chip surface was regenerated with NaCl (1 M) at a flow rate of 100 ⁇ L/min for 1 min followed by two consecutive 1-min washes with the running buffer at a flow rate of 100 ⁇ L/min
  • double subtractions from the reference surface (Fc1) and the blank were performed to the sensorgrams obtained at varying concentrations of Keap1 Ketch domain.
  • the slopes of the initial association phase from the corrected SPR sensorgrams were calculated by fitting to the linear model in BIAevaluation software 4.1 and plotted against the concentration of Keap1 Ketch domain protein.
  • Lt is the total concentration of Keap1 Ketch domain and fb is the fraction of the Keap1 Ketch domain protein bound at a given concentration (x) of Nrf2 protein or Nrf2 peptide.
  • the direct inhibitors identified using Keap1 Ketch domain also bind to the full length Keap1 protein with similar affinity and are capable of disrupting the protein-protein interaction between full length Keap1 and Nrf2 as shown in FIG. 4 .
  • the activity observed with Keap1 Ketch domain represents the activity for the full length Keap1.
  • cell-based assays including the ARE-luciferase reporter assays in HepG2 and SW480 cell lines, in the evaluation of various isothiocyanates and curcumin analogs.
  • the activity of LH601A, LH602, and their simple analogs in cell-based assays has also helped guide lead development efforts.
  • the two leads have relatively large molecular weights, which are not unexpected considering that we are targeting protein-protein interactions that often require effective inhibitors to be larger in molecular size.
  • Lead 2 also has a large cLogP as well as a 4-hydroxyaniline structure alert for potential reactive metabolite formation.
  • Working on both leads focused on target binding affinity, ARE-inducing activity, physicochemical, pharmaceutical, and ADME properties.
  • This section illustrates crystallization and structure characterization of the Keap1 Kelch domain bound to small molecule compounds identified through the high throughput screening.
  • the structure of the isolated human Keap1 Kelch domain has previously determined to 1.35 ⁇ resolution and the complex of the human Keap1 Kelch domain bound to the 16mer Nrf2 peptide to 1.5 ⁇ resolution.
  • This protein is highly purified and suitable for cocrystallization efforts.
  • the Keap1 Kelch domain ( ⁇ 10 mg/mL) and various inhibitors are mixed in solution prior to crystallization attempts at varying stoichiometries, typically with five to ten-fold molar excess of an inhibitor, as solubility permits.
  • Protein-inhibitor complexes are screened for crystallization using a sparse matrix screen, such as those in commercially available kits (Hampton Research).
  • Crystallization screens are performed using a Gryphon robot (Art Robbins), which allows 96 conditions to be screened with 70 ⁇ L of sample, at three different ratios of protein to well buffer. Hits from crystallization screens are used to improve crystal morphology and increase crystal size.
  • the structure solution proceeds via molecular replacement using the previously determined structure of the human Keap1 Kelch domain. Presence of bound inhibitor is confirmed via difference Fourier maps. Successful crystallization in novel space groups (different from the isolated domain) is also be indicative of successful complex formation, as the crystal lattice of the Keap1 Kelch domain occludes the expected binding site of the inhibitor.
  • the refinement of the structure proceeds using standard methods. The resulting structure(s) is compared and contrasted with the structures of the isolated (uncomplexed) Keap1 Kelch domain and with the Keap1 Kelch domain-Nrf2 peptide complex to assess structural changes and key binding determinants of the inhibitors.
  • inhibitors and various additives are assessed in, e.g., DMSO, ⁇ -octyl glucoside, and PEG 400. These compounds are used to solubilize the inhibitors in concentrated stocks that are then diluted (typically 10-fold) by mixing with the protein. The appropriate additive, concentrations of stock solutions and various dilutions are assessed on a case-by-case basis for each inhibitor.
  • This section illustrates the approach to evaluate selected compounds in vivo for their oral bioavailability, pharmacokinetic profiles, and effects on the expression of ARE-mediated genes and carcinogenesis in TRAMP mouse model of prostate cancer to establish feasibility of using direct inhibitors of Keap1-Nrf2 interaction as antioxidant inflammation modulators.
  • Potent inhibitors are subjected to in vivo studies in mice to investigate their oral bioavailability, preliminary pharmacokinetic profiles, and effects on prostate carcinogenesis and ARE-mediated gene expression. The latter will be performed using qPCR and Western blotting of tissues taken from mice treated with potent inhibitors of Keap1-Nrf2 interaction.
  • the mouse model has been used to demonstrate the induction of Nrf2-mediated expression of oxidative stress and inflammation markers by the chemopreventive and anti-inflammatory indirect Keap1-Nrf2 inhibitors.
  • the test compounds are orally bioavailable, can reach their biologically active concentration, and have sufficiently long half-lives (sufficient exposure) to reach their site of action. Since these are standard parameters required of candidate compounds advanced to in vivo studies, a person skilled in the art would be able to use them to assess the maximum tolerated doses, oral bioavailability, pharmacokinetic profile of test compounds before evaluating their effects on carcinogenesis and relevant gene expression in TRAMP mouse model of prostate cancer.
  • the in vivo studies can be expanded in the future to include other animal models of oxidative stress like unilateral ureteral obstruction model of chronic kidney disease.
  • TRAMP mouse is an autochthonous transgenic animal model of prostate cancer that recapitulates the whole spectrum of human prostate tumorigenesis from the earliest PIN lesions to androgen-independent disease. It represents an excellent model of prostate carcinogenesis of which the prostate tissue progresses into the different stages of tumors and exhibiting histological and molecular features to human PCa.
  • This transgenic mouse model has been used to investigate the chemopreventive properties of many natural product and synthetic indirect inhibitors of Keap1-Nrf2 interaction. The effects of the direct inhibitors of Keap1-Nrf2 interaction are being tested on both the carcinogenesis process and the expression of Nrf2-mediated antioxidant gene expression.
  • Nrf2-mediated genes including Nrf2, GSTm2, NQO1, UGT1A1, HO-1, SOD1 in tissues and organs taken from drug-treated TRAMP mice.
  • this invention represents an interdisciplinary approach towards discovery of potential drug candidates for a variety of oxidative stress-related diseases. It involves hit discovery through HTS, hit to lead, lead development, and (preclinical) pharmacological evaluation.
  • the structures of human Keap1 Kelch domain and its complex with the 16-mer Nrf2 peptide have been solved through crystallization, and cocrystallization conditions for Keap1 Kelch domain.
  • the two small molecule leads are under investigation and are used for cocrystal structure analysis and characterization of the human Keap1 Kelch domain complex with the small molecule inhibitors.
  • HepG2-ARE-C8 The human hepatoma HepG2 cells stably transfected with the pARE-TI-luciferase construct (HepG2-ARE-C8) have been used to evaluate the activity of many naturally occurring chemopreventive chemicals in inducing ARE genes, and the TRAMP mouse model of prostate cancer has been used to test the chemopreventive properties of natural product indirect inhibitors of Keap1-Nrf2 interaction.
  • This section illustrates the approach to determine the activity of synthesized analogs in a series of biochemical assays and cell-based assays to obtain structure-activity relationship and to elucidate the role of Keap1-Nrf2-ARE pathway in oxidative stress and inflammation.
  • the approach includes studying mechanism of action of the lead compounds and their analogs by measuring their biological activity as direct inhibitors of Keap1-Nrf2 interaction in a series of direct binding and cell-based assays, including fluorescence-based binding assays, SPR-based solution competition assay, and ARE-luciferase reporter assay; determining the specificity of the direct inhibitors of Keap1-Nrf2 interaction by measuring the lack of inhibition of the ubiquitous NF- ⁇ B signaling pathway, including NF- ⁇ B-luciferase reporter assay and inhibition of nitric oxide production in LPS-stimulated RAW 264.7 cells; and directing the lead development efforts through structure-activity relationship analysis to obtain compounds with enhanced binding affinity, improved physicochemical and pharmaceutical properties, and decreased development liabilities.
  • Major assays include:
  • the first of these assays is an FP assay that uses a fluorescently labeled Nrf2 peptide amide as the probe and directly measures the displacement of fluorescent probe from the Nrf2-binding site of Keap1 Kelch domain by inhibitors via changes in fluorescence anisotropy. This was the assay used to screen the MLPCN library and discover the two potent hits used as leads in this project and dose-response curves of the two hits are shown in FIG. 3B .
  • the second of the fluorescence-based assays is a time-resolved fluorescence energy transfer (TR-FRET) assay which uses an in situ labeling method we recently developed to attach a lanthanide chelate to Keap1 Kelch domain as the FRET donor and fluorescein-labeled Nrf2 peptide as the acceptor.
  • TR-FRET time-resolved fluorescence energy transfer
  • ARE-luciferase or ARE ⁇ -lactamase reporter assay to study the effect of isothiocyanates and the compounds already synthesized on the expression of ARE genes.
  • the HepG2-C8 cells used in this assay were selected after stable transfection of HepG2 cells with pARE-TI-luciferase construct using a FuGENETM 6 method and the CellSensor® ARE-b1a HepG2 cell line is commercially available from Invitrogen.
  • PathHunter® nuclear translocation assay kit from DiscoveRx and this assay is been used to demonstrate nuclear translocation of Nrf2 upon inhibition of Keap1-Nrf2 interaction by our direct inhibitors.
  • the PathHunter® assay uses Enzyme Fragment Complementation (EFC) technology with U2OS cells expressing two complementing fragments of ⁇ -Gal in different cellular compartments and quantitate using chemiluminescence the activity of ⁇ -Gal, which corresponds to the amount of Nrf2 translocating into the nucleus upon disruption of Keap1-Nrf2 interaction.
  • EFC Enzyme Fragment Complementation
  • qPCR Quantitative real-time polymerase chain reaction
  • Nrf2-mediated genes including endogenous Nrf2, GSTm2, UGT1A1, NQO1, HO-1, and SOD1
  • Western blotting is used to investigate the protein expressions of these biomarkers; these are routinely performed on cells and tissues from culture and animals.
  • the total RNA is reverse-transcribed to cDNA by TaqMan Reverse Transcription Reagents.
  • SYBR Green fluorescence is used to measure the product of qPCR.
  • Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is used as the housekeeping gene, and the Applied Biosystems 7900HT Fast Real-Time PCR System is used to detect quantitatively the induction of mRNA of Nrf2, GSTm2, NQO1, UGT1A1, HO-1, SOD1 as we reported previously.
  • Applied Biosystems 7900HT Fast Real-Time PCR System is used to detect quantitatively the induction of mRNA of Nrf2, GSTm2, NQO1, UGT1A1, HO-1, SOD1 as we reported previously.
  • For Western blotting to determine protein expression cells is treated similarly with test compounds but for a longer duration (e.g., 24 h) before they are collected, lysed, and analyzed for protein expression. The proteins are separated by SDSPAGE and transferred onto nitrocellulose membranes. Membranes are probed using the different mono- or polyclonal antibodies (targeting the various proteins) and the respective secondary antibodies. The bands are visualized and quantified by BioR
  • Keap1-Nrf2 interaction such as curcumin and isothiocyanates have been shown to have inhibitory effect on the activity of NF- ⁇ B in cells induced by cytokines like TNF ⁇ and the nitric oxide production in RAW 264.7 cells stimulated by LPS through attenuation of the ubiquitous NF- ⁇ B signaling pathway. Since we are developing direct and specific inhibitors of Keap1-Nrf2 interaction, they are not expected to interfere with the NF- ⁇ B signaling pathway, thus would unlikely have any effect on NF- ⁇ B activity and nitric oxide production in cells.
  • This approach is to develop the chemical structure of two lead compounds and to obtain potent Keap1-Nrf2 inhibitors with mid-to-low nanomolar binding affinity to Keap1 Kelch domain and good physicochemical and pharmaceutical properties including solubility, membrane permeability and without obvious developmental issues.
  • LH601A Based on the comparison of structure features and properties of the two leads in Table 1, we first chose LH601A as a lead for development. Based on our docking studies, the carboxylate of LH601A is directly interacting with the Arg415 residue in the Nrf2 peptide binding site of Keap1 Kelch domain. Thus, most replacements selected for cis-2-carbonyl cyclohexane-1-carboxylic acid contain a carboxylate as shown in FIG. 5 .
  • the carboxylate can also be replaced with carboxylate bioisosteres such as tetrazole, 3-hydroxyisoxazole, hydrated trifluoromethyl ketone, sulfonamide, and hydroxamic acid.
  • the aryl group can also be varied between 5 and 6-membered rings and fused ring systems, and the saturated ring in tetrahydro-isoquinoline can be replaced with other 5- or 6-membered rings shown in FIG. 5 .
  • LH602 provides a second scaffold the bicyclic naphthalene.
  • LH602 was resynthesized and has been confirmed to have an IC 50 of 3 ⁇ M and a Kd of 1.7 ⁇ M to Keap1 Ketch domain.
  • similar reactions to those shown in Schemes 1-3 can be used. New routes are also explored to obtain compounds with unique replacements.
  • Hit 1 there are three chiral centers in Hit 1.
  • Hit 1 according to Scheme 1 by reacting 1-phthalimidomethyl tetrahydroisoquinoline (( ⁇ ) ⁇ 4) with cis-cyclohexanedicarboxylic anhydride, we obtained an expected mixture of four stereoisomers (5, LH601) based on our chiral HPLC analyses on a Chiralcel OD-R column while the Hit 1 sample from the NIH MLPCN library contains 90% of one major diastereomer (probably due to repeated recrystallization in the commercial process). LH601 mixture was shown to be two fold less active than the Hit 1 sample obtained from the NIH MLPCN library.
  • LH601A/B was separated into diastereomers (LH601A/B and LH601C/D) using flash silica gel chromatography.
  • LH601A/B was shown to contain the active isomer in Hit 1 and was further separated into the two enantiomers LH601A and LH601B by preparative normal phase chiral separation on a Chrialcel OD column.
  • the activities of LH601A, LH601B and LH601C/D were then compared to that of Hit 1 in our FP and SPR assay. As shown in the SPR dose-response curves in FIG.
  • VCD vibrational circular dichroism
  • 3E demonstrate the structurally specific ligand-target interaction between LH601A and Keap1.
  • the strong binding affinity of LH601A over its stereoisomers provides further evidence of its true direct binding to Keap1 and supports its use as a lead for structure development.
  • the imine intermediate 3 was converted to the (S)-teterhydroisoquinoline 4 (see, e.g., Roszkowski, P., et al., Tetrahedron: Asymmetry , 2006, 17, 1415-1419).
  • the (S)-4 has alternatively been resolved from the (R)-4 using ( ⁇ )-dibenzoyl L-tartaric acid (( ⁇ )DBTA).
  • the meso anhydride cis-cyclohexane-dicarboxylic anhydride was converted to (1S,2R)-2-(benzyloxycarbonyl)cyclohexane carboxylic acid ((R,S)-10) with benzyl alcohol in the presence of quinine.
  • Simple amide bond coupling between the optically active (S)-4 and ((R,S)-10 produced the benzyl protected (SRS)-11, which upon hydrogenolysis gave the desired enantiomer (SRS)-5 (LH601A).
  • Hit 2 provides a second chemotype with a different scaffold for our lead development efforts, especially if we can eliminate or replace 4-hydroxyaniline moiety—the source of concern for reactive metabolite formation.
  • One advantage of Hit 2 is its simpler stereochemistry with only one chiral center. Because of the two adjacent carbonyl groups, the only chiral center could not be easily controlled unless at least one of the carbonyl groups is bioisosterically replaced.
  • Hit 2 ID LH602
  • Scheme 3 we accomplished the resynthesis of Hit 2 (ID LH602) using the series of reactions outlined in Scheme 3.
  • N-Phthaloyl Glycine (4 g, 19.5 mmol) and Phenethylamine (2.65 mL, 21.5 mmol) were taken together and CH 3 CN (100 mL) was added.
  • POCl 3 (7.25 mL, 78 mmol) was added drop wise and the resulting solution was heated at 85° C. for 24 hrs, then cooled to RT and concentrated under reduced pressure.
  • the obtained dark residue was diluted with DCM (100 mL) and washed with aq. NaHCO3 (100 mL) and water (100 mL), then followed by brine (100 mL).
  • LH603 [2-(1-((1,3-dioxoisoindolin-2-yl)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)benzoic acid] (SM-II-93)
  • LH605 [4-(1-((1,3-dioxoisoindolin-2-yl)methyl)-3,4-dihydroisoquinolin-2(1H)-yl)-4-oxobutanoic acid] (SM-II-99)
  • LH601 [cis-2-(1-((1,3-dioxoisoindolin-2-yl)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)cyclo hexanecarboxylic acid] (SM-II-120)
  • Cis 1,2 Cyclohexanedicarboxylic anhydride and chiral S-amine (resolved using 2,3 Dibenzoyl-L-tartaric acid) were used.
  • HBTU was added to a solution of LH601A in acetonitrile and stirred for 30 min at room temperature. Then anhydrous ammonium was bubble into the reaction mixture for 1 min. The reaction mixture was concentrated and the residue was purified by column chromatography using acetonitrile (0 to 20%) in DCM as eluent to give the designed product.
  • the chiral S-amine (resolved using 2,3 Dibenzoyl-L-tartaric acid) (13.5 mg, 0.046 mmol) was dissolved in 1 mL, of DCM and 1 mL of water was added. NaHCO 3 and NaCl (50 mg each) were added to the biphasic mixture and cyclohexanecarbonyl chloride (18 ⁇ L 0.138 mmol) was added slowly ands stirred at RT vigorously for 1 hr, The organic layer separated and washed with waster and brine (2 mL each), dried and concentrated. The obtained precipitate was filtered and washed with hexane and dried to get 9 mg (50%) of product.
  • N-Boc protected-phthalimidomethyl-1,2,3,4-tetrahydro-isoquinoline (2.5 g, 6.3 mmol) was dissolved in CH 3 CN (25 mL) and anhydrous NH 2 .NH 2 (63 mmol, 3.2 mL) was added slowly. The reaction mixture was heated at 85° C. for 24 hrs and solvent was evaporated under reduced pressure. The obtained purple color solid was partitioned with DCM (25 mL) and water (25 mL) and organic layer was washed again with water and brine, and then concentrated.
  • N-Boc protected methylamino-1,2,3,4-tetrahydroisoquinoline (0.1-0.35 mmol) was dissolved in 1-3 mL of Toluene and anhydride (1.5 eq) was added and stirred at 50° C. for 1-3 hrs, then heated at 80° C. up to 24 hrs. The solvent was evaporated and crude was purified via column chromatography (ISCO) using Ethyl acetate (0 to 30%) in Hexane as eluent.
  • Impure final products were purified via column chromatography (ISCO) using Ethyl acetate (0 to 100% with 1% AcOH) in Hexane as eluent. Some diastereomeric pairs were separated during chromatography (ISCO) using CH 3 CN (0 to 20% with 1% AcOH) in DCM.
  • LH728 [cis-2-(1-((5-nitro-1,3-dioxoisoindolin-2-yl)methyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)cyclohexanecarboxylic acid] (SM-III-175)
  • LH751 [cis-2-((2-(2-carboxycyclohexanecarbonyl)-1,2,3,4-tetrahydroisoquinolin-1-yl)methyl)-1,3-dioxoisoindoline-5-carboxylic acid](SM-IV-15)
  • LH671A and LH671B Prepared by following the general synthetic procedure
  • LH674AB cis-2-((S)-1-((1,3-dioxoisoindolin-2-yl)methyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)cyclohexanecarboxylic acid
  • a mixture of phthalic anhydride and beta-alanine were heated to 190-200° C. in a sealed tube. Stirred for additional 30 min at 190-200° C. after the solids totally melted. The reaction mixture was cooled to room temperature and 50% EtOH/water was added. The resulted suspension was filtered and the white precipitate was further washed with 50% EtOH/water, and then dried under vacuum overnight to give the N-phthaloyl-beta-alanine. Phosphoryl chloride was added to a suspension of N-phthaloyl-beta-alanine and phenylethylamine in acetionitrile. The suspension was stirred for 6 h under reflux. Acetonitrile and phosphoryl chloride was removed by vacuum. The reaction mixture was then diluted with DCM and washed with sat NaHCO 3 solution, water and brine, then dried over anhydrous Na 2 SO 4 .
  • the filtrate was concentrated and the residue was purified by column chromatography using acetonitrile (0 to 20%) in DCM as eluent to afford the imine compound.
  • Acetic acid and sodium triacetoxyborohydride were added to a solution of imine compound in DCM and stirred for 1 h at room temperature.
  • the reaction mixture was then washed with sat NaHCO 3 solution, water and brine, then dried over anhydrous Na 2 SO 4 .
  • the filtration was concentrated and the residue was purified by column chromatography using acetonitrile (0 to 50%) in DCM as eluent to give the amine compound.
  • Cis-1,2-Cyclohexanedicarboxylic anhydride was added to a solution of amine compound in p-xylene at 50° C. and stirred for 3 h.
  • the reaction mixture was concentrated under vacuum and the residue was purified by column chromatography using acetonitrile (0 to 20% with 1% AcOH) in DCM as eluent to give the designed product.
  • Two diastereomers were separated by HPLC using water plus 0.1% TFA and acetonitrile plus 0.1% TFA as solvents.
  • LH705AB cis-2-((R)-1-(2-(1,3-dioxoisoindolin-2-yl)ethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)cyclohexanecarboxylic acid
  • LH705AB Prepared by following the above synthetic procedure and diastereomers were obtained by HPLC separation (mixture of 2 isomers)
  • LH705CD cis-2-((S)-1-(2-(1,3-dioxoisoindolin-2-yl)ethyl)-1,2,3,4-tetrahydroisoquinoline-2-carbonyl)cyclohexanecarboxylic acid
  • (1R,2S)-2-(benzyloxycarbonyl)cyclohexanecarboxylic acid was prepared from the meso anhydride cis-cyclohexane-dicarboxylic anhydride by reaction with benzyl alcohol at low temperature in the presence of quinidine (Christov, C.; et al., Chem Med Chem 2011, 6, 131-140).
  • Keap1 Kelch domain was measured using SA chip immobilized with biotin-16mer Nrf2 peptide (300 RU) when 40 nM of Keap1 Kelch domain was incubated in the presence of test compounds at two concentrations (5 ⁇ M and 50 ⁇ M) at room temperature. Each solution of Keap1 Kelch domain was injected through Fc1 and Fc2 with a 1-min association time and a 3-min dissociation time at 25° C.

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