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WO2024229267A1 - Diaryl urea-based compounds and related compositions and methods of use in the treatment of diabetes mellitus and neurodegenerative disease - Google Patents

Diaryl urea-based compounds and related compositions and methods of use in the treatment of diabetes mellitus and neurodegenerative disease Download PDF

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WO2024229267A1
WO2024229267A1 PCT/US2024/027498 US2024027498W WO2024229267A1 WO 2024229267 A1 WO2024229267 A1 WO 2024229267A1 US 2024027498 W US2024027498 W US 2024027498W WO 2024229267 A1 WO2024229267 A1 WO 2024229267A1
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Jessica Sonia FORTIN
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    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
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    • C07C275/00—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups
    • C07C275/28—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
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    • C07C275/40—Derivatives of urea, i.e. compounds containing any of the groups, the nitrogen atoms not being part of nitro or nitroso groups having nitrogen atoms of urea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by nitrogen atoms not being part of nitro or nitroso groups
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    • C07C335/16—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton
    • C07C335/20—Derivatives of thiourea having nitrogen atoms of thiourea groups bound to carbon atoms of six-membered aromatic rings of a carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups
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    • C07D209/30—Indoles; Hydrogenated indoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to carbon atoms of the hetero ring
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    • C07D277/62—Benzothiazoles
    • C07D277/64—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2
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    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/12—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms
    • C07D295/135—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly or doubly bound nitrogen atoms with the ring nitrogen atoms and the substituent nitrogen atoms separated by carbocyclic rings or by carbon chains interrupted by carbocyclic rings
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    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the present disclosure relates to diaryl urea-based compounds, pharmaceutical compositions comprising same, their use in the prophylactic and therapeutic treatment of diabetes mellitus in humans and felines and their use in the treatment of neurodegenerative disease, such as diseases involving tubulin-associated unit and alpha-synuclein protein aggregation.
  • Amyloidosis is a term encompassing a wide range of protein misfolding disorders characterized by the accumulation of toxic protein aggregates. Notable examples include Alzheimer’s disease, Parkinson’s disease, and type 2 diabetes. Diabetes mellitus, also known as type 2 diabetes (T2D), is one disorder in which about 70% of cases involve the accumulation of misfolded islet amyloid protein (IAPP) affecting both humans and other species, including the cat. As of 2021, 537 million people worldwide are living with T2D, with this number expected to rise to 783 million by the year 2045.
  • IAPP misfolded islet amyloid protein
  • IAPP also known as amylin
  • IAPP is normally co-secreted with insulin by pancreatic P- cells.
  • Normal IAPP is a hormone that signals satiety, with its main function being insulin and glucagon inhibition.
  • IAPP misfolds and aggregates in the islets of Langerhans, P-cell mass and function both decrease, suggesting intermediate IAPP species (oligomers) are cytotoxic toward pancreatic P-cells.
  • IAPP is a prone-to-aggregate protein comprised of 37 amino acid residues. Of these, the hydrophobic core spanning residues 20-29 has been suggested as the region most likely to promote aggregation, especially in humans.
  • IAPP has been identified in a multitude of mammals other than humans and cats, such as the Chinese hamster, cow, hare, degu, tamarin, mouse, raccoon, and baboon. Differences in the genetic sequences encoding IAPP correlate with its ability to form fibrils. For instance, rodent IAPP does not have a propensity to aggregate because its 20-29 residue core contains more proline, which can disassemble beta sheet conformations.
  • Human IAPP (hlAPP) and feline IAPP (flAPP) share the most similarities in IAPP sequence (Fig. 1) and a high propensity for aggregation, oftentimes leading to T2D in both species.
  • Silybin A and Silybin B two components derived from the milk thistle plant.
  • Silybin B was shown to be the more potent of the two compounds, interacting heavily with the hlAPP core and reducing its toxicity.
  • Neurodegenerative diseases such as Alzheimer’s (AD) and Parkinson’s (PD) can trace their origin to protein fibrillization, the process by which a misfolded protein develops fibrils (Mahmoudi et al., Nanoscale 5(7): 2570-2588 (2013)).
  • NFTs neurofibrillary tangles
  • the isoforms vary by the presence or absence of inserts encoded by the Tau gene (Zhong et al., J. Biol. Chem. 287(24): 20711-20719 (2012)).
  • the six isoforms (0N3R, 0N4R, 1N3R, 1N4R, 2N3R, 2N4R) are transcribed in the human brain by alternative splicing and are expressed in different ratios during neurodegeneration resulting in tau pathological effects (Boyarko et al., Front Neurosci 15: 702788 (2021)).
  • Each isoform is distinguished by two criteria: the absence, presence, or partial presence of two 29-amino acid inserts encoded by exons 2 and 3 resulting in ON (no insert), IN (one insert), or 2N (two inserts) tau; and the presence (4R) or absence (3R) of the R2 domain of the microtubule binding domain region (MTBR) in addition to the already present Rl, R3, and R4 domains (Boyarko et al., supra).
  • Tau isoforms are differentially expressed across the hippocampus during various stages of development; in fetal stages only the 0N3R isoform is expressed, while all six isoforms are expressed in adulthood (Sergeant et al., Biochim Biophy Acta 1739(2-3): 179-197 (2005)). In healthy adults the ratio of 3R to 4R isoforms is approximately 1 (Boyarko et al., supra, and Bachmann et al., Front Neurosci 15: 643115 (2021)).
  • R 1 and R 2 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy.
  • Halo can be F, I, Cl, or Br.
  • Mono-halo methyl can be -C(H2)F, -C(H2)I, - C(H 2 )C1 or -C(H 2 )Br.
  • Di-halo methyl can be -C(H)F 2 , -C(H)I 2 , -C(H)C1 2 or -C(H)Br 2 .
  • Tri- halo methyl can be -CF3, -CI3, -CCI3 or -CBrs.
  • the Ci-Ce alkyl can be -CH3.
  • the Ci-Ce alkoxy can be -OCH3.
  • the compound can have the structure: .
  • the compound can have the structure:
  • R 1 and R 2 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO 2 , a Ci-Ce alkyl, and a Ci-Ce alkoxy; and R 10 is heteroaryl (pyridinