WO2020118183A1 - 2,4-diaminopyrimidine bicycles for treating cancer - Google Patents

2,4-diaminopyrimidine bicycles for treating cancer Download PDF

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WO2020118183A1
WO2020118183A1 PCT/US2019/064937 US2019064937W WO2020118183A1 WO 2020118183 A1 WO2020118183 A1 WO 2020118183A1 US 2019064937 W US2019064937 W US 2019064937W WO 2020118183 A1 WO2020118183 A1 WO 2020118183A1
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alkyl
optionally substituted
compound according
hydroxy
ring
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Tarun M. Kapoor
Tommaso CUPIDO
Rudolf PISA
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Kapoor Tarun M
Cupido Tommaso
Pisa Rudolf
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    • 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
    • C07D403/12—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 linked by a chain containing hetero atoms as chain links
    • 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
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/08—Bridged systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
    • C07D495/04—Ortho-condensed systems

Definitions

  • AAA+ proteins ATPases Associated with diverse cellular Activities
  • AAA+ proteins share a common conserved module of approximately 230 amino acid residues. This is a large, functionally diverse protein family belonging to the AAA superfamily of ring-shaped P-loop NTPases, which exert their activity through the energy-dependent remodeling or translocation of macromolecules.
  • AAA+ proteins couple chemical energy provided by ATP hydrolysis to conformational changes which are transduced into mechanical force exerted on a
  • R 1 and R 2 are chosen independently from hydrogen, halogen, halo(Ci-C 4 )alkyl, (Ci-C 4 )alkyl, (Ci-C 4 )acyl, hydroxy(Ci-C 4 )alkyl, hydroxy, (Ci-C 4 )alkoxy, halo(Ci-C 4 )alkoxy, carboxy, (Ci- C 4 )alkoxycarbonyl, carboxamido, cyano, acetoxy, nitro, amino, (Ci-C 4 )alkylamino, di(Ci- C 4 )alkylamino, (Ci-C 4 )alkylthio, (Ci-C 4 )alkylsulfonylamino, (Ci-C 4 )alkylsulfmyl, and (Ci- C 4 )alkylsulfonyl;
  • the invention relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a compound described herein.
  • Spastin is a AAA+ protein which, in an ATP-dependent manner, severs microtubules to control filament number. In current models, spastin plays critical roles in neurogenesis, axonal regeneration, and cell division. Thus far, no chemical inhibitors have been reported for spastin.
  • Five constructs were active and revealed up to ⁇ 3-fold changes in K1 / 2. Only one construct, with a mutation in sensor-II (S689R), was found to be an inactive enzyme.
  • the range in K1/2 values for the active spastin mutants is comparable to the range observed for the five wild-type AAA+ proteins, indicating that it should be possible to selectively inhibit spastin— or any of the five members of the AAA+ superfamily.
  • aliphatic hydrocarbons examples include isopropyl, 2-butenyl, 2-butynyl, cyclopentyl, norbomyl, etc.
  • Aromatic hydrocarbons include benzene (phenyl), naphthalene (naphthyl), anthracene, etc.
  • Suitable pharmaceutically acceptable non-toxic acids or bases including inorganic acids and bases and organic acids and bases.
  • salts may be prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids.
  • Suitable pharmaceutically acceptable acid addition salts for the compounds of the present invention include acetic, adipic, alginic, ascorbic, aspartic, benzenesulfonic (besylate), benzoic, boric, butyric, camphoric, camphorsulfonic, carbonic, citric, ethanedisulfonic, ethanesulfonic, ethylenediaminetetraacetic, formic, fumaric, glucoheptonic, gluconic, glutamic, hydrobromic, hydrochloric, hydroiodic, hydroxynaphthoic, isethionic, lactic, lactobionic, laurylsulfonic, maleic, malic, mandelic, methanesulfonic, mucic, naph
  • the formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular), rectal and topical (including dermal, buccal, sublingual and intraocular) administration.
  • parenteral including subcutaneous, intradermal, intramuscular, intravenous and intraarticular
  • topical including dermal, buccal, sublingual and intraocular
  • the most suitable route may depend upon the condition and disorder of the recipient.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association a compound of formula I or a
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide sustained, delayed or controlled release of the active ingredient therein.
  • Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti -oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient.
  • Formulations for parenteral administration also include aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents.
  • the formulations may be presented in unit- dose of multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example saline, phosphate-buffered saline (PBS) or the like, immediately prior to use.
  • a sterile liquid carrier for example saline, phosphate-buffered saline (PBS) or the like, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • ACN acetonitrile
  • DCM dichloromethane
  • DIEA diisopropylethylamine
  • EtOAc ethyl acetate
  • eq equivalents
  • step 3 The intermediate from step 3 was first treated with a mixture of TFA:DCM:H20 (4.5:5:0.5; 3 mL) for 30 min. The solvent was evaporated and the residue was triturated with diehtyl ether and filtered to recovered an off-white precipitate. The precipitate was then treated with 0.1 M KOH in MeOLfLhO (7:3; 5 mL) for 1 h at 40 °C.
  • cells expressing recombinant proteins were collected by centrifugation, resuspended in the protein-specific lysis buffer (Buffer A), and lysed with an Emulsiflex-C5 homogenizer (Avestin) through 5-6 cycles at 10,000-15,000 psi.
  • the homogenized lysate was centrifuged at 40,000 rpm for 45 to 60 min using a Ti45 rotor in a Beckman Coulter Optima LE- 80K ultracentrifuge. All recombinant proteins were purified using multi-step strategies, which involved combinations of affinity, ion exchange and size exclusion chromatography.
  • affinity matrices were washed with at least 50 volumes of washing buffers (Buffer B).
  • Buffer B washing buffers
  • proteins were loaded onto specific columns (see below), which were pre-equilibrated in low-salt buffers (Buffers C), and eluted in a gradient ofhigh-salt buffers (Buffers D).
  • the fractions from these columns that were enriched in the protein of interest were pooled, concentrated using an appropriate (see below) size Amicon® Ultra centrifugal filter (Millipore), filtered through a 0.22-pm Millex-GP PES membrane (Millipore SLGP033RS), and injected onto size-exclusion chromatography columns equilibrated in Buffers E. Fractions containing the protein of interest were recovered, pooled and concentrated to a minimum of 1.0 mg/mL. The concentrated protein samples were recovered in 1.5 ml eppendorf tubes, glycerol was added to a final 15% (v/v), and the solutions centrifuged 20,000 for 10 min. The supernatant was collected and snap-frozen in liquid nitrogen and stored at -80 °C. The buffers used are listed below the specific protein purification procedure.
  • spastin wild-type and mutants
  • a previously published procedure Ziolkowska and Roll-Mecak“In Vitro Microtubule Severing Assays” Methods in Molecular Biology 1046, 323-334 (2013)
  • the clarified lysate containing GST-spastin was incubated with a GSTrap 4B matrix (GE Healthcare) and eluted in Buffer B supplemented with 20 mM reduced glutathione.
  • the protein solution was incubated with PreScission protease (0.1 mg/mL) for 8-12 h, and diluted 1 :2.5 with Buffer C and loaded into a CaptoS cation exchange column (GE Healthcare) equilibrated in 95% Buffer C and 5% Buffer B.
  • Fractions eluted from the ion exchange chromatography column were pooled, concentrated 10-fold with an Amicon Ultra 30K MWCO centrifugal filter, and further purified over a 10/300 Superdex 200 column (GE Healthcare) in Buffer E.
  • the eluate containing purified spastin was pooled, concentrated using an Amicon Ultra 3 OK MWCO centrifugal filter to at least 1 mg/mL and stored at -80 °C.
  • Buffer A 50 mM K-HEPES, 150 mM KC1, 5 mM MgCh, 5 mM dithiothreitol (DTT, Goldbio), 0.5 mM phenylmethanesulfonyl fluoride (PMSF), 5 U/mL benzonase, and complete EDTA-free protease inhibitors (Roche), pH 7.5.
  • Buffer B 50 mM K- HEPES, 400 mM KC1, 5 mM MgCh, 5 mM DTT, 0.1 mM PMSF, pH 7.5.
  • Buffer C 25 mM Na- MES, 5% (w/v) glycerol, 5 mM MgCh, 5 mM DTT, pH 6.5.
  • Buffer D 25 mM Na-MES, 2 M NaCl, 5% (w/v) glycerol, 5 mM MgCh, 5 DTT, pH 6.5.
  • Buffer E 25 mM K-HEPES, 300 mM KC1, 10% (w/v) glycerol, 5 mM MgCh, 5 mM DTT, pH 7.5.
  • GST-FIGL1 (aa 295-674).
  • the clarified lysate from cells expressing GST-FIGL1 was incubated with sepharose glutathione beads (GE Healthcare) for lh and eluted using Buffer B supplemented with 10 mM reduced glutathione. Fractions containing the proteins were pooled, diluted with 3 volumes of Buffer C and purified using a 16/60 MonoQ GL column (GE
  • the protein fractions were pooled, concentrated 10-fold using an Amicon® Ultra 50K MWCO centrifugal filter, and further purified over a 10/300 Superdex 200 column (GE Healthcare) in Buffer E. Fractions from size-exclusion column were concentrated to a minimum of 1 mg/mL using an Amicon® Ultra 30K MWCO centrifugal filter, and stored.
  • Buffer A 50 mM Tris-HCl, 150 mM NaCl, 5 mM MgCh , 10 mM DTT, 0.5 mM PMSF, 0.01% (v/v) triton X-100, 5 U/mL benzonase, and complete EDTA-free protease inhibitors (Roche), pH 7.5.
  • Buffer B 50 mM Tris-HCl, 300 mM NaCl, 5 mM MgCh , 10 mM DTT, 0.1 mM PMSF, pH 7.5.
  • Buffer C 50 mM Tris-HCl, 75 mM NaCl, 5 mM MgCh, 5 mM DTT, pH 8.5.
  • Buffer D 50 mM Tris-HCl, 1 M NaCl, 5 mM MgCh, 5 mM DTT, pH 8.5.
  • Buffer E 20 mM Tris-HCl, 150 mM NaCl, 5 mM MgCh, 5 mM DTT, 5% (w/v) glycerol, pH 7.5.
  • MBP-Katanin The clarified lysate from cells expressing MBP-katanin was filtered through a 0.22-pm Millex-GP PES membrane and loaded onto MBP trap column (GE).
  • the column was washed with 30 column volumes of Buffer B and eluted with a Buffer B supplemented with 10 mM maltose.
  • the eluate was diluted 1 :2 with Buffer C and loaded on a 16/60 MonoQ GL ion exchange column and eluted with a gradient of Buffer D.
  • Combined MonoQ fractions were further purified on a 10/300 Superdex 200 gel filtration column in Buffer E.
  • the fractions containing purified MBP-katanin protein were frozen in liquid nitrogen and stored at -80°C.
  • Buffer A 50 mM Tris-HCl, 250 mM NaCl, 5 mM MgCh, 100 pM ATP (Sigma, 2383), 5 mM DTT, 1 mM PMSF and complete EDTA-free protease inhibitors (Roche), pH 8.0.
  • Buffer B 50 mM Tris-HCl, 250 mM NaCl, 5 mM MgCh, 100 pM ATP, 5 mM DTT, pH 7.5.
  • Buffer C 20 mM Tris-HCl, 100 mM NaCl, 1 mM MgCh, 5 mM DTT, 100 pM ATP, pH 7.5.
  • Buffer D 20 mM Tris-HCl, 500 mM NaCl, 1 mM MgCh, 5 mM DTT, 100 pM ATP, pH 7.5.
  • Buffer E 20 mM K-HEPES, 250 mM NaCl, 5 mM MgCh, 5 mM DTT, and 10% (w/v) glycerol, pH 7.5.
  • VCP/p97 The clarified lysate from cells expressing His6-VCP/p97 was incubated with Ni-NTA beads (Qiagen) for 40 min, and the beads were extensively washed using Buffer B. The protein was eluted with Buffer B supplemented with 300 mM imidazole, diluted 1 :2 in Buffer C, loaded onto a Mono Q column 5/50 GL (GE Healthcare) and fractioned over a gradient with Buffer D. Protein was eluted at approximately 350 mM NaCl.
  • Buffer A 50 mM Tris-HCl, 250 mM NaCl, 20 mM imidazole, 5 mM MgCh , 1 mM //7.s(2-carboxyethyl (phosphine (TCEP, Soltec Ventures), 0.5 mM ATP, 5 U/mL benzonase, and complete EDTA-free protease inhibitors (Roche), pH 8.0.
  • Buffer B 50 mM Tris- HCl, 400 mM NaCl, 20 mM imidazole, 5 mM MgCh, 1 mM TCEP, pH 7.5.
  • Buffer C 50 mM K- HEPES, 150 mM NaCl, 5 mM MgCh, 2 mM TCEP, pH 7.5.
  • Buffer D 50 mM K-HEPES, 1 M NaCl, 5 mM MgCh, 2 mM TCEP, pH 7.5.
  • Buffer E 25 mM K-Hepes 150 mM NaCl, 5 mM MgCh, 2 mM TCEP, 5% (w/v) glycerol, pH 7.5.
  • PCH2 The clarified lysate from cells expressing GST-PCH2 was incubated with sepharose glutathione beads (GE Healthcare) for 1 h and the beads were washed with 50 volumes of Buffer B and then with 5 volumes of Buffer A without complete EDTA-free protease inhibitor cocktail.
  • PreScission protease was diluted to 0.1 mg/mL in a buffer containing 50 mM Tris-HCl, 250 mM NaCl, 5 mM MgCh , 10 mM DTT, pH 8.0, 0.1 mM PMSF, 10 pM reduced glutathione and the beads were incubated with this solution for 12 h.
  • the protein-containing solution was recovered by filtration and incubated with fresh sepharose glutathione beads for 1 h. Beads were removed by centrifugation at 300g-, 5 min twice and the solution was concentrated 20-fold using an Amicon® Ultra 30K MWCO centrifugal filter, filtered through a 0.22-mih Millex-GP PES membrane and further purified over a 10/300 Superdex 75 column (GE Healthcare) in Buffer E. Fractions containing purified protein from the size-exclusion column were concentrated to a minimum of 1 mg/mL using an Amicon® Ultra 30K MWCO centrifugal filter and stored at -80 °C.
  • Buffer A 50 mM Tris-HCl, 250 mM NaCl, 5 mM MgCh , 10 mM DTT,
  • Buffer B 50 mM Tris-HCl, 250 mM NaCl, 5 mM MgCh , 10 mM DTT, pH 8.0, 0.1 mM PMSF, 10 pM reduced glutathione, pH 8.0.
  • Buffer E 50 mM Tris-HCl, 150 mM NaCl, 5 mM MgCh , 5 mM DTT, 10 % (w/v) glycerol, pH 8.0.
  • ATPase activity of AAA+ proteins was determined in the steady-state conditions using an enzymatic ATP-regeneration system. This system is coupled to the oxidation of NADH, which is stoichiometric to the hydrolysis of ATP, and can be followed by monitoring the decrease in the fluorescence signal at 440 nm. This same assay was employed to measure variation of the ATPase rate at different ATP concentrations as well as inhibition of ATPase activity at different compound concentrations.
  • the protein sample was diluted in assay buffer, and NADH (Sigma, N7410) was added to final concentration of 200-175 mM, (in the case of FIGL1, spastin, and katanin) or 100150 mM (in the case of VCP/p97 and PCH2).
  • NADH Sigma, N7410
  • phosphoenol pyruvic acid monopotassium salt Sigma, P7127
  • D-lactic dehydrogenase Sigma, L3888
  • pyruvate kinase lyophilized powder, Sigma, P9136
  • Assay buffer conditions for the different AAA+ proteins were established (FIGL1), or adapted (spastin, VCP/p97, PCH2, katanin) from conditions reported in previous studies to optimize enzymes’ specific activities.
  • the final concentration of proteins was wild-type spastin 80-100 nM, katanin 80 nM, FIGL1 50 nM, VCP/p97 450 nM and PCH2 340 nM, for measuring ATPase rate at different ATP concentrations. Protein concentrations were chosen to allow for robust measurement of enzymatic rate over a range of MgATP concentrations (0.027 - 3 mM) during a maximum assay time window of 90 min.
  • Spastin wild type and mutants 25 mM K-HEPES, 225 mM KC1, 25 mM K2HPO4, 5 mM MgCh, 2.5 mM DTT, 0.1 mg/mL BSA, pH 7.5, 0.05% v/v triton X-100.
  • Katanin 25 mM K- HEPES, 70 mM KC1, 25 mM K 2 HP0 4 , 5 mM MgCh, 2.5 mM DTT, 0.1 mg/mL BSA, pH 7.5.
  • FIGL1 25 mM Na-MES, 70 mM KOAc, 25 mM K 2 HP0 4 , 5 mM Mg(OAc) 2 , 1 mM TCEP, 0.1 mg/ml BSA, pH 6.5.
  • VCP/p97 50 mM K-HEPES, 25 mM KC1, 25 mM K 2 HP0 4 , 15 mM MgCh, 1 mM TCEP, pH 7.5.
  • PCH2 25 mM TRIS-HCl, 150 mM KC1, 25 mM K 2 HP0 4 , 5 mM MgCh, 2.5 mM DTT, 0.1 mg/mL BSA, 0.025% (v/v) triton-X100, pH 8.5.
  • recombinant spastin 85 nM was diluted in a buffer containing 25 mM K-HEPES, 225 mM KC1, 25 mM K 2 HP0 4 , 5 mM MgCh, 2.5 mM DTT, 0.1 mg/mL bovine serum albumin, and 2 to 0.02 mM of compound 5 (1% final DMSO concentration).
  • ATPase reactions were initiated by adding MgATP (0.044 to 3 mM). For concentrations of compound equal to 2 pM the enzymatic parameters could not be calculated.
  • V ATPase rate
  • Vmax represents the maximum ATPase rate and x represents the ATP concentration.
  • Results are shown in Table 1 in which +++ indicates ATPase activity of 0-10% of
  • control indicates ATPase activity of 11-40% of control; + indicates ATPase activity of 41-70% of control; and - indicates ATPase activity of 71-100% of control.
  • the data represented is for compound concentrations of 10 mM.
  • VCP/97 neurodegenerative diseases such as Alzheimer’s disease.
  • the compounds that are selective for VCP/97 can be used as anticancer agents.
  • Thermofisher (SelectScreen) using the Z’-LYTE assay.
  • the Z’-LYTE biochemical assay employs a fluorescence-based, coupled-enzyme format and is based on the differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage.
  • the peptide substrate is labeled with two fluorophores— one at each end— that make up a FRET pair.
  • the kinase transfers the gamma-phosphate of ATP to a single tyrosine, serine or threonine residue in a synthetic FRET-peptide.
  • a site-specific protease recognizes and cleaves non-phosphorylated FRET- peptides.
  • Phosphorylation of FRET-peptides suppresses cleavage by the ThermoFisher proprietary development Reagent. Cleavage disrupts FRET between the donor (i.e., coumarin) and acceptor (i.e., fluorescein) fluorophores on the FRET-peptide, whereas uncleaved, phosphorylated FRET-peptides maintain FRET.
  • a ratiometric method which calculates the ratio (the Emission Ratio) of donor emission to acceptor emission after excitation of the donor fluorophore at 400 nm, is used to quantitate reaction progress, according to the equation:
  • Emission Ratio (Coumarin Emission, 445nm) / (Fluorescein Emission, 520 nm).
  • the maximum Emission Ratio is established by the 0% phosphorylation control (100% Inhibition Control), which contains no ATP and therefore exhibits no kinase activity. This control yields 100% cleaved peptide in the Development Reaction.
  • the 100% Phosphorylation Control which consists of a synthetically phosphorylated peptide of the same sequence as the peptide substrate, is designed to allow for the calculation of percent phosphorylation. This control yields a very low percentage of cleaved peptide in the Development Reaction.
  • the minimum Emission Ratio in a screen is established by the 0% Inhibition Control, which contains active kinase. This control is designed to produce a 10-50% phosphorylated peptide in the kinase reaction.
  • Percent phosphorylation is calculated according to the equation: ( l-[(Emission Ratio x F ioo%) - Cioo%] / (Co%- Cioo%) + [Emission Ratio x (Fioo%- Fo%)] ⁇ *100.
  • Cioo% average Coumarin emission signal of 100% phosphorylation control
  • Co % average Coumarin emission signal of the 0% phosphorylation control
  • Fioo % average fluorescein emission signal of 100% phosphorylation control
  • Fo % average fluorescein emission signal of 0%
  • TrkA-C inhibitors ⁇ !- (% Phos the sample / % Phos of the 0% inhibition control) ⁇ *100.
  • Table 2 shows four examples of TrkA-C inhibitors. Compounds that are selective for TrkA-C inhibition can be used for treating solid tumors. In particular, those that have a neurotrophic receptor tyrosine kinase gene fusion.
  • Penicillin/Streptomycin (GIbco cat# 15070063)). 30 uL of assay media containing 4000-5000 cells were layered in a white 384-well plate (Greiner Cat# 781075). After 12-16 hours, compound dilutions were prepared in 100% DMSO and added to the assay media (1% DMSO,

Abstract

A genus of quinazoline and pyrrolo[3,2-d]pyrimidine derivatives is disclosed. The compounds are of the genus (I). The compounds selectively inhibit subfamilies of AAA+ proteins and TrkA-C. They are useful in the treatment of Alzheimer's disease and as anticancer agents.

Description

2,4-DIAMINOPYRIMEDINE BICYCLES FOR TREATING CANCER
GOVERNMENT RIGHTS STATEMENT
[0001] This invention was made with Government support under contracts numbers ROl GM98579 and ROl GM65933 from the National Institutes of Health. The Government has certain rights in the invention.
FIELD OF THE INVENTION
[0002] The invention relates to quinazoline and pyrrol o[3,2-i/]pyri mi dine derivatives that selectively inhibit subfamilies of AAA+ proteins and TrkA-C. The compounds are useful as anticancer agents and as probes of the function of AAA+ protein-dependent systems.
BACKGROUND OF THE INVENTION
AAA+ Proteins
[0003] ATPases Associated with diverse cellular Activities (AAA+ proteins) share a common conserved module of approximately 230 amino acid residues. This is a large, functionally diverse protein family belonging to the AAA superfamily of ring-shaped P-loop NTPases, which exert their activity through the energy-dependent remodeling or translocation of macromolecules. AAA+ proteins couple chemical energy provided by ATP hydrolysis to conformational changes which are transduced into mechanical force exerted on a
macromolecular substrate. They are functionally and organizationally diverse, and vary in activity, stability, and mechanism. Members of the AAA family are found in all organisms, and they are essential for many cellular functions. They are involved in processes such as DNA replication, protein degradation, membrane fusion, microtubule severing, peroxisome biogenesis, signal transduction and the regulation of gene expression.
[0004] Thus far, selective chemical inhibitors have been reported for only 3 of the -100 AAA+ proteins in eukaryotes. Selective and potent chemical inhibitors are available only for VCP/p97, a regulator of proteolysis, Mdnl, an essential protein needed for ribosome assembly, and dynein, a microtubule-based motor protein. Moreover, the discovery of these inhibitors has relied on serendipity, and the available data do not suggest a rational strategy to design selective chemical inhibitors for any of the other AAA+ proteins.
[0005] To devise a rational strategy for designing inhibitors of AAA+ proteins one may focus on the active site of these enzymes. Differences in the amino acids that comprise the nucleotide binding site of AAA+ proteins can be exploited to develop selective inhibitors. Nucleotide binding in these proteins occurs at the interface of two AAA domains, the conserved structural unit that defines this superfamily. Five proteins whose AAA domains are similar at the level of primary sequence (up to 50% sequence identity) are: (1) VCP/p97, (2) katanin, (3) spastin, (4) FIGL1, and (5) PCH2. Katanin and spastin are two microtubule-severing proteins; FIGL1 and PCH2, are related to DNA repair and cell cycle regulation. Analysis of the steady-state nucleotide hydrolysis rates of recombinant forms of these five AAA+ proteins revealed up to ~3.5-fold differences in the KI/2 for ATP hydrolysis. This suggests differences in how these proteins interact with the nucleotide at their active sites.
[0006] As expected for a conserved active site, most of the residues in these five AAA+ proteins are invariant. Only four amino acids, located in motifs that are known as the N-terminal loop (N- loop), the phosphate-binding loop (P-loop) and in the sensor-II motif, vary significantly across these proteins. If one can devise small molecules that distinguish among the five proteins, one may selectively inhibit different enzyme pathways.
TrkA
[0007] Tropomyosin receptor kinase A (TrkA; also known as high affinity nerve growth factor receptor, neurotrophic tyrosine kinase receptor type 1, or TRK1 -transforming tyrosine kinase protein) is a membrane-bound receptor that, upon neurotrophin binding, phosphorylates itself (autophosphorylation) and members of the MAPK pathway. The presence of this kinase leads to cell differentiation and may play a role in specifying sensory neuron subtypes. Mutations in this gene have been associated with congenital insensitivity to pain with anhidrosis, self-mutilating behaviors, intellectual disability and/or cognitive impairment and certain cancers.
[0008] TrkA is the high affinity catalytic receptor for the neurotrophin, Nerve Growth Factor, or "NGF". As such, it mediates the multiple effects of NGF, which include neuronal differentiation and avoidance of programmed cell death. TrkA is of interest in the treatment of cancer because of the identification of TrkA, TrkB and TrkC gene fusions and other oncogenic alterations in a number of tumor types.
SUMMARY OF THE INVENTION
[0009] In one aspect, the invention relates to 4-(pyrazol-3-yl)amino-2-aminopyrimidine bicycles of formula I:
Figure imgf000004_0001
wherein:
A is a five- or six-membered heteroaryl ring, or, a six-membered aryl ring;
Y is carbon or nitrogen;
R1 and R2 are chosen independently from hydrogen, halogen, halo(Ci-C4)alkyl, (Ci-C4)alkyl, (Ci-C4)acyl, hydroxy(Ci-C4)alkyl, hydroxy, (Ci-C4)alkoxy, halo(Ci-C4)alkoxy, carboxy, (Ci- C4)alkoxycarbonyl, carboxamido, cyano, acetoxy, nitro, amino, (Ci-C4)alkylamino, di(Ci- C4)alkylamino, (Ci-C4)alkylthio, (Ci-C4)alkylsulfonylamino, (Ci-C4)alkylsulfmyl, and (Ci- C4)alkylsulfonyl;
R3 and R4 are independently selected from hydrogen, (Ci-C4)alkyl, optionally substituted phenyl, optionally substituted heteroaryl, and CH2R10, or, taken together with the nitrogen to which they are attached, R3 and R4 may form an optionally substituted five-, six- or seven-membered heterocyclic ring, wherein said heterocyclic ring contains one or more heteroatoms, and wherein said optional substituents are selected from halogen, halo(Ci-C4)alkyl, (Ci-C4)acyl, hydroxy(Ci- C4)alkyl, hydroxy, (Ci-C4)alkoxy, halo(Ci-C4)alkoxy, carboxy, (Ci-C4)alkoxycarbonyl, carboxamido, amino, (Ci-C4)alkylamino, di(Ci-C4)alkylamino, (Ci-C4)alkylsulfonylamino, (Ci- C4)alkylsulfmyl, (Ci-C4)alkylaminocarbonyl, (Ci-Cio)hydrocarbyl and (Ci-C4)alkylsulfonyl; with the proviso that no carbon atom of said five-, six-, or seven-membered ring formed by R3 and R4 may carry more than one substituent;
R5 is chosen from hydrogen, halogen, (Ci-C4)acyl, hydroxy, (Ci-C4)alkoxy, halo(Ci-C4)alkoxy, carboxy, (Ci-C4)alkoxycarbonyl, carboxamido, amino, (Ci-C4)alkylamino, di(Ci-C4)alkylamino, optionally substituted (Ci-Cio)hydrocarbyl, optionally substituted aryl, and optionally substituted heteroaryl, wherein said optional substituents on aryl and heteroaryl are selected from halogen, halo(Ci-C4)alkyl, (Ci-C4)alkyl, cyano, acetoxy, nitro, (Ci-C4)acyl, hydroxy(Ci-C4)alkyl, hydroxy, (Ci-C4)alkoxy, halo(Ci-C4)alkoxy, carboxy, (Ci-C4)alkoxycarbonyl, carboxamido, amino, (Ci-C4)alkylamino, di(Ci-C4)alkylamino, (Ci-C4)alkylsulfonylamino, (Ci- C4)alkylsulfmyl, and (Ci-C4)alkylsulfonyl.
R10 is an optionally substituted heterocycle or an optionally substituted six-membered aryl, wherein said optional substituents are selected from cyano, amino, alkylamino, dialkylamino, hydroxy, cyano(Ci-C4)alkyl, carboxy(Ci-C4)alkyl, carboxamido(Ci-C4)alkyl, amino(Ci-C4)alkyl, alkylamino(Ci-C4)alkyl, dialkylamino(Ci-C4)alkyl, and hydroxy(Ci-C4)alkyl. In the structure depicted above, it is intended that the bicyclic ring system formed by ring A and the ring containing Y-N-N is maximally unsaturated; thus the dotted circle represents unsaturation.
[0010] In another aspect, the invention relates to pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a compound described herein.
[0011] In another aspect, the invention relates to a method for selectively inhibiting spastin comprising exposing spastin to a compound described herein.
[0012] In another aspect, the invention relates to a method for selectively inhibiting tropomyosin receptor kinase A comprising exposing tropomyosin receptor kinase A to a compound described herein.
[0013] In another aspect, the invention relates to a method for selectively inhibiting tropomyosin receptor kinase B comprising exposing tropomyosin receptor kinase B to a compound described herein. [0014] In another aspect, the invention relates to a method for selectively inhibiting tropomyosin receptor kinase C comprising exposing tropomyosin receptor kinase C to a compound described herein.
DETAILED DESCRIPTION OF THE INVENTION
[0015] As discussed above, most of the residues in VCP/p97, katanin, spastin, FIGL1, and PCH2 proteins are invariant. Only four amino acids, located in motifs that are known as the N-terminal loop (N-loop), the phosphate-binding loop (P-loop) and in the sensor-II motif, vary significantly across these proteins.
[0016] Spastin is a AAA+ protein which, in an ATP-dependent manner, severs microtubules to control filament number. In current models, spastin plays critical roles in neurogenesis, axonal regeneration, and cell division. Thus far, no chemical inhibitors have been reported for spastin. We noted that the non-conserved residues of spastin’ s ATP -binding site have diverged with respect to the other AAA+ proteins. At each of these residue positions we introduced an amino acid found at an equivalent site in the other four AAA+ proteins. Five constructs were active and revealed up to ~3-fold changes in K1/2. Only one construct, with a mutation in sensor-II (S689R), was found to be an inactive enzyme. The range in K1/2 values for the active spastin mutants is comparable to the range observed for the five wild-type AAA+ proteins, indicating that it should be possible to selectively inhibit spastin— or any of the five members of the AAA+ superfamily.
[0017] Compounds of the formula I:
Figure imgf000006_0001
I
have been found to selectively inhibit spastin. In some embodiments, R3 and R4, together with the nitrogen to which they are attached, form an optionally substituted five-, six-, or seven- membered ring. In some embodiments, A is a fused optionally substituted benzene, pyridine, thiophene, or pyrrole ring. In some embodiments, A is a benzene ring and the bicyclic system is a quinazoline. The quinazoline may be optionally substituted with halogen, methyl,
trifluoromethyl, methoxy, trifluoromethoxy, hydroxy or amino. In some embodiments, A is a pyridine, thiophene, or pyrrole ring, optionally substituted with methyl. In a preferred
embodiment, A is an optionally substituted pyrrole ring. When A is a pyridine ring, it may be fused to form a pyrido[2,3-d]pyrimidine, optionally substituted with fluoro, trifluoromethyl, or methyl. When A is a pyrrole ring, it may be fused to form a 7H-pyrrolo[2,3-d]pyrimidine or a pyrrolo[l,2-f [l,2,4]triazine, optionally substituted with fluoro, trifluoromethyl, or methyl. When A is a thiophene ring, it may be fused to form a thieno[3,2-d]pyrimidine, optionally substituted with fluoro, trifluoromethyl, or methyl.
[0018] In some embodiments, R5 is chosen from (Ci-C3)hydrocarbyl, (C4-Cio)hydrocarbyl and heteroaryl, and the hydrocarbyls or heteroaryl may be optionally substituted with one or two substituents selected from halogen, methyl, trifluoromethyl, methoxy, trifluoromethoxy, hydroxy and amino. In some embodiments R5 is chosen from (C3-C6)alkyl, (C3-C6)cycloalkyl, phenyl, furanyl, and benzyl optionally substituted with one or two substituents selected from halogen, methyl, trifluoromethyl, methoxy, trifluoromethoxy, hydroxy and amino.
[0019] In one subgenus, in which R5 is cyclopropyl, t-butyl and R3 is hydrogen, R4 may be optionally substituted benzyl. In another subgenus, R3 and R4 form an optionally substituted five- or six-membered monocycle. In yet another subgenus, R3 and R4 form an optionally substituted diazabicycloheptane, diazabicyclooctane or diazabicyclononane.
[0020] In some embodiments, R3 and R4 form a piperazine, homopiperazine, piperidine, morpholine or pyrrolidine ring optionally substituted with (Ci-C4)alkyl or hydroxy(Ci-C4)alkyl. In a some of these embodiments, R3 and R4 form a piperazine ring substituted with methyl, ethyl, or hydroxymethyl. In preferred embodiments, R3 and R4 form a 2-substituted piperazin-l-yl ring or 3, 5 -di substituted piperazin-l-yl ring substituted with (Ci-C4)alkyl or hydroxy(Ci-C4)alkyl. [0021] In some preferred embodiments, A is a benzene ring optionally substituted with halogen and R5 is chosen from (Ci-C6)alkyl, (C3-C6)cycloalkyl, and benzyl optionally substituted with halogen, methyl, or trifluorom ethyl. In other preferred embodiments, A is a pyrrole ring optionally substituted with halogen.
[0022] In summary, the invention relates to:
[0023] [1] A compound of formula I.
[0024] [2] A compound according to [1] above wherein A is a five-membered heteroaryl ring.
[0025] [3] A compound according to [1] above wherein A is a six-membered heteroaryl ring.
[0026] [4] A compound according to [1] above wherein A is a six-membered aryl ring.
[0027] [5] A compound according to [1] and [4] above wherein A is a benzene ring optionally substituted with halogen, methyl, trifluoromethyl, methoxy, trifluoromethoxy, hydroxy or amino.
[0028] [6] A compound according to [1] and [2] above wherein A is a pyrrole ring optionally substituted with fluoro, trifluoromethyl, or methyl.
[0029] [7] A compound according to [1] and [3] above wherein A is a pyridine ring optionally substituted with fluoro, trifluoromethyl, or methyl.
[0030] [8] A compound according to [1] and [2] above wherein A is a thiophene ring optionally substituted with fluoro, trifluoromethyl, or methyl.
[0031] [9] A compound according to any of [1] through [8] above wherein Y is carbon.
[0032] [10] A compound according to any of [1] through [8] above wherein Y is nitrogen.
[0033] [11] A compound according to any of [1] through [10] above wherein R1 is hydrogen.
[0034] [12] A compound according to any of [1] through [10] above wherein R1 is halo.
[0035] [13] A compound according to any of [1] through [10] above wherein R1 is methoxy. [0036] [14] A compound according to any of [1] through [10] above wherein R1 is trifluoromethoxy.
[0037] [15] A compound according to any of [1] through [14] above wherein R2 is hydrogen.
[0038] [16] A compound according to any of [1] through [14] above wherein R2 is halo.
[0039] [17] A compound according to any of [1] through [14] above wherein R2 is methoxy.
[0040] [18] A compound according to any of [1] through [14] above wherein R2 is
trifluoromethoxy.
[0041] [19] A compound according to any of [1] through [18] above wherein R3 and R4, taken together with the nitrogen to which they are attached, forms a six-membered heterocyclic ring.
[0042] [20] A compound according to any of [1] through [18] above wherein R3 and R4, taken together with the nitrogen to which they are attached, forms a five-membered heterocyclic ring.
[0043] [21] A compound according to any of [1] through [18] above wherein R3 and R4, taken together with the nitrogen to which they are attached, forms a seven-membered heterocyclic ring.
[0044] [22] A compound according to any of [19] through [21] above wherein the heterocyclic ring formed by R3 and R4 is optionally substituted with one or more (Ci-C4)alkyl groups.
[0045] [23] A compound according to any of [19] through [22] above wherein the heterocyclic ring formed by R3 and R4 is optionally substituted with one or more hydroxy(Ci-C4)alkyl groups.
[0046] [24] A compound according to any of [1] through [18] above wherein at least one of R3 and R4 is CH2R10, wherein R10 is an optionally substituted heterocycle or an optionally substituted six-membered aryl, and wherein said optional substituents are selected from cyano, amino, alkylamino, dialkylamino, hydroxy, cyano(Ci-C4)alkyl, carboxy(Ci-C4)alkyl,
carboxamido(Ci-C4)alkyl, amino(Ci-C4)alkyl, alkylamino(Ci-C4)alkyl, dialkylamino(Ci- C4)alkyl, and hydroxy(Ci-C4)alkyl.
[0047] [25] A compound according to any of [1] through [18] and [24] above wherein at least one of R3 and R4 is hydrogen. [0048] [26] A compound according to any of [1] through [18] and [24] above wherein at least one of R3 and R4 is (Ci-C4)alkyl.
[0049] [27] A compound according to any of [1] through [18] and [24] through [25] above wherein at least one of R3 and R4 is optionally substituted phenyl, wherein the optional substituents are selected from (Ci-Cio)hydrocarbyl, (Ci-Cio)alkoxy(Ci-C4)alkyl, halogen, halo(Ci-C4)alkyl, (Ci-C4)acyl, hydroxy(Ci-C4)alkyl, hydroxy, (Ci-C4)alkoxy, halo(Ci-C4)alkoxy, carboxy, (Ci-C4)alkoxycarbonyl, carboxamido, amino, (Ci-C4)alkylamino, di(Ci-C4)alkylamino, (Ci-C4)alkylsulfonylamino, (Ci-C4)alkylsulfmyl, (Ci-C4)alkylaminocarbonyl, and (Ci- C4)alkylsulfonyl.
[0050] [29] A compound according to any of [1] through [18] and [24] through [25] above wherein at least one of R3 and R4 is optionally substituted phenyl, wherein the optional substituents are selected from (Ci-Cio)hydrocarbyl, (Ci-Cio)alkoxy(Ci-C4)alkyl, halogen, halo(Ci-C4)alkyl, (Ci-C4)acyl, hydroxy(Ci-C4)alkyl, hydroxy, (Ci-C4)alkoxy, halo(Ci-C4)alkoxy, carboxy, (Ci-C4)alkoxycarbonyl, carboxamido, amino, (Ci-C4)alkylamino, di(Ci-C4)alkylamino, (Ci-C4)alkylsulfonylamino, (Ci-C4)alkylsulfmyl, (Ci-C4)alkylaminocarbonyl, and (Ci- C4)alkylsulfonyl.
[0051] [28] A compound according to any of [1] through [18] and [24] through [25] above wherein at least one of R3 and R4 is optionally substituted heteroaryl, wherein the optional substituents are selected from (Ci-Cio)hydrocarbyl, (Ci-Cio)alkoxy(Ci-C4)alkyl, halogen, halo(Ci-C4)alkyl, (Ci-C4)acyl, hydroxy(Ci-C4)alkyl, hydroxy, (Ci-C4)alkoxy, halo(Ci-C4)alkoxy, carboxy, (Ci-C4)alkoxycarbonyl, carboxamido, amino, (Ci-C4)alkylamino, di(Ci-C4)alkylamino, (Ci-C4)alkylsulfonylamino, (Ci-C4)alkylsulfmyl, (Ci-C4)alkylaminocarbonyl, and (Ci- C4)alkylsulfonyl.
[0052] [29] A compound according to any of [1] through [28] above wherein R5 is (Ci- Cio)hydrocarbyl.
[0053] [30] A compound according to any of [1] through [28] above wherein R5 is (C4- Cio)hydrocarbyl. [0054] [31] A compound according to any of [1] through [28] above wherein R5 is (Ci- C3)hydrocarbyl.
[0055] [32] A compound according to any of [1] through [28] above wherein R5 is (C2- C5)hydrocarbyl.
[0056] [33] A compound according to any of [1] through [28] above wherein R5 is (C3- C4)hydrocarbyl.
[0057] [34] A compound according to any of [1] through [28] above wherein R5 is cyclopropyl, t-butyl or phenyl.
[0058] [35] A compound according to any of [1] through [28] above wherein R5 is cyclopropyl or t-butyl.
[0059] [36] A compound according to any of [19] through [22] and [29] through [35] above wherein the heterocyclic ring formed by R3 and R4 is optionally substituted with one or more (Ci-Cio)oxaalkyl groups.
[0060] [37] A compound according to any of [19] through [22] and [29] through [35] above wherein the heterocyclic ring formed by R3 and R4 is optionally substituted with one or more (C2-Cs)oxaalkyl groups.
[0061] Throughout this specification the terms and substituents retain their definitions.
[0062] Ci to C10 hydrocarbyl includes alkyl, cycloalkyl, polycycloalkyl, alkenyl, alkynyl, aryl and combinations thereof. Examples include benzyl, phenethyl, cyclohexylmethyl, adamantyl, camphoryl and naphthyl ethyl. Hydrocarbyl refers to any substituent comprised of hydrogen and carbon as the only elemental constituents. Aliphatic hydrocarbons are hydrocarbons that are not aromatic; they may be saturated or unsaturated, cyclic, linear or branched. Examples of aliphatic hydrocarbons include isopropyl, 2-butenyl, 2-butynyl, cyclopentyl, norbomyl, etc. Aromatic hydrocarbons include benzene (phenyl), naphthalene (naphthyl), anthracene, etc.
[0063] Unless otherwise specified, alkyl (or alkylene) is intended to include linear or branched saturated hydrocarbon structures and combinations thereof. Alkyl refers to alkyl groups from 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms.
Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, s-butyl, t-butyl and the like.
[0064] Cycloalkyl is a subset of hydrocarbon and includes cyclic hydrocarbon groups of from 3 to 8 carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, norbomyl and the like.
[0065] Unless otherwise specified, the term“carbocycle” is intended to include ring systems in which the ring atoms are all carbon but of any oxidation state. Thus (C3-C10) carbocycle refers to both non-aromatic and aromatic systems, including such systems as cyclopropane, benzene and cyclohexene; (Cs-Co) carbopolycycle refers to such systems as norbornane, decalin, indane and naphthalene. Carbocycle, if not otherwise limited, refers to monocycles, bicycles and polycycles.
[0066] Heterocycle means an aliphatic or aromatic carbocycle residue in which from one to four carbons is replaced by a heteroatom selected from the group consisting of N, O, and S. The nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. Unless otherwise specified, a heterocycle may be non-aromatic (heteroaliphatic) or aromatic (heteroaryl). Examples of heterocycles include pyrrolidine, pyrazole, pyrrole, indole, quinoline, isoquinoline, tetrahydroisoquinoline, benzofuran, benzodioxan, benzodioxole (commonly referred to as methylenedioxyphenyl, when occurring as a substituent), tetrazole, morpholine, piperazine, piperidine, thiazole, pyridine, pyridazine, pyrimidine, thiophene, furan, oxazole, oxazoline, isoxazole, dioxane, tetrahydrofuran and the like. Examples of heterocyclyl residues include piperazinyl, piperidinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyrazinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolyl, quinuclidinyl, isothiazolidinyl, benzimidazolyl, thiadiazolyl, benzopyranyl, benzothiazolyl, benzoxazolyl, tetrahydrofuryl, tetrahydropyranyl, thienyl (also historically called thiophenyl), benzothienyl, thiamorpholinyl, oxadiazolyl, triazolyl and tetrahydroquinolinyl.
[0067] Hydrocarbyloxy refers to groups of from 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms attached to the parent structure through an oxygen. Alkoxy is a subset of hydrocarbyloxy and includes groups of a straight or branched configuration. Examples include methoxy, ethoxy, propoxy, isopropoxy and the like. Lower- alkoxy refers to groups containing one to four carbons. The term "halogen" means fluorine, chlorine, bromine or iodine atoms.
[0068] Unless otherwise specified, acyl refers to formyl and to groups of 1, 2, 3, 4, 5, 6, 7 and 8 carbon atoms of a straight, branched, cyclic configuration, saturated, unsaturated and aromatic and combinations thereof, attached to the parent structure through a carbonyl functionality. Examples include acetyl, benzoyl, propionyl, isobutyryl and the like. Lower-acyl refers to groups containing one to four carbons. The double bonded oxygen, when referred to as a substituent itself is called“oxo”.
[0069] Oxaalkyl refers to alkyl residues in which one or more carbons (and their associated hydrogens) have been replaced by oxygen. Examples include methoxypropoxy, 3,6,9- trioxadecyl and the like. The term oxaalkyl is intended as it is understood in the art [see Naming and Indexing of Chemical Substances for Chemical Abstracts, published by the American Chemical Society, 196, but without the restriction of 127(a)], i.e. it refers to compounds in which the oxygen is bonded via a single bond to its adjacent atoms (forming ether bonds); it does not refer to doubly bonded oxygen, as would be found in carbonyl groups.
[0070] As used herein, the term“optionally substituted” may be used interchangeably with “unsubstituted or substituted”. The term“substituted” refers to the replacement of one or more hydrogen atoms in a specified group with a specified radical. For example, substituted alkyl, aryl, cycloalkyl, heterocyclyl etc. refer to alkyl, aryl, cycloalkyl, or heterocyclyl wherein one or more H atoms in each residue are replaced with halogen, haloalkyl, alkyl, acyl, alkoxyalkyl, hydroxy lower alkyl, carbonyl, phenyl, heteroaryl, benzenesulfonyl, hydroxy, lower alkoxy, haloalkoxy, oxaalkyl, carboxy, alkoxycarbonyl [-C(=0)0-alkyl], alkoxycarbonylamino [ HNC(=0)0-alkyl], aminocarbonyl (also known as carboxamido) [-C(=0)NH2],
alkylaminocarbonyl [-C(=0)NH-alkyl], cyano, acetoxy, nitro, amino, alkylamino, dialkylamino, (alkyl)(aryl)aminoalkyl, alkylaminoalkyl (including cycloalkylaminoalkyl), dialkylaminoalkyl, dialkylaminoalkoxy, heterocyclylalkoxy, mercapto, alkylthio, sulfoxide, sulfone, sulfonylamino, alkylsulfmyl, alkylsulfonyl, acyl ami noalkyl, acylaminoalkoxy, acylamino, amidino, aryl, benzyl, heterocyclyl, heterocyclylalkyl, phenoxy, benzyloxy, heteroaryl oxy, hydroxyimino, alkoxyimino, oxaalkyl, aminosulfonyl, trityl, amidino, guanidino, ureido, benzyloxyphenyl, and benzyloxy.“Oxo” is also included among the substituents referred to in“optionally substituted”; it will be appreciated by persons of skill in the art that, because oxo is a divalent radical, there are circumstances in which it will not be appropriate as a substituent (e.g. on phenyl), whereas on others (e.g. camphor) it will be appropriate. In one embodiment, 1, 2, or 3 hydrogen atoms are replaced with a specified radical. In the case of alkyl and cycloalkyl, more than three hydrogen atoms can be replaced by fluorine; indeed, all available hydrogen atoms could be replaced by fluorine. Preferred substituents are halogen, (Ci-C4)alkyl, (Ci-C4)alkoxy, (Ci-C4)fluoroalkyl, (Ci-C4)fluoroalkoxy, hydroxy, amino, (Ci-C4)alkylamino, di(Ci-C4)alkylamino, (Ci- C4)acylamino, (Ci-C4)fluoroalkyl and (Ci-C4)fluoroalkoxy.
[0071] Substituents Rn are generally defined when introduced and retain that definition throughout the specification and in all independent claims.
[0072] Preparation of compounds can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be readily determined by one skilled in the art. Suitable groups for that purpose are discussed in standard textbooks in the field of chemistry, such as Protective Groups in Organic Synthesis by T.W. Greene and P.G.M.Wuts [John Wiley & Sons, New York, 1999], in Protecting Group Chemistry , 1st Ed., Oxford University Press, 2000; and in March’s Advanced Organic chemistry: Reactions, Mechanisms, and Structure , 5th Ed., Wiley-Interscience
Publication, 2001.
[0073] The compounds described herein will, in some instances, particularly when R3 and R4 form a piperazine, piperidine, morpholine or pyrrolidine ring optionally substituted with (Ci- C4)alkyl, contain an asymmetric center and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms which may be defined in terms of absolute stereochemistry as (R)- or (S)-. The present invention is meant to include all such possible isomers as racemates, optically pure forms and intermediate mixtures. Optically active (R)- and (S)- isomers may be prepared using homo-chiral synthons or homo-chiral reagents, or optically resolved using conventional techniques. All tautomeric forms are intended to be included. The graphic representations of racemic, ambiscalemic and scalemic or enantiomerically pure compounds used herein are taken from Maehr T Chem. Ed. 62. 114-120 (1985): solid and broken wedges are used to denote the absolute configuration of a chiral element; wavy lines indicate disavowal of any stereochemical implication which the bond it represents could generate; solid and broken bold lines are geometric descriptors indicating the relative configuration shown but denoting racemic character; and wedge outlines and dotted or broken lines denote enantiomerically pure compounds of indeterminate or unconfirmed absolute configuration. The term "enantiomeric excess" is related to the older term "optical purity" in that both are measures of the same phenomenon. The value of ee will be a number from 0 to 100, zero being racemic and 100 being pure, single enantiomer. A compound which in the past might have been called 98% optically pure is now more precisely described as 96% ee.; in other words, a 90% e.e. reflects the presence of 95% of one enantiomer and 5% of the other in the material in question. When a single enantiomer is referred to, it will be presumed to be >90% ee.
[0074] As used herein, the terms“treatment” or“treating,” or“palliating” or“ameliorating” refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological systems associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder. For prophylactic benefit, the compositions may be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological systems of a disease, even though a diagnosis of this disease may not have been made. For example, treating osteoporosis may involve administering the compounds to a patient at risk of developing osteoporosis to diminish the likelihood and/or severity of the condition.
[0075] As used herein, and as would be understood by the person of skill in the art, the recitation of“a compound” - unless expressly further limited - is intended to include salts of that compound. Thus, for example, the recitation“a compound of the formula:
Figure imgf000016_0001
would include salts:
Figure imgf000016_0002
wherein X is any counterion. In a particular embodiment, the term“compound of formula” refers to the compound or a pharmaceutically acceptable salt thereof.
[0076] The term“pharmaceutically acceptable salt” refers to salts prepared from
pharmaceutically acceptable non-toxic acids or bases including inorganic acids and bases and organic acids and bases. When the compounds of the present invention are basic, salts may be prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids. Suitable pharmaceutically acceptable acid addition salts for the compounds of the present invention include acetic, adipic, alginic, ascorbic, aspartic, benzenesulfonic (besylate), benzoic, boric, butyric, camphoric, camphorsulfonic, carbonic, citric, ethanedisulfonic, ethanesulfonic, ethylenediaminetetraacetic, formic, fumaric, glucoheptonic, gluconic, glutamic, hydrobromic, hydrochloric, hydroiodic, hydroxynaphthoic, isethionic, lactic, lactobionic, laurylsulfonic, maleic, malic, mandelic, methanesulfonic, mucic, naphthylenesulfonic, nitric, oleic, pamoic, pantothenic, phosphoric, pivalic, polygalacturonic, salicylic, stearic, succinic, sulfuric, tannic, tartaric acid, teoclatic, p-toluenesulfonic, and the like. When the compounds contain an acidic side chain, suitable pharmaceutically acceptable base addition salts for the compounds of the present invention include, but are not limited to, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, arginine, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium cations and carboxylate, sulfonate and phosphonate anions attached to alkyl having from 1 to 20 carbon atoms.
[0077] Also provided herein is a pharmaceutical composition comprising a compound disclosed above, or a pharmaceutically acceptable salt form thereof, and a pharmaceutically acceptable carrier or diluent.
[0078] While it may be possible for the compounds of formula I to be administered as the raw chemical, it is preferable to present them as a pharmaceutical composition. According to a further aspect, the present invention provides a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically carriers thereof and optionally one or more other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
[0079] The formulations include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intraarticular), rectal and topical (including dermal, buccal, sublingual and intraocular) administration. The most suitable route may depend upon the condition and disorder of the recipient. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing into association a compound of formula I or a
pharmaceutically acceptable salt thereof ("active ingredient") with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both and then, if necessary, shaping the product into the desired formulation. [0080] Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be presented as a bolus, electuary or paste.
[0081] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, lubricating, surface active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide sustained, delayed or controlled release of the active ingredient therein.
[0082] Formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti -oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient. Formulations for parenteral administration also include aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The formulations may be presented in unit- dose of multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of a sterile liquid carrier, for example saline, phosphate-buffered saline (PBS) or the like, immediately prior to use.
Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
EXPERIMENTAL SECTION
[0083] Solvents were purchased from Sigma- Aldrich. Reactions were run in capped round bottom flasks or screw capped glass vials stirred with Teflon®-coated magnetic stir bars.
Evaporation of solvents was accomplished using a Biichi rotary evaporator, equipped with a dry ice-acetone condenser, at 5-50 mm Hg (25-50°C). Experiments were monitored by thin layer chromatography or liquid chromatography mass spectrometry. The maintenance of 40°C to 150°C reaction temperatures was accomplished by the use of an oil bath. Products obtained as solids or oils were dried under vacuum.
[0084] Compounds were purified using silica gel column chromatography, as necessary.
Analytical TLC was performed using Whatman 250 micron aluminum backed UV F254 precoated silica gel flexible plates. Subsequent to elution, ultraviolet illumination at 254 nm allowed for visualization of UV active materials. Staining with basic potassium permanganate solution or iodine vapors allowed for further visualization.
[0085] Proton nuclear magnetic resonance spectra (1H-NMR) were recorded on Bruker Avance DPX400, spectrometer operating at 400.13 MHz, carrying a BBFO probe, or a Bruker Avance II MHz spectrometer (600 MHz) with a 5 mm TXI probe. All observed proton absorptions are reported as 6 in units of parts per million (ppm) relative to tetramethylsilane (6 0.0) using the signal of tetramethylsilane itself or the residual solvent signal as an internal standard:
dimethylsulfoxide-d6 (6 2.50, quintet at 25°C. Multiplicities are reported as follows: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), dt (doublet of triplets), td (triplet of doublets), m (multiplet), or br. s. (broad signal). Coupling constants are reported as a J value in Hertz (Hz). The number of protons (n) for a given resonance is indicated by nH, and is based on spectral integration values. Liquid chromatography mass spectral analyses were obtained using a Waters Acquity H-Class UPLC/MS with QDa mass spectrometer. The system used a el
Photodiode Array Detector detector, and a Symmetry® C18 (3.5 micron) 2.1 x 50 mm column for separation (mobile phase for positive mode: solvent A: water with 0.1% formic acid, solvent B: acetonitrile with 0.1% formic acid). Values are reported in units of mass to charge (m/z).
[0086] The following abbreviations are used in this section: ACN = acetonitrile; DCM = dichloromethane; DIEA = diisopropylethylamine; EtOAc = ethyl acetate; eq = equivalents;
EtOH = ethanol; HC1 = hydrochloric acid; HEX = hexane; MeOH = methanol; MTBE = methyl tert-butyl ether; rt = room temperature; TFA = trifluoroacetic acid; THF = tetrahydrofuran.
[0087] A general synthetic scheme for the synthesis of substituted 2,4-dichloroquinazoline, which is the common starting material for most syntheses, is known in the art as shown below:
Figure imgf000020_0001
[0088] In general, synthetic procedure A was employed for the synthesis of compounds.
Synthetic Procedure A
Figure imgf000020_0002
Synthesis of Compound 3: [N2-benzyl-N4-(5-cyclopropyl-lH-pyrazol-3-yl)quinazoline-2,4- diamine].
Figure imgf000021_0001
To a stirring solution of 5-cyclopropyl-lH-pyrazol-3-amine (62 mg, 0.5mmol) and DIEA (1 eq) in ACN (2 mL) in a round bottom glass flask at rt, a solution of 2,4-dichloro-quinazoline (100 mg, 0.5mmol) and DIEA (1 eq) in ACN (2 mL) was added dropwise. After 48 hours an equal volume of water was added and the mixture stirred for 2 h at rt. The product was recovered by filtration and washed in a sequential manner with the following solvents: 1- cold ACN, 2- water: ACN (80:20), 3- HC1 in water (pH 3.0), 4- water: ACN (80:20), 5- sodium hydroxide in water (pH 10.0), 6- hexane. After drying, a solid was recovered: 2-chloro-N-(5-cyclopropyl-lH-pyrazol-3- yl)quinazolin-4-amine (102 mg, 72%). MS-ESI [M+l]: expected 285.7, found 286.4. Step 2. Without further purification, 2-chloro-N-(5-cyclopropyl-lH-pyrazol-3-yl)quinazolin-4-amine from step 1 (40 mg, 0.13 mmol) was dissolved in n-butanol (1 mL), and a solution of benzylamine (1.5 eq) in n-butanol (0.5 mL) was added to the mixture. 37% HC1 in water (10 pL) was added and the reaction was heated to 110°C while stirring. Once starting material was consumed (about 3 h), the mixture was cooled to rt and hexane (0.5 mL) was added. The precipitated product was recovered by filtration, and washed in a sequential manner with the following solvents: 1- hexane, 2- diluted aqueous sodium hydroxide (pH 9.0), 3- diluted HC1 in water (pH 2.5), 4- MeOH, and then dried to obtain compound 3 (35 mg, 73%) as a hydrochloride salt. ¾ NMR (400 MHz, DMSO-d6) 6 13.30 (s, 1H), 12.60 (s, 1H), 11.45 (s, 1H), 8.75 (s, 1H), 8.63 (d, J = 8.2 Hz, 1H), 7.83 (t, J = 7.7 Hz, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.46 - 7.27 (m, 5H), 6.14 (s, 1H), 1.83 (s, 1H), 0.88 (d, J = 7.9 Hz, 2H), 0.77 (s, 1H), 0.47 (s, 1H). MS-ESI [M+l]: expected 357.4, found 357.2.
Synthesis of compound 5: [(R)-N-(5-(tert-butyl)-lH-pyrazol-3-yl)-2-(2-methyl-piperazin-l- yl)quinazolin-4-amine] .
Figure imgf000022_0001
To a stirring solution of 5-(tert-butyl)-lH-pyrazol-3-amine (80 mg, 0.57 mmol) and DIEA (1 eq) in ACN (2.5 mL) in a round bottom glass flask at rt, a solution of 2,4-dichloro-quinazoline (100 mg, 0.5 mmol) and DIEA (1 eq) in ACN (2 mL) was added dropwise and the mixture was stirred for 36 h. Then, an equal volume of distilled water was added while stirring at rt. After 30 min, a precipitate was collected, filtered and washed in a sequential manner with the following solvents: 1- EhCfACN (50:50), 2- sodium hydroxide in water (pH 10), 3- H20:ACN (50:50), and finally dried to obtain N-(5-(tert-butyl)-lH-pyrazol-3-yl)-2-chloroquinazolin-4-amine. MS-ESI [M+l]: expected 302.8, found 302.2. Step 2. Without further purification, 50 mg of this intermediate (0.17 mmol) were dissolved in dry n-butanol (2 mL) and 10 pL of 37% HC1 were added while stirring at rt. Then, tert-butyl (R)-3-methylpiperazine-l-carboxylate (0.25 mmol, 1.5 eq) was dissolved in n-butanol (0.5 mL) and added to the mixture. The reaction was heated to 100°C for 5 h while stirring and then cooled down to rt. The excess solvent was evaporated under reduced pressure, and the product of the reaction was washed with hexane, and dried. The solid residue was washed with HC1 in water (pH 3) for 15 min while stirring. A precipitate was collected, dissolved in a mixture of DCM:TFA:H20 (5:4.5:0.5) and stirred for 1 h at rt. The solvent was evaporated under reduced pressure and the product washed with MTBE twice. A precipitate was collected, dissolved in aqueous sodium hydroxide (pH 12) and extracted with EtOAc. The organic solution was washed with brine, dried over MgSCE, and filtered. The volume of EtOAc solution containing the desired product was reduced to 1.0 mL and the product precipitated by adding 20 pL of concentrated HC1 (37%), washed once with EtOAc, once with acetone, and finally dried to obtain compound 6 as a hydrochloride salt, in a 21% total yield. ¾ NMR (400 MHz, DMSO-76) d 13.36 (s, 1H), 11.54 (s, 1H), 10.04 (d, J= 10.5 Hz, 1H), 9.73 (d, 7= 11.0 Hz, 1H), 8.66 (d, J= 8.3 Hz, 1H), 8.26 (d, J= 8.4 Hz, 1H), 7.87 (t, J= 7.8 Hz, 1H), 7.49 (t, J= 7.7 Hz, 1H), 6.45 (s, 1H), 5.19 (s, 1H), 4.92 (d, J= 14.6 Hz, 1H), 3.63 (t, J= 13.5 Hz, 1H), 3.33 (d, J = 15.5 Hz, 3H), 3.13 (d, = 11.3 Hz, 1H), 1.50 (d, J= 7.0 Hz, 3H), 1.32 (s, 9H). MS-ESI [M+l]: expected for C20H27N7365.5, found 366.
Synthesis of compound 6: [(R)-N-(5-(tert-butyl)-lH-pyrazol-3-yl)-2-(2-methyl-piperazin-l-yl)- 7H-pyrrolo[2,3-d]pyrimidin-4-amine].
Figure imgf000023_0001
To a stirring solution of 5-(tert-butyl)-lH-pyrazol-3-amine (80 mg, 0.57 mmol) in ethanol (2.5 mL) and 5 M sodium hydroxide (0.05 mL) in a round bottom glass flask, a solution of 2,4- dichloro-7H-pyrrolo[2,3-d]pyrimidine (100 mg, 0.53 mmol) in dry ethanol (2 mL) was added. The mixture was heated at reflux for 12 h while stirring. Then, the solvent was evaporated under reduced pressure and the residue was washed several times with diluted HC1 in water (pH 3.0), and then with diluted sodium hydroxide in water (pH 10). The solid was dried, dissolved in ethyl acetate and purified using flash silica gel chromatography, using a gradient of HEX:EtOAc to obtain N-(5-(tert-butyl)-lH-pyrazol-3-yl)-2-chloro-7H-pyrrolo[2,3-d]pyrimidin-4-amine. MS- ESI [M+l]: expected 290.8, found 291.4. 50 mg of the intermediate from above (-0.17 mmol) was dissolved in dry n-butanol (2 mL). Tert-butyl (R)-3-methylpiperazine-l-carboxylate (-2 eq) and 10 mΐ of DIEA were added, and the reaction heated at 110°C in a closed vessel while stirring until consumption of starting material (3-4 h). The solvent was evaporated and the solid residue was washed with diluted HC1 in water (pH 2.0) once, and extracted with DCM. The organic solution was washed with diluted sodium hydroxide in water (pH 12), brine, dried over MgSCL, and filtered. Then, the solvent was evaporated, and the residue was dissolved in a mixture of DCM:TFA:H20 (5:4.5:0.5) while stirring at rt for lh. The solvent was evaporated under a nitrogen current and the product washed with diethyl ether twice and precipitated in diethyl ether overnight at rt. A solid was collected by centrifugation, dissolved in diluted sodium hydroxide in water (pH 12), and extracted with EtOAc. The organic layer containing the desired product was washed with brine, dried over MgS04, and filtered. After evaporation of the solvent under reduced pressure, the residue was dried to afford (R)-N-(5-(tert-butyl)-lH-pyrazol-3-yl)-2-(2- methylpiperazin-l-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine as free base. ¾ NMR (600 MHz, DMSO-d6) 5 11.93 (s, 1H), 10.91 (s, 1H), 9.50 (s, 1H), 6.63 (m, 3H), 4.46 (dd, J = 33.1, 11.8 Hz, 2H), 2.92 (d, J = 10.8 Hz, 1H), 2.71 (dt, J = 30.1, 10.2 Hz, 3H), 2.40 (t, J = 11.2 Hz, 1H), 1.30 (s, 9H), 1.02 (d, J = 6.0 Hz, 3H). MS-ESI [M+l]: expected for CisHieNs 354.5, found 355.
Synthesis of compound 7:
Figure imgf000024_0001
To a stirring solution of 2,4-dichloro-7H-pyrrolo[2,3-d]pyrimidine (300 mg, -1.6 mmol) in DCM (10 mL) at rt, a solution of tosyl chloride (1.2 eq) was added dropwise, and the mixture was stirred for 2 hrs. The crude reaction was washed with 5% NaHC03, extracted with EtOAc, dried over Mg2S04, and purified with silica gel chromatography in a gradient of Hexane :EtO Ac to obtain 2,4-dichloro-7-tosyl-7H-pyrrolo[2,3-d]pyrimidine. To a stirring solution of 2,4- dichloro-7-tosyl-7H-pyrrolo[2,3-d]pyrimidine (200 mg, -0.54 mmol) in tert-butanol (5 mL) a solution of 5-(tert-butyl)-lH-pyrazol-3-amine (1.2 eq) in isopropanol was added, followed by DIEA (2.5 eq), and the solution was heated at 60 °C for 3 hrs while stirring. The solvent was evaporated and the crude residue was purified by silica gel chromatography in a gradient of Hexane:EtOAc to obtain N-(5-(tert-butyl)-lH-pyrazol-3-yl)-2-chloro-7-tosyl-7H-pyrrolo[2,3- d]pyrimidin-4-amine. 100 mg of the N-(5-(tert-butyl)-lH-pyrazol-3-yl)-2-chloro-7-tosyl-7H- pyrrolo[2,3-d]pyrimidin-4-amine was dissolved in n-butanol (2 mL). Tert-butyl (R)-2- isopropylpiperazine-l-carboxylate (~2 eq) and 15 mΐ of DIEA were added, and the reaction heated at 110°C while stirring, until consumption of starting material (3-4 h). The solvent was evaporated and the residue was purified with silica gel chromatography to afford tert-butyl (R)- 4-(4-((5-(tert-butyl)-lH-pyrazol-3-yl)amino)-7-tosyl-7H-pyrrolo[2,3-d]pyrimidin-2-yl)-2- isopropylpiperazine-l-carboxylate. The intermediate from step 3 was first treated with a mixture of TFA:DCM:H20 (4.5:5:0.5; 3 mL) for 30 min. The solvent was evaporated and the residue was triturated with diehtyl ether and filtered to recovered an off-white precipitate. The precipitate was then treated with 0.1 M KOH in MeOLfLhO (7:3; 5 mL) for 1 h at 40 °C. The crude was extracted with EtOAc, dried over MgiSCL and purified using preparative TLC, using neutral alumina with DGVTMeOH as solvent (9: 1), to obtain (R)-N-(5-(tert-butyl)-lH-pyrazol-3-yl)-2- (3-isopropylpiperazin-l-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine, compound 7. 1H NMR (600 MHz, DMSO-d6) d 11.93 (s, 1H), 10.91 (s, 1H), 9.45 (s, 1H), 6.64 (m, 3H), 4.49 (dd, J = 65.1, 12.3 Hz, 2H), 4.04 (q, J = 7.1 Hz, 1H), 3.61 (m, 1H), 2.72 (m, 1H), 1.77 (m, 1H), 1.62 (m, 1H), 1.29 (s, 9H), 0.96 (dd, J = 2.3, 4.7 Hz). MS-ESI [M+l]: expected for C20H30N8 382.5, found 383.5. 13C NMR (600 MHz, DMSO-d6) d 159.44, 153.61, 152.50, 152.38, 148.65, 118.37, 100.01, 99.76, 96.92, 60.67, 48.61, 45.95, 45.61, 31.45, 31.19, 30.46, 19.70, 19.42.
[0089] The following compounds in Table A were synthesized via appropriate reagents using conditions as in the examples described above.
Table A.
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
[0090] The following additional compounds were made by the methods described above:
Figure imgf000030_0001
Figure imgf000031_0001
[0091] Protein expression. All recombinant proteins were expressed in Escherichia coli Rosetta cells (Stratagene) grown in LB medium. Protein expression was induced at O.D.600= 0.6-0.8 with 0.5 mM IPTG (Goldbio), and the cultures were grown at 16 °C for 12-16 h.
[0092] Common procedure for protein purification. All purification steps were performed at 4°C ice. Protein concentration was estimated using a Bradford assay (BioRad), and bovine serum albumin (BSA) standards. Molecular weight and purity of the recombinant protein samples were analyzed by SDS-PAGE, followed by staining with Coomassie blue (BioRad). Specific buffers and purification conditions were optimized in order to obtain higher protein yield and higher protein specific activity.
[0093] Briefly, cells expressing recombinant proteins were collected by centrifugation, resuspended in the protein-specific lysis buffer (Buffer A), and lysed with an Emulsiflex-C5 homogenizer (Avestin) through 5-6 cycles at 10,000-15,000 psi. The homogenized lysate was centrifuged at 40,000 rpm for 45 to 60 min using a Ti45 rotor in a Beckman Coulter Optima LE- 80K ultracentrifuge. All recombinant proteins were purified using multi-step strategies, which involved combinations of affinity, ion exchange and size exclusion chromatography. After incubation with clarified cell homogenate, the affinity matrices were washed with at least 50 volumes of washing buffers (Buffer B). For ion exchange chromatography, proteins were loaded onto specific columns (see below), which were pre-equilibrated in low-salt buffers (Buffers C), and eluted in a gradient ofhigh-salt buffers (Buffers D). The fractions from these columns that were enriched in the protein of interest were pooled, concentrated using an appropriate (see below) size Amicon® Ultra centrifugal filter (Millipore), filtered through a 0.22-pm Millex-GP PES membrane (Millipore SLGP033RS), and injected onto size-exclusion chromatography columns equilibrated in Buffers E. Fractions containing the protein of interest were recovered, pooled and concentrated to a minimum of 1.0 mg/mL. The concentrated protein samples were recovered in 1.5 ml eppendorf tubes, glycerol was added to a final 15% (v/v), and the solutions centrifuged 20,000 for 10 min. The supernatant was collected and snap-frozen in liquid nitrogen and stored at -80 °C. The buffers used are listed below the specific protein purification procedure.
[0094] Spastin (wild-type and mutants). For purification of wild type and mutant spastin proteins a previously published procedure (Ziolkowska and Roll-Mecak“In Vitro Microtubule Severing Assays” Methods in Molecular Biology 1046, 323-334 (2013)) was modified. Briefly, the clarified lysate containing GST-spastin was incubated with a GSTrap 4B matrix (GE Healthcare) and eluted in Buffer B supplemented with 20 mM reduced glutathione. The protein solution was incubated with PreScission protease (0.1 mg/mL) for 8-12 h, and diluted 1 :2.5 with Buffer C and loaded into a CaptoS cation exchange column (GE Healthcare) equilibrated in 95% Buffer C and 5% Buffer B. Fractions eluted from the ion exchange chromatography column were pooled, concentrated 10-fold with an Amicon Ultra 30K MWCO centrifugal filter, and further purified over a 10/300 Superdex 200 column (GE Healthcare) in Buffer E. The eluate containing purified spastin was pooled, concentrated using an Amicon Ultra 3 OK MWCO centrifugal filter to at least 1 mg/mL and stored at -80 °C.
[0095] Spastin buffers. Buffer A: 50 mM K-HEPES, 150 mM KC1, 5 mM MgCh, 5 mM dithiothreitol (DTT, Goldbio), 0.5 mM phenylmethanesulfonyl fluoride (PMSF), 5 U/mL benzonase, and complete EDTA-free protease inhibitors (Roche), pH 7.5. Buffer B: 50 mM K- HEPES, 400 mM KC1, 5 mM MgCh, 5 mM DTT, 0.1 mM PMSF, pH 7.5. Buffer C: 25 mM Na- MES, 5% (w/v) glycerol, 5 mM MgCh, 5 mM DTT, pH 6.5. Buffer D: 25 mM Na-MES, 2 M NaCl, 5% (w/v) glycerol, 5 mM MgCh, 5 DTT, pH 6.5. Buffer E: 25 mM K-HEPES, 300 mM KC1, 10% (w/v) glycerol, 5 mM MgCh, 5 mM DTT, pH 7.5.
[0096] GST-FIGL1 (aa 295-674). The clarified lysate from cells expressing GST-FIGL1 was incubated with sepharose glutathione beads (GE Healthcare) for lh and eluted using Buffer B supplemented with 10 mM reduced glutathione. Fractions containing the proteins were pooled, diluted with 3 volumes of Buffer C and purified using a 16/60 MonoQ GL column (GE
Healthcare). The protein fractions were pooled, concentrated 10-fold using an Amicon® Ultra 50K MWCO centrifugal filter, and further purified over a 10/300 Superdex 200 column (GE Healthcare) in Buffer E. Fractions from size-exclusion column were concentrated to a minimum of 1 mg/mL using an Amicon® Ultra 30K MWCO centrifugal filter, and stored.
[0097] GST-FIGL1 buffers. Buffer A: 50 mM Tris-HCl, 150 mM NaCl, 5 mM MgCh, 10 mM DTT, 0.5 mM PMSF, 0.01% (v/v) triton X-100, 5 U/mL benzonase, and complete EDTA-free protease inhibitors (Roche), pH 7.5. Buffer B: 50 mM Tris-HCl, 300 mM NaCl, 5 mM MgCh, 10 mM DTT, 0.1 mM PMSF, pH 7.5. Buffer C: 50 mM Tris-HCl, 75 mM NaCl, 5 mM MgCh, 5 mM DTT, pH 8.5. Buffer D: 50 mM Tris-HCl, 1 M NaCl, 5 mM MgCh, 5 mM DTT, pH 8.5. Buffer E: 20 mM Tris-HCl, 150 mM NaCl, 5 mM MgCh, 5 mM DTT, 5% (w/v) glycerol, pH 7.5.
[0098] MBP-Katanin. The clarified lysate from cells expressing MBP-katanin was filtered through a 0.22-pm Millex-GP PES membrane and loaded onto MBP trap column (GE
Healthcare). The column was washed with 30 column volumes of Buffer B and eluted with a Buffer B supplemented with 10 mM maltose. The eluate was diluted 1 :2 with Buffer C and loaded on a 16/60 MonoQ GL ion exchange column and eluted with a gradient of Buffer D. Combined MonoQ fractions were further purified on a 10/300 Superdex 200 gel filtration column in Buffer E. The fractions containing purified MBP-katanin protein were frozen in liquid nitrogen and stored at -80°C.
[0099] MBP-katanin buffers. Buffer A: 50 mM Tris-HCl, 250 mM NaCl, 5 mM MgCh, 100 pM ATP (Sigma, 2383), 5 mM DTT, 1 mM PMSF and complete EDTA-free protease inhibitors (Roche), pH 8.0. Buffer B: 50 mM Tris-HCl, 250 mM NaCl, 5 mM MgCh, 100 pM ATP, 5 mM DTT, pH 7.5. Buffer C: 20 mM Tris-HCl, 100 mM NaCl, 1 mM MgCh, 5 mM DTT, 100 pM ATP, pH 7.5. Buffer D: 20 mM Tris-HCl, 500 mM NaCl, 1 mM MgCh, 5 mM DTT, 100 pM ATP, pH 7.5. Buffer E: 20 mM K-HEPES, 250 mM NaCl, 5 mM MgCh, 5 mM DTT, and 10% (w/v) glycerol, pH 7.5.
[0100] VCP/p97. The clarified lysate from cells expressing His6-VCP/p97 was incubated with Ni-NTA beads (Qiagen) for 40 min, and the beads were extensively washed using Buffer B. The protein was eluted with Buffer B supplemented with 300 mM imidazole, diluted 1 :2 in Buffer C, loaded onto a Mono Q column 5/50 GL (GE Healthcare) and fractioned over a gradient with Buffer D. Protein was eluted at approximately 350 mM NaCl. Combined MonoQ fractions were further concentrated using an Amicon Ultra 50K MWCO centrifugal filter, and the concentrated protein sample was filtered though a 0.22pm Millex-GP PES membrane and loaded on a 10/300 Superdex 200 column (GE Healthcare) in Buffer E. Fractions from size-exclusion column were concentrated to a minimum of 1 mg/ml using an Amicon® Ultra 50K MWCO centrifugal filter and concentrated protein sample was stored at -80 °C.
[0101] VCP/p97 buffers. Buffer A: 50 mM Tris-HCl, 250 mM NaCl, 20 mM imidazole, 5 mM MgCh, 1 mM //7.s(2-carboxyethyl (phosphine (TCEP, Soltec Ventures), 0.5 mM ATP, 5 U/mL benzonase, and complete EDTA-free protease inhibitors (Roche), pH 8.0. Buffer B: 50 mM Tris- HCl, 400 mM NaCl, 20 mM imidazole, 5 mM MgCh, 1 mM TCEP, pH 7.5. Buffer C: 50 mM K- HEPES, 150 mM NaCl, 5 mM MgCh, 2 mM TCEP, pH 7.5. Buffer D: 50 mM K-HEPES, 1 M NaCl, 5 mM MgCh, 2 mM TCEP, pH 7.5. Buffer E: 25 mM K-Hepes 150 mM NaCl, 5 mM MgCh, 2 mM TCEP, 5% (w/v) glycerol, pH 7.5.
[0102] PCH2. The clarified lysate from cells expressing GST-PCH2 was incubated with sepharose glutathione beads (GE Healthcare) for 1 h and the beads were washed with 50 volumes of Buffer B and then with 5 volumes of Buffer A without complete EDTA-free protease inhibitor cocktail. PreScission protease was diluted to 0.1 mg/mL in a buffer containing 50 mM Tris-HCl, 250 mM NaCl, 5 mM MgCh, 10 mM DTT, pH 8.0, 0.1 mM PMSF, 10 pM reduced glutathione and the beads were incubated with this solution for 12 h.
The protein-containing solution was recovered by filtration and incubated with fresh sepharose glutathione beads for 1 h. Beads were removed by centrifugation at 300g-, 5 min twice and the solution was concentrated 20-fold using an Amicon® Ultra 30K MWCO centrifugal filter, filtered through a 0.22-mih Millex-GP PES membrane and further purified over a 10/300 Superdex 75 column (GE Healthcare) in Buffer E. Fractions containing purified protein from the size-exclusion column were concentrated to a minimum of 1 mg/mL using an Amicon® Ultra 30K MWCO centrifugal filter and stored at -80 °C.
[0103] PCH2 buffers. Buffer A: 50 mM Tris-HCl, 250 mM NaCl, 5 mM MgCh, 10 mM DTT,
0.5 mM PMSF, 0.005% (v/v) triton X-100, MgATP 0.5 mM, 5 U/ml benzonase, and complete EDTA-free protease inhibitors (Roche), pH 7.5. Buffer B: 50 mM Tris-HCl, 250 mM NaCl, 5 mM MgCh, 10 mM DTT, pH 8.0, 0.1 mM PMSF, 10 pM reduced glutathione, pH 8.0. Buffer E: 50 mM Tris-HCl, 150 mM NaCl, 5 mM MgCh, 5 mM DTT, 10 % (w/v) glycerol, pH 8.0.
[0104] ATPase activity of AAA+ proteins was determined in the steady-state conditions using an enzymatic ATP-regeneration system. This system is coupled to the oxidation of NADH, which is stoichiometric to the hydrolysis of ATP, and can be followed by monitoring the decrease in the fluorescence signal at 440 nm. This same assay was employed to measure variation of the ATPase rate at different ATP concentrations as well as inhibition of ATPase activity at different compound concentrations. Briefly, the protein sample was diluted in assay buffer, and NADH (Sigma, N7410) was added to final concentration of 200-175 mM, (in the case of FIGL1, spastin, and katanin) or 100150 mM (in the case of VCP/p97 and PCH2). Then, phosphoenol pyruvic acid monopotassium salt (Sigma, P7127), D-lactic dehydrogenase (Sigma, L3888) and pyruvate kinase (lyophilized powder, Sigma, P9136) were added to a final concentration of 1 mM, 40 U/mL and 40 U/mL, respectively for measuring ATPase rate, or 1 mM, 30 U/mL and 30 U/mL, respectively for compound testing.
[0105] In the case of ATPase rate measurement at different ATP concentrations, 27 pL of these mixtures were added to single wells in a flat bottom 384 well black polystyrene plate (Greiner Bio One, Wemmel, Belgium, catalog # 781090), and the reaction was initiated by adding 3 pL of 10X MgATP solutions in ddH20 (pH 7.0, 0.027 - 3 mM final). The final volume of these steady- state ATPase reactions was 30 pL.
[0106] Compounds were typically tested in a high-throughput assay format. Briefly, in a flat bottom 384 well black polystyrene plate (Greiner Bio One, Wemmel, Belgium, catalog # 781090) 11.75 pL of the appropriate assay buffer, depending of the AAA+ protein to be tested, was dispensed. Compounds were dispensed into the assay plates as DMSO solutions (0.25 pL) using a Janus 384 MDT NanoHead (Perkin Elmer). For collection of structural -activity relationship data, 0.25 pL of a 0.96 mM (10 pM final) or a 0.192 mM (2 pM final) solution of the compounds was dispensed and the liquid was collected at the bottom of each well by
centrifugation for 30 s at 180g. For the concentration-response analysis 11 serial dilutions of the compounds in DMSO from 3 mM to 0.31 mM were added using the same procedure. Then, 6 pL of the mixtures containing diluted protein samples, NADH, phosphoenol pyruvic acid monopotassium salt, D-lactic dehydrogenase, and pyruvate kinase, at the appropriate
concentrations, was added to the assay wells. The liquid was collected at the bottom of the well by centrifugation for 30 s at 180g and reactions were initiated by adding 6 pL of 2 mM MgATP in ddH20 (pH 7.0) to each well (0.5 mM final). The final volume of the steady-state ATPase reactions was 24 pL for compound testing.
[0107] Assay buffer conditions for the different AAA+ proteins were established (FIGL1), or adapted (spastin, VCP/p97, PCH2, katanin) from conditions reported in previous studies to optimize enzymes’ specific activities. The final concentration of proteins was wild-type spastin 80-100 nM, katanin 80 nM, FIGL1 50 nM, VCP/p97 450 nM and PCH2 340 nM, for measuring ATPase rate at different ATP concentrations. Protein concentrations were chosen to allow for robust measurement of enzymatic rate over a range of MgATP concentrations (0.027 - 3 mM) during a maximum assay time window of 90 min. For compound testing, which is carried out at a fixed MgATP concentration of 0.5 mM, minimal adjustment of these concentrations was employed (spastin 60-90 nM, FIGL1 75 nM, Katanin 75 nM, VCP/p97 430 nM, and PCH2 270 nM).
[0108] To dilute the protein sample for the measurement of the ATPase rate at different ATP concentrations the following buffers were used:
[0109] Spastin wild type and mutants: 25 mM K-HEPES, 225 mM KC1, 25 mM K2HPO4, 5 mM MgCh, 2.5 mM DTT, 0.1 mg/mL BSA, pH 7.5, 0.05% v/v triton X-100. Katanin: 25 mM K- HEPES, 70 mM KC1, 25 mM K2HP04, 5 mM MgCh, 2.5 mM DTT, 0.1 mg/mL BSA, pH 7.5. FIGL1 : 25 mM Na-MES, 70 mM KOAc, 25 mM K2HP04, 5 mM Mg(OAc)2, 1 mM TCEP, 0.1 mg/ml BSA, pH 6.5. VCP/p97: 50 mM K-HEPES, 25 mM KC1, 25 mM K2HP04, 15 mM MgCh, 1 mM TCEP, pH 7.5. PCH2: 25 mM TRIS-HCl, 150 mM KC1, 25 mM K2HP04, 5 mM MgCh, 2.5 mM DTT, 0.1 mg/mL BSA, 0.025% (v/v) triton-X100, pH 8.5.
[0110] Since we observed that in buffers containing 25 mM K2HP04 a fine white precipitate formed after prolonged incubation at rt when pH>7.5, we used (NH4)2S04 (20 mM) as substitute for K2HP04 in compound inhibition assays, which required longer times. When compounds were tested, 0.005% (v/v) triton-X100 was included to the buffers, if not present already, to prevent aggregation of compounds during the assay.
[0111] For the measurement of the ATPase rates and the apparent enzymatic parameters of spastin in the presence of different concentrations of compound, recombinant spastin (85 nM) was diluted in a buffer containing 25 mM K-HEPES, 225 mM KC1, 25 mM K2HP04, 5 mM MgCh, 2.5 mM DTT, 0.1 mg/mL bovine serum albumin, and 2 to 0.02 mM of compound 5 (1% final DMSO concentration). ATPase reactions were initiated by adding MgATP (0.044 to 3 mM). For concentrations of compound equal to 2 pM the enzymatic parameters could not be calculated.
[0112] For all analyses, the time course of fluorescence decrease was measured using a Synergy NEO Microplate Reader (lec = 340 nm, 440 nm emission filter). The fluorescence values were plotted against time and fit by linear regression to obtain a rate of fluorescence decrease. The rate from a control reaction with no ATP (background rate of fluorescence decrease) was subtracted from all rates. The ATPase rate was calculated from the background corrected rates of fluorescence decrease using a calibration curve relating the presence of ADP to the NADH fluorescence signal decrease, which is mediated by the enzymatic ATP regeneration system. Percent inhibition of the ATPase activity was calculated by normalizing the rate of fluorescence decrease in the presence of the compounds to DMSO control.
[0113] Enzyme parameters Ki/2, kcat and Hill coefficients (h) for the recombinant enzymes were determined by fitting the rates at each ATP concentration (n=3 independent measurements) to the equation 1 (Hill equation) using Prism v. 6.0 (GraphPad Software Inc), and then averaging the fitting values. V = ATPase rate
Figure imgf000038_0001
Vmax represents the maximum ATPase rate and x represents the ATP concentration.
Analysis of concentration-response was performed as follows. For each experiment percent inhibition versus concentration of compound were plotted and data were fit using a sigmoidal dose-response curve model and equation (2) in Prism to find the IC50. x is the compound concentration, 'max' reflects the maximum percent activity, and 'min' the minimum percent activity as fit by the data. Three independent experiments were performed for each condition.
