WO2024254218A2 - Modulateurs d'interaction protéine-protéine complexe de canal fgf14:nav1.6 à petites molécules non peptidiques - Google Patents

Modulateurs d'interaction protéine-protéine complexe de canal fgf14:nav1.6 à petites molécules non peptidiques Download PDF

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WO2024254218A2
WO2024254218A2 PCT/US2024/032668 US2024032668W WO2024254218A2 WO 2024254218 A2 WO2024254218 A2 WO 2024254218A2 US 2024032668 W US2024032668 W US 2024032668W WO 2024254218 A2 WO2024254218 A2 WO 2024254218A2
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compound
alkyl
formula
ring
cycloalkyl
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WO2024254218A3 (fr
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Jia Zhou
Fernanda LAEZZA
Pingyuan WANG
Nolan M. DVORAK
Paul A. WADSWORTH
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University of Texas System
University of Texas at Austin
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University of Texas at Austin
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • 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

Definitions

  • the field of the invention relates generally to modulators of FGF14:NAV1.6 channel complex protein-protein interaction. More specifically, the field of invention relates to non-peptide small molecule modulators of FGF14:NAV1.6 channel complex protein-protein interaction modulators.
  • One aspect of the invention pertains to a compound of Formula I or a pharmaceutically acceptable salt thereof, wherein:
  • R 1 is H, alkyl, alkoxy, halogen, cyano, amino, hydroxyl, NO2, CF 3 or -OCF 3 ;
  • A is an aryl ring or a heteroaryl ring (e.g., pyridine), wherein ring A is fused to the C4 and C5 of the 5 -membered N-heterocycle ring moiety of Formula I, e,g, as shown the compounds exemplified below;
  • R 2 is H, alkyl, alkoxy, halogen, -CO2R 10 , -CChMe or hydroxyl;
  • R 3 is H, alkyl, alkoxy, halogen or hydroxyl; or R 2 and R 3 together form a 3-6 membered cycloalkyl ring;
  • R 4 is H, alkyl, alkoxy, halogen, cyano, hydroxyl or NT 1 ! 2 ;
  • T 1 is H, alkyl
  • R 5 is H, alkyl, aryl, cycloalkyl (e.g., is a bridged 6-14-membered bicyclic cycloalkyl, with 1-3 carbon length “bridge”); or heteroaryl, wherein each ring is optionally substituted with one or more groups selected independently from: H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, benzyl, alkoxy (e.g., OMe, 3-OMe), halogen, cyano (e.g., 3-CN), nitro, amino, amido (e.g., 3-amido hydroxyl, -COOR 8 (e.g., -COOMe), -CONHR 9 , CHF2, CF 3 or -OCF 3 , wherein alkyl is optionally substituted with one or more substituents chosen from: hydroxyl, cyan, amino, or halogen;
  • R 6 , and R 7 are independently chosen from H, alkyl, F, CHF2, CF 3 , etc., or R 6 and R 7 taken together form a 3-7 membered ring (e.g., cyclopropane);
  • R 8 is independently chosen from: H, alkyl, aryl, cycloalkyl, benzyl; and R 9 is independently chosen from: H, alkyl, aryl, cycloalkyl, benzyl, or substituted benzyl.
  • R 10 is alkyl (e.g., Cl-C6-alkyl), aryl, or alkylaryl.
  • Another aspect of the invention pertains to a compound of the Formula (II) or a pharmaceutically acceptable salt thereof, wherein:
  • R 4 is hydroxyl or NT 1 T 2 ; wherein T 1 is H or alkyl; and wherein T 2 is independently chosen from H, alkyl, cycloalkyl, benzyl, allyl, hydroxyl-alkyl; or T 1 and T 2 together form a 4-12 membered cycloalkyl ring or cycloheteralkyl ring, wherein the 4-12 membered cycloalkyl ring is optionally substituted with one or more groups selected independently from alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, benzyl, alkoxy, halogen, cyan, nitro, amino, hydroxyl, CHF2, CF 3 or -OCF 3 ;
  • R 5 is an aryl ring or a cycloalkyl ring, wherein each ring is optionally substituted with one or more groups selected independently from: H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, benzyl, alkoxy, halogen, cyano, nitro, amino, hydroxyl, -COOR 8 , -CONHR 9 , CHF2, CF 3 or -OCF 3 , wherein alkyl is optionally substituted with one or more chosen substituents chosen from: hydroxyl, cyano, amino, or halogen;
  • R 8 is independently chosen from: H, alkyl, aryl, cycloalkyl, or benzyl;
  • R 8 is independently chosen from: H, alkyl, aryl, cycloalkyl, benzyl, or substituted benzyl.
  • a further aspect of the invention pertains to a compound of Formula III, or a pharmaceutically acceptable salt thereof, wherein:
  • a yet further aspect of the invention pertains to compound of Formula IV, or a pharmaceutically acceptable salt thereof, wherein: wherein A, n, R 1 , and R 5 are defined as for the compound of Formula I.
  • a yet further aspect of the invention pertains to compound of Formula V, or a pharmaceutically acceptable salt thereof, wherein: wherein A, n, R 1 , R 5 , T 1 , and T 2 are defined as for the compound of Formula I.
  • a yet further aspect of the invention pertains to a compound of Formula VI, or a pharmaceutically acceptable salt thereof, wherein: wherein R 1 , R 4 , and R 5 are defined as for the compound of Formula I.
  • a yet further aspect of the invention pertains to a compound of Formula VII, or a pharmaceutically acceptable salt thereof, wherein: wherein R 1 and R 5 are defined as for the compound of Formula I.
  • a yet further aspect of the invention pertains to a compound of Formula VIII, or a pharmaceutically acceptable salt thereof, wherein: Formula VIII wherein R 1 , R 5 , T 1 , and T 2 are defined as for the compound of Formula I.
  • a yet further aspect of the invention pertains to a compound of Formula IX, or a pharmaceutically acceptable salt thereof, wherein:
  • R 1 , R 5 , T 1 , and T 2 are defined as for the compound of Formula I.
  • a yet further aspect of the invention pertains to a compound of Formula X, or a pharmaceutically acceptable salt thereof, wherein: wherein A is a heteroaryl ring, n, R 1 , R 4 and R 5 are defined as for the compound of Formula I.
  • a yet further aspect of the invention pertains to a method of treating a disease or condition in a patient comprising administering to the patient a therapeutically effective amount of a compound of disclosed herein or a pharmaceutically acceptable salt thereof.
  • treatment of said disease or condition involves modulating a FGF14:NAV1.6 channel complex protein-protein interaction.
  • FIG. 1 High throughput screening against the FGF14:Navl.6 complex.
  • FIG. 1A Summary of the LCA primary screen of -45,000 compounds showing hits based on a Z-score criterion.
  • FIG. IB Compounds are excluded based on the CellTiter Blue (CTB) cell viability fluorescence assay.
  • FIG. 1C Counter screen of non-toxic hits against the luciferase enzyme.
  • FIG. ID Dose-response of repurchased top hits after triplicate rescreen and prioritization based on drug-like properties.
  • CTB CellTiter Blue
  • FIG. 2A Selectivity and orthogonal validation of hits.
  • FIG. 2A The top 16 hits were screened (50 pM) against the FGF13-la:Navl.6, FGF13-lB:Navl.6, and FGF14:Navl.2 complexes, as well as the FGF14 dimer, using LCA. Compounds that modulated complex assembly by > 25% were excluded from further studies.
  • FIG. 2B The remaining 5 selective hits were then quantitatively assessed using SPR. Binding sensorgrams are shown for FGF14 (left) and Navi.6 (middle) and normalized steadystate saturation plots (right) reveal differences in compound: protein binding affinities.
  • FIG. 4A - FIG 4H Dose-responses of top compounds. Note that PW01028 displayed nanomolar potency in the LCA dose-response studies.
  • FIG. 6A - 6D Dose-responses of top compounds. Note that PW01028 displayed nanomolar potency in the LCA dose-response studies.
  • FIG. 7. Third generation of PW1028 analogs were synthesized and single concentration activity screening was tested.
  • FIG. 8 Dose-responses of top compound.
  • FIG. 9 The binding modes and molecular docking of compound PW01028.
  • FIG. 9A Docking of PW01028 (magenta) into the binding pocket of FGF14 in 3D view. Important residues are drawn in sticks. Hydrogen bonds are shown as dashed magenta lines, while TI ⁇ TI interactions are shown as dashed blue lines.
  • FIG. 9B Docking of compound PW01028 into the binding pocket of FGF14 in 2D view. Important residues and key interactions are presented in a similar fashion.
  • FIG. 10 Ligand binding studies of Compound PW01028.
  • FIG. 10A Ligand binding studies of Compound PW01028.
  • FIG. 11 Compound PW01028 inhibits shifts voltage-dependence of steadystate inactivation of Na+ currents and increases firing in MSN in an-FGF14 dependent manner.
  • FIG. 11 A Traces of Na peak currents (left column, asterisk) from cells treated with vehicle (0.1% DMSO; black) or compound PW01028 (1 pM; blue) elicited using the depicted voltage-clamp protocol (inset). The traces on the right represent a fraction of inactivated channels.
  • FIG. 11B Voltage-dependence of steady-state inactivation curves for the indicated experimental groups. Data were fitted using the Boltzmann equations.
  • FIG. 11C Comparison of V1/2 of steady-state inactivation between indicated groups.
  • FIG. 12 compound PW01028 inhibits shifts voltage-dependence of steady-state inactivation of Na+ currents and increases firing in MSN in an-FGF14 dependent manner.
  • FIG. 12A Traces of Na peak currents (left column, asterisk) from cells treated with vehicle (0.1% DMSO; black) or compound PW01028 (1 pM; blue) elicited using the depicted voltage-clamp protocol (inset).
  • the traces on the right represent a fraction of inactivated channels
  • FIG. 12B Voltage-dependence of steady-state inactivation curves for the indicated experimental groups. Data were fitted using the Boltzmann equations.
  • the term “about” refers to a ⁇ 10% variation from the nominal value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.
  • pharmaceutically acceptable salt refers to those salts of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without undue toxicity, and the like.
  • pharmaceutically acceptable salt may include acetate, hydrobromide, hydrochloride, sulfate, bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate, glucoheptonate, lactobionate and laurylsulphonate salts, and the like. (See S. M. Barge et al., "Pharmaceutical Salts," J. Pharm. Sci., 66: 1-19 (1977), which is incorporated herein by
  • HBTU refers to 3-[Bis(dimethylamino)methyliumyl]-3H- benzotriazol-1 -oxide hexafluorophosphate (also known as 2-(lH-benzotriazol-l-yl)- 1 , 1 , 3 ,3 -tetramethyluronium hexafluorophosphate) .
  • HOBt refers the following structure, known as 1- hydroxybenzotriazole, (including hydrates and polymorphs, thereof):
  • DIPEA N,N-Diisopropylethylamine (also known as Htinig’s base, DIPEA, and ethyldiisopropylamine).
  • DCM di chloromethane
  • TFA trifluoroacetic acid
  • alkyl refers to both straight and branched chain radicals, and cyclic alkyl groups.
  • the alkyl group has 1-12 carbons.
  • the alkyl group has 1-7 carbons.
  • the alkyl group has 1-6 carbons.
  • the alkyl group has 1-4 carbons.
  • alkyl may include methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4- dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, and dodecyl.
  • alkyl is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group.
  • halogenated alkyl or “haloalkyl” specifically refers to an alkyl group that is substituted with one or more halide, e.g., fluorine, chlorine, bromine, or iodine.
  • the term “monohaloalkyl” specifically refers to an alkyl group that is substituted with a single halide, e.g., fluorine, chlorine, bromine, or iodine.
  • polyhaloalkyl specifically refers to an alkyl group that is independently substituted with two or more halides, i.e. each halide substituent need not be the same halide as another halide substituent, nor do the multiple instances of a halide substituent need to be on the same carbon.
  • alkoxyalkyl specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below.
  • aminoalkyl specifically refers to an alkyl group that is substituted with one or more amino groups.
  • hydroxyalkyl specifically refers to an alkyl group that is substituted with one or more hydroxy groups.
  • cycloalkyl refers to both unsubstituted and substituted cycloalkyl moieties
  • the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g., an “alkylcycloalkyl.”
  • a substituted alkoxy can be specifically referred to as, e.g., a “halogenated alkoxy”
  • a particular substituted alkenyl can be, e.g., an “alkenylalcohol,” and the like.
  • the practice of using a general term, such as “cycloalkyl,” and a specific term, such as “alkylcycloalkyl,” is not meant to imply that the general term does not also include the specific term.
  • cycloalkyl includes bicyclic ring systems.
  • the bicyclic ring system may be in the form of a bridged, fused, or spiro form.
  • heteroalkyl by itself or in combination with another term, means, unless otherwise stated, a linear or branched chain having at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, S, P, and Si.
  • the heteroatoms are selected from the group consisting of O, and N.
  • the heteroatom(s) may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Up to two heteroatoms may be consecutive.
  • alkylene refers to straight and branched chain alkyl linking groups, i.e., an alkyl group that links one group to another group in a molecule.
  • alkylene may include -(CH2) n — where n is 2-8.
  • aryl means a polyunsaturated hydrocarbon substituent.
  • Aryl groups can be monocyclic or polycyclic (e.g., 2 to 3 rings that are fused together or linked covalently).
  • Non-limiting examples of aryl and heteroaryl rings are phenyl, naphthyl, pyranyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyrazolyl, pyridinyl, furanyl, thiophenyl, thiazolyl, imidazolyl, isoxazolyl, and the like.
  • heteroaryl refers to groups having 5 to 14 ring atoms
  • heteroaryl groups include 1,2, 3 -triazole, 1,2,4-triazole, 5-amino 1,2,4-triazole, imidazole, oxazole, isoxazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, 3 -amino- 1,2,4- oxadiazole, 1,2,5-oxadiazole, 1,3,4-oxadiazole, pyridine, 2-aminopyridine, 4- aminopyridine, 2-aminoimidazoline, and 4-aminoimidazoline.
  • amino refers to an -NH2 group.
  • An “amido” group refers to an -CONH2 group.
  • An alkylamido group refers to an -CONHR group wherein R is as defined above.
  • a dialkylamido group refers to an - CONRR' group wherein R and R' are as defined above.
  • halogen or “halo” as used herein by itself or as part of another group refers to chlorine, bromine, fluorine or iodine.
  • hydroxy or “hydroxyl” as used herein by itself or as part of another group refers to an — OH group.
  • alkoxy group refers to an -O-alkyl group wherein “alkyl” is as defined above.
  • the alkyl group has 1-12 carbons.
  • the alkyl group has 1-7 carbons.
  • the alkyl group has 1-6 carbons.
  • the alkyl group has 1-4 carbons.
  • a “thio” group refers to an -SH group.
  • heterocycle or “heterocyclic ring”, as used herein except where noted, represents a stable 5- to 7-membered monocyclic-, or stable 7- to 11 -membered bicyclic heterocyclic ring system, any ring of which may be saturated or unsaturated, and which consists of carbon atoms and from one to three heteroatoms selected from the group consisting of N, O and S, and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quatemized, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. Rings may contain one oxygen or sulfur, one to three nitrogen atoms, or one oxygen or sulfur combined with one or two nitrogen atoms.
  • the heterocyclic ring may be attached at any heteroatom or carbon atom
  • alkylamino refers to an amino group which is substituted with one alkyl group having from 1 to 6 carbon atoms.
  • dialkylamino refers to an amino group which is substituted with two alkyl groups, each having from 1 to 6 carbon atoms.
  • arylamine or “arylamino” as used herein by itself or as part of another group refers to an amino group which is substituted with an aryl group, as defined above.
  • arylalkyl denotes an alkyl group substituted with an aryl group, for example, Ph-CH 2 -, etc.
  • Optionally substituted groups may include one or more substituents independently selected from: halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, oxo, carbamoyl, alkyl, heteroalkyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkylsulfonyl, arylsulfonyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.
  • the optional substituents may be further substituted with one or more substituents independently selected from: halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl ( — C(O)NR2), unsubstituted alkyl, unsubstituted heteroalkyl, alkoxy, alkylthio, alkylamino, (alkyl)2amino, alkylsulfinyl, alkyl sulfonyl, aryl sulfonyl, unsubstituted cycloalkyl, unsubstituted heterocyclyl, unsubstituted aryl, or unsubstituted heteroaryl.
  • substituents independently selected from: halogen, nitro, cyano, hydroxy, amino, mercapto, formyl, carboxy, carbamoyl ( — C(O)NR2), unsubstituted alkyl, unsubstit
  • Psychiatric disorders are neural circuitry alterations that lead to malfunctioning in high-order psychological domains.
  • new biological signatures that break free of traditional clinical classifications - spanning longitudinally across disorders in lieu of a single domain-disease view - are required.
  • PPI Protein-protein interactions
  • Their highly specific and flexible interfaces could make protein-channel interactions ideal targets for development of molecular probes.
  • MSN medium spiny neurons
  • NAc nucleus accumbens
  • FGF14 fibroblast growth factor 14
  • FGF14:Na v 1.6 complex assembly is critical to regulate the firing rate of NAc targets by controlling GABA release onto the ventral tegmental area and ventral pallidum via the dopaminergic receptors type 1 direct (DI) and type 2 indirect (D2) pathways, respectively.
  • probes that modulate MSN activity by fine-tuning FGF14:Na v 1.6 complex assembly would be valuable for interrogating the circuitry affected in many neuropsychiatric disorders.
  • Novel probes are needed that serve as precise in vivo measures of mechanisms hypothesized to underlie circuit deficits in mental disorders. Probes developed from molecular knowledge will provide the foundation for a paradigm shift in neuropsychopharmacology, leading to rigorous mechanistic hypotheses and future medications.
  • the nucleus accumbens located within the ventral striatum, serves as the central point of brain reward circuitry, controlling goal-directed behaviors and motivation for reward for natural stimuli and drug of abuse.
  • motivation is a fundamental dimension of behavior that cuts across diagnostic boundaries. Lack of motivation is a prevalent symptom in a number of neuropsychiatric illnesses such as schizophrenia, major depressive disorder, and neurodevelopmental disorders. It is therefore critical to develop adequate molecular tools to probe the circuits and the behaviors associated with motivation.
  • MSNs comprise 95% of the NAc neuronal population, are formed exclusively by GABAergic cells, and give rise to the only output of the NAc.
  • FGF14 was established as a functionally relevant component of the Navi.6 channel complex and a primary determinant of excitability in MSNs. Genetic silencing of either FGF 14 or Navi .6 affects MSN firing and plasticity in the NAc, and leads to changes in motivated behaviors associated with depression, anxiety, addiction, schizophrenia and psychosis, thereby demonstrating a link between the FGF14:Navl.6 complex and a full range affective disorders.
  • Fig. 1A shows results of the LCA primary screen of -45,000 compounds showing hits based on a Z-score criterion.
  • Fig. IB summarizes exclusion of compounds based on the CellTiter Blue (CTB) cell viability fluorescence assay.
  • Fig. 1C shows results of counter screen of non-toxic hits against the luciferase enzyme.
  • Fig. ID shows dose-response of repurchased top hits after triplicate rescreen and prioritization based on drug-like properties.
  • compound 7605086 displayed the most favorable drug-like properties for CNS drug development. To improve the potency and aqueous solubility of compound 7605086, substituents of the parental scaffold were replaced, producing probe compound
  • compound PW01028 modulated FGF14:Navl.6 complex formation with an IC50 of -250 nM.
  • surface plasmon resonance (SPR) showed that compound PW01028 displayed nanomolar binding affinity to FGF14 with evidence for ligand binding to the FGF14 R117 residue, a previously identified “hot-spot” at the FGF14:Navl.6 PPI interface.
  • compound PW01028 was shown to exacerbate FGF14-mediated regulatory effects on Navi.6 channel inactivation, causing a marked (+ 15 mV) depolarizing shift in the voltage-dependence of steady-state inactivation of the transient Na+ current.
  • the mechanism of action (MOA) of compound PW01028 was recapitulated in MSNs of the
  • Counter screening assays Cell viability assay.
  • the CellTiter-Blue® (CTB) Cell Viability Assay is a widely used, reliable fluorescent method for monitoring cell viability in HTS.
  • the assay is based on the ability of living cells to convert a redox dye (resazurin) into a fluorescent end-product (resorufin). Nonviable cells rapidly lose metabolic capacity and thus do not generate a fluorescent signal.
  • the assay is used in our pipeline as the first counter screening assay to eliminate toxic compounds. It is initiated by dispensing the proper amount of CTB reagent (i.e., 10 pL) per well in 384-well plates.
  • Fluorescence is then read after 16 hrs, and cut- offs are set at a Z-score of ⁇ -3 to identify and exclude toxic compounds (Fig. IB).
  • the assay was typically run at a single, maximal concentration of the test compound calculated from the IC50. Additional details about the use of this assay can be found in previous publications.
  • SPR Surface plasmon resonance
  • Analogues were then tested using SPR against FGF14 and Navl.6-C-terminal tail purified proteins, the gold standard for protein binding studies.
  • SPR experiments were used to quantitatively assess the binding affinity of analogues, serving as both a secondary bioactivity assay to determine accurate binding affinities for proteimligand interactions via KD, K on and K o ff, as well as to assess the compound’s ability to disrupt PPI.
  • the reliability of the model, goodness of fit, and reported kinetic and binding constants were validated by varying flow rates, immobilization densities, or different chips.
  • Statistical analysis was conducted by visually inspecting the goodness of fit of the data from the residuals. Additionally, c2 was used as a global measure for residual noise; standard deviation was used to report errors in measurements by repeating the experiment at least three times.
  • Figs. 2A and 2B summarize selectivity and orthogonal validation of hits from the screening assay.
  • the top 16 hits were screened (50 pM) against the FGF13-la:Navl.6, FGF13-lB:Navl.6, and FGF14:Navl.2 complexes, as well as the FGF14 dimer, using LCA. Compounds that modulated complex assembly by > 25% were excluded from further studies.
  • the remaining 5 selective hits were then quantitatively assessed using SPR. Binding sensorgrams are shown for FGF14 (left) and Navi.6 (middle) and normalized steady-state saturation plots (right) reveal differences in compound: protein binding affinities.
  • Confirmatory assays Automated and manual patch-clamp electrophysiology in heterologous cells. Patch-clamp electrophysiology is a rigorous, sophisticated confirmatory and orthogonal assay that serves to assess compound functional activity, mechanism of action, specificity, and selectivity toward various Nav isoforms. It is highly predictive of in vivo efficacy of compounds.
  • compounds are tested in cells stably expressing FGF 14 and Navi .6 or other relevant iFGF:Nav channel pairs. Initially screened with an automated planar patch-clamp system, compounds were tested with an 8-point dose-response in 384-well plates with a throughput of >2,000 data sets/week.
  • the top 16 hits were screened (50 pM) against the FGF13- la:Navl.6, FGF13-lB:Navl.6, and FGF14:Navl.2 complexes, as well as the FGF14 dimer, using LCA. Compounds that modulated complex assembly by > 25% were excluded from further studies.
  • FGF14 stands out among iFGFs as a potent, specific, and diverse modulator of Nav channels, especially Navi.6.
  • FGF14 suppressed or augmented Navl.6-encoded currents and channel availability, depending on its N-terminal spliced isoform.
  • genetic deletion of FGF14 caused suppression of Navl.6-mediated persistent and/or resurgent currents, while its overexpression increased Na + current peak amplitude and channel availability. This suggested a complex functional role for FGF14 in modulating native Na + currents that can be utilized to develop allosteric Navi.6 modulators as probes for excitability.
  • HPLC analysis conditions Waters pBondapak C18 (300 x 3.9 mm); flow rate 0.5 mL/min; UV detection at 270 and 254 nm; linear gradient from 10% acetonitrile in water to 100% acetonitrile in water in 20 min followed by 30 min of the last-named solvent (0.1% TFA was added into both acetonitrile and water). All biologically evaluated compounds were > 95% pure.
  • Reagents and conditions (a) tert-butyl 2-bromoacetate, K2CO3, DMF, rt, overnight, 96%. (b) TFA, rt, 4 h, quant, (c) m-toluidine, EDCI, DMAP, DMF, rt, overnight, 82%. (d) NaBH 4 , MeOH, 0 °C, 1 h, 79%.
  • Reagents and conditions (a) tert-butyl 2-bromoacetate, K2CO3, DMF, rt, overnight, 93%. (b) TFA, rt, 4 h, quant, (c) (1) m-toluidine, EDCI, DMAP, DMF, rt, overnight, 86%.
  • Reagents and conditions (a) (1) corresponding substituted aniline, EDCI, DMAP, DMF, rt, overnight; (2) NaBH4, MeOH, 0 °C, 30 min, 34%-82% for two steps.
  • Reagents and conditions (a) hexamethylenetetramine, H2O, AcOH, reflux, 6 h, (b) tert-butyl 2-bromoacetate, K2CO3, DMF, rt, overnight, 96%. (b) TFA, rt, 4 h, quant, (c) m-toluidine, EDCI, DMAP, DMF, rt, overnight, 82%. (d) NaBF , MeOH, 0 °C, 1 h, 79%.
  • Reagents and conditions (a) tert-butyl 2-bromoacetate, K2CO3, DMF, rt, overnight, 90%. (b) TFA, rt, 4 h, quant, (c) (1) m-toluidine, EDCI, DMAP, DMF, rt, overnight; (2) NaBH 4 , MeOH, 0 °C, 1 h, 79%. [0160] tert- Butyl 2-(5-cyano-3-formyl-lZ7-indol-l-yl)acetate (PW01010).
  • Reagents and conditions (a) methyl 2-bromopropanoate, K2CO3, DMF, rt, overnight, 94%. (b) LiOH, MeOH, rt, overnight, 92%. (c) (1) m-toluidine, EDCI, DMAP, DMF, rt, overnight; (2) NaBH 4 , MeOH, 0 °C, 1 h, 60%.
  • R 1 is H, alkyl, alkoxy, halogen, cyano, amino, hydroxyl, NO2, CF 3 or -OCF 3 ;
  • A is an aryl ring or a heteroaryl ring (e.g., pyridine), wherein ring A is fused to the C4 and C5 of the 5 -membered N-heterocycle ring moiety of Formula I, e,g, as shown the compounds exemplified below;
  • R 2 is H, alkyl, alkoxy, halogen, -CO2R 10 , -CChMe or hydroxyl;
  • R 3 is H, alkyl, alkoxy, halogen or hydroxyl; or R 2 and R 3 together form a 3-6 membered cycloalkyl ring;
  • R 4 is H, alkyl, alkoxy, halogen, cyano, hydroxyl or NT 1 ! 2 ;
  • T 1 is H, alkyl
  • R 5 is H, alkyl, aryl, cycloalkyl (e.g., is a bridged 6-14-membered bicyclic cycloalkyl, with 1-3 carbon length “bridge”); or heteroaryl, wherein each ring is optionally substituted with one or more groups selected independently from: H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, benzyl, alkoxy (e.g., OMe, 3-OMe), halogen, cyano (e.g., 3-CN), nitro, amino, amido (e.g., 3-amido hydroxyl, -COOR 8 (e.g., -COOMe), -CONHR 9 , CHF2, CF 3 or -OCF 3 , wherein alkyl is optionally substituted with one or more substituents chosen from: hydroxyl, cyan, amino, or halogen; R 6 , and R 7 are independently chosen from H, alkyl, F
  • R 8 is independently chosen from: H, alkyl, aryl, cycloalkyl, benzyl; and
  • R 9 is independently chosen from: H, alkyl, aryl, cycloalkyl, benzyl, or substituted benzyl.
  • R 10 is alkyl (e.g., Cl-C6-alkyl), aryl, or alkylaryl.
  • R 4 is hydroxyl or NT 1 ! 2 ; wherein T 1 is H or alkyl; and wherein T 2 is independently chosen from H, alkyl, cycloalkyl, benzyl, allyl, hydroxyl-alkyl; or T 1 and T 2 together form a 4-12 membered cycloalkyl ring or cycloheteralkyl ring, wherein the 4-12 membered cycloalkyl ring is optionally substituted with one or more groups selected independently from alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, benzyl, alkoxy, halogen, cyan, nitro, amino, hydroxyl, CHF2, CF 3 or -OCF 3 ;
  • R 5 is an aryl ring or a cycloalkyl ring, wherein each ring is optionally substituted with one or more groups selected independently from: H, alkyl, cycloalkyl, alkenyl, aryl, heteroaryl, benzyl, alkoxy, halogen, cyano, nitro, amino, hydroxyl, -COOR 8 , -CONHR 9 , CHF2, CF 3 or -OCF 3 , wherein alkyl is optionally substituted with one or more chosen substituents chosen from: hydroxyl, cyano, amino, or halogen;
  • R 8 is independently chosen from: H, alkyl, aryl, cycloalkyl, or benzyl;
  • R 8 is independently chosen from: H, alkyl, aryl, cycloalkyl, benzyl, or substituted benzyl.
  • R 4 is hydroxyl.
  • R 4 is NT X T 2 .
  • T 1 is H.
  • R 5 is an aryl ring.
  • aryl ring is substituted with one or more substituents selected independently from: H, alkyl, benzyl, alkoxy, halogen, cyano, nitro, amino, hydroxyl, -COOR 8 , -CONHR 9 , CHF2, CF 3 or -OCF 3 , wherein alkyl is optionally substituted with one or more chosen substituents chosen from: hydroxyl, cyano, amino, or halogen;
  • R 8 is H, alkyl; and R 9 is independently chosen from: H, alkyl, aryl, cycloalkyl, benzyl, or substituted benzyl.
  • a method of treating a disease or condition in a patient comprising administering to the patient a therapeutically effective amount of a compound of any of the preceding embodiments or a pharmaceutically acceptable salt thereof.

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  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
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Abstract

La présente invention concerne de nouveaux modulateurs d'interaction protéine-protéine complexe de canal FGF14:NAV1.6 à petites molécules de la structure générale selon la formule (I), et ainsi que leur préparation et leur utilisation.
PCT/US2024/032668 2023-06-05 2024-06-05 Modulateurs d'interaction protéine-protéine complexe de canal fgf14:nav1.6 à petites molécules non peptidiques Pending WO2024254218A2 (fr)

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EP0287196B1 (fr) * 1987-02-18 1994-11-23 Beecham Group Plc Dérivés de l'indole, un procédé pour leur préparation et compositions pharmaceutiques les contenant
CA2043741C (fr) * 1990-06-07 2003-04-01 Kiyofumi Ishikawa Derives de peptides antagonistes d'endotheline
RU2313524C2 (ru) * 2003-05-09 2007-12-27 Ф.Хоффманн-Ля Рош Аг МИТИЛИНДОЛЫ И МЕТИЛПИРРОЛОПИРИДИНЫ, ФАРМАЦЕВТИЧЕСКАЯ КОМПОЗИЦИЯ, ОБЛАДАЮЩАЯ АКТИВНОСТЬЮ α-1-АДРЕНЕРГИЧЕСКИХ АГОНИСТОВ
US9487483B2 (en) * 2012-06-28 2016-11-08 Novartis Ag Complement pathway modulators and uses thereof
RS59934B1 (sr) * 2015-06-09 2020-03-31 Abbvie Inc Modulatori nuklearnih receptora (ror) za lečenje inflamatornih i autoimunih oboljenja

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