(Y max - Y min}
V ~ % ATPase rata relative to D S0 control“ (Y min) * - ;— h
1 * IQ f-AAx
[0114] Results are shown in Table 1 in which +++ indicates ATPase activity of 0-10% of
Figure imgf000038_0002
control; ++ indicates ATPase activity of 11-40% of control; + indicates ATPase activity of 41-70% of control; and - indicates ATPase activity of 71-100% of control. The data represented is for compound concentrations of 10 mM.
TABLE 1
Figure imgf000038_0003
Figure imgf000039_0001
n.d. = not determined
[0115] The compounds provided herein that are selective for spastin can be used for
neurodegenerative diseases such as Alzheimer’s disease. The compounds that are selective for VCP/97 can be used as anticancer agents.
[0116] Compounds provided herein have been shown to inhibit tropomyosin receptor kinase A, tropomyosin receptor kinase B , and tropomyosin receptor kinase C .
[0117] In vitro kinase testing was performed by Thermofisher (SelectScreen) using the Z’-LYTE assay. The Z’-LYTE biochemical assay employs a fluorescence-based, coupled-enzyme format and is based on the differential sensitivity of phosphorylated and non-phosphorylated peptides to proteolytic cleavage. The peptide substrate is labeled with two fluorophores— one at each end— that make up a FRET pair. In the primary reaction, the kinase transfers the gamma-phosphate of ATP to a single tyrosine, serine or threonine residue in a synthetic FRET-peptide. In the secondary reaction, a site-specific protease recognizes and cleaves non-phosphorylated FRET- peptides. Phosphorylation of FRET-peptides suppresses cleavage by the ThermoFisher proprietary development Reagent. Cleavage disrupts FRET between the donor (i.e., coumarin) and acceptor (i.e., fluorescein) fluorophores on the FRET-peptide, whereas uncleaved, phosphorylated FRET-peptides maintain FRET. A ratiometric method, which calculates the ratio (the Emission Ratio) of donor emission to acceptor emission after excitation of the donor fluorophore at 400 nm, is used to quantitate reaction progress, according to the equation:
Emission Ratio = (Coumarin Emission, 445nm) / (Fluorescein Emission, 520 nm).
[0118] Compounds were screened in 1% DMSO (final). All peptide/kinase mixtures were diluted to a 2X working concentration in a buffer containing 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgC12, 1 mM EGTA. All ATP solutions were diluted to a 4X working concentration in the same buffer. Assays were conducted in a black 384-well plate (Coming Cat. #4514). Assay reagent were added in the following order: 1. 100 nL of test compound 100X in 100% DMSO; 2. 2.4 uL of buffer; 3. 5 uL of 2X peptide/kinase mixture; 4. 2.5 uL 4X ATP solution. Plates were shaken for 30 seconds and incubated for 60 minutes at room temperature. Next, 5 uL of development reagent solution was added, plate were shaken for 30 seconds and incubated for 60 minutes at room temperature, after which fluorescence was read.
[0119] Data were analyzed as follows: the maximum Emission Ratio is established by the 0% phosphorylation control (100% Inhibition Control), which contains no ATP and therefore exhibits no kinase activity. This control yields 100% cleaved peptide in the Development Reaction. The 100% Phosphorylation Control, which consists of a synthetically phosphorylated peptide of the same sequence as the peptide substrate, is designed to allow for the calculation of percent phosphorylation. This control yields a very low percentage of cleaved peptide in the Development Reaction. The minimum Emission Ratio in a screen is established by the 0% Inhibition Control, which contains active kinase. This control is designed to produce a 10-50% phosphorylated peptide in the kinase reaction.
[0120] Percent phosphorylation (% Phos) is calculated according to the equation: ( l-[(Emission Ratio x F ioo%) - Cioo%] / (Co%- Cioo%) + [Emission Ratio x (Fioo%- Fo%)] } *100. Where Cioo% = average Coumarin emission signal of 100% phosphorylation control; Co% = average Coumarin emission signal of the 0% phosphorylation control; Fioo% = average fluorescein emission signal of 100% phosphorylation control; Fo% = average fluorescein emission signal of 0%
phosphorylation control. Percent inhibition were calculated according to the equation: %
Inhibition = { !- (% Phos the sample / % Phos of the 0% inhibition control)} *100. [0121] Table 2 shows four examples of TrkA-C inhibitors. Compounds that are selective for TrkA-C inhibition can be used for treating solid tumors. In particular, those that have a neurotrophic receptor tyrosine kinase gene fusion.
TABLE 2
Figure imgf000041_0001
[0122] Compound 7 has exhibited an EC50 of 0.33 mM in the following TrkA cell proliferation assay:
[0123] Inhibition of KM12 cell proliferation was measured using the CellTiter-Glo®
luminescence-based assay (Promega Cat# G7570). Briefly, KM12 cells were grown in assay media (RPMI1640 supplemented with 10% FBS, 2mM glutamine and 50 U/ml
Penicillin/Streptomycin (GIbco cat# 15070063)). 30 uL of assay media containing 4000-5000 cells were layered in a white 384-well plate (Greiner Cat# 781075). After 12-16 hours, compound dilutions were prepared in 100% DMSO and added to the assay media (1% DMSO,
40 uM as the highest concentration) and 10 uL of this solution was added to the wells containing the cells. After 72 hours, 25 uL of CellTiter-Glo® was added to the wells, plates were shaken for 20 minutes at room temperature and the luminescence signal was measured. The average luminescence value corresponding to no inhibition of cell growth (100% control) or complete inhibition of cell growth (0% control) were measured from wells in which cells were incubated with DMSO alone, or 0.1% sodium dodecyl sulfate, respectively. Percent growth inhibition of cell proliferation was calculated according to the equation: [(luminescence of the sample - 0% control) / (100% control - 0% control)] x 100.
[0124]

Claims

1. A compound of formula
Figure imgf000042_0001
wherein
A is a five- or six-membered heteroaryl ring, or, a six-membered aryl ring;
Y is carbon or nitrogen;
R1 and R2 are chosen independently from hydrogen, halogen, halo(Ci-C4)alkyl, (Ci- C4)alkyl, (Ci-C4)acyl, hydroxy(Ci-C4)alkyl, hydroxy, (Ci-C4)alkoxy, halo(Ci-C4)alkoxy, carboxy, (Ci-C4)alkoxycarbonyl, carboxamido, cyano, acetoxy, nitro, amino, (Ci-C4)alkylamino, di(Ci-C4)alkylamino, (Ci-C4)alkylthio, (Ci-C4)alkylsulfonylamino, (Ci-C4)alkylsulfmyl, and (Ci-C4)alkylsulfonyl;
R3 and R4, taken together with the nitrogen to which they are attached, may form an optionally substituted six-, five-, or seven-membered heterocyclic ring, wherein said heterocyclic ring contains one or more heteroatoms, or, R3 and R4 are independently selected from hydrogen, (Ci-C4)alkyl, optionally substituted phenyl, optionally substituted heteroaryl, and CH2R10, and wherein said optional substituents are selected from (Ci-Cio)hydrocarbyl, (Ci-Cio)alkoxy(Ci- C4)alkyl, halogen, halo(Ci-C4)alkyl, (Ci-C4)acyl, hydroxy(Ci-C4)alkyl, hydroxy, (Ci-C4)alkoxy, halo(Ci-C4)alkoxy, carboxy, (Ci-C4)alkoxycarbonyl, carboxamido, amino, (Ci-C4)alkylamino, di(Ci-C4)alkylamino, (Ci-C4)alkylsulfonylamino, (Ci-C4)alkylsulfmyl, (Ci- C4)alkylaminocarbonyl, and (Ci-C4)alkylsulfonyl;
with the proviso that no carbon atom of said six-, five-, or seven-membered ring formed by R3 and R4 may carry more than one substituent; R5 is chosen from optionally substituted (C4-Cio)hydrocarbyl, hydrogen, halogen, (Ci- C4)acyl, hydroxy, (Ci-C4)alkoxy, halo(Ci-C4)alkoxy, carboxy, (Ci-C4)alkoxycarbonyl, carboxamido, amino, (Ci-C4)alkylamino, di(Ci-C4)alkylamino, optionally substituted (Ci- C3)hydrocarbyl, optionally substituted aryl, and optionally substituted heteroaryl, wherein said optional substituents on (C4-Cio)hydrocarbyl, (Ci-C3)hydrocarbyl, aryl, and heteroaryl are selected from halogen, halo(Ci-C4)alkyl, (Ci-C4)alkyl, cyano, acetoxy, nitro, (Ci-C4)acyl, hydroxy(Ci-C4)alkyl, hydroxy, (Ci-C4)alkoxy, halo(Ci-C4)alkoxy, carboxy, (Ci- C4)alkoxycarbonyl, carboxamido, amino, (Ci-C4)alkylamino, di(Ci-C4)alkylamino, (Ci- C4)alkylsulfonylamino, (Ci-C4)alkylsulfmyl, and (Ci-C4)alkylsulfonyl; and
R10 is an optionally substituted heterocycle or an optionally substituted six-membered aryl, wherein said optional substituents are selected from cyano, amino, alkylamino, dialkylamino, hydroxy, cyano(Ci-C4)alkyl, carboxy(Ci-C4)alkyl, carboxamido(Ci-C4)alkyl, amino(Ci-C4)alkyl, alkylamino(Ci-C4)alkyl, dialkylamino(Ci-C4)alkyl, and hydroxy(Ci- C4)alkyl.
2. A compound according to claim 1 wherein R3 and R4 form an optionally substituted five- or six-membered monocycle.
3. A compound according to claim 2 wherein R3 and R4 form an optionally substituted piperazine, homopiperazine, piperidine, morpholine or pyrrolidine ring.
4. A compound according to claim 3 wherein R3 and R4 form a piperazine ring substituted with methyl, ethyl, or hydroxymethyl.
5. A compound according to claim 2 wherein R3 and R4 form a 2-substituted piperazin-l-yl ring substituted with (Ci-C4)alkyl or hydroxy(Ci-C4)alkyl.
6. A compound according to claim 2 wherein R3 and R4 form a 3, 5 -di substituted piperazin-l-yl ring substituted with (Ci-C4)alkyl or hydroxy(Ci-C4)alkyl.
7. A compound according to claim 1 wherein: R3 and R4, together with the nitrogen to which they are attached, form an optionally substituted five-, six-, or seven-membered ring.
8. A compound according to claim 7 wherein A is an optionally substituted benzene, pyridine, thiophene, or pyrrole ring.
9. A compound according to claim 8 wherein A is a benzene ring optionally substituted with halogen, methyl, trifluoromethyl, methoxy, trifluoromethoxy, hydroxy or amino.
10. A compound according to claim 8 wherein A is a pyridine, thiophene, or pyrrole ring, optionally substituted with fluoro, trifluoromethyl, or methyl.
11. A compound according to claim 10 wherein A is an optionally substituted pyrrole ring.
12. A compound according to claim 10 wherein A is:
(a) a pyridine ring fused to form a pyrido[2,3-d]pyrimidine;
(b) a pyrrole ring fused to form a pyrrolo[l,2-f][l,2,4]triazine or a pyrrolo[2,3- djpyrimidine; or
(c) a thiophene ring fused to form a thieno[3,2-d]pyrimidine;
all optionally substituted with methyl.
13. A compound according to claim 7 wherein R5 is chosen from (Ci-Cio)hydrocarbyl and heteroaryl, said hydrocarbyl or heteroaryl optionally substituted with one or two substituents selected from halogen, methyl, trifluoromethyl, methoxy, trifluoromethoxy, hydroxy and amino.
14. A compound according to claim 13 wherein R5 is chosen from (C3-C6)alkyl, (C3- C6)cycloalkyl, phenyl, furanyl, and benzyl, each optionally substituted with one or two substituents selected from halogen, methyl, trifluoromethyl, methoxy, trifluoromethoxy, hydroxy and amino.
15. A compound according to claim 7 wherein R5 is cyclopropyl, t-butyl or phenyl.
16. A compound according to any of claims 2 to 6 and 13 to 15 wherein A is a benzene ring optionally substituted with halogen.
17. A compound according to any of claims 2 to 6 and 13 to 15 wherein A is a pyrrole ring optionally substituted with halogen.
18. A compound according to claim 16 wherein R5 is chosen from (Ci-C6)alkyl, (C3- C6)cycloalkyl, and benzyl optionally substituted with halogen, methyl, or trifluorom ethyl.
19. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a compound according to any of claims 1-15.
20. A method for inhibiting spastin comprising exposing spastin to a compound according to any of claims 1-15.
21. A method for inhibiting TrkA, TrkB, or TrkC comprising exposing TrkA, TrkB, or TrkC to a compound according to claim 11.
22. A method according to claim 21, wherein the compound is selected from:
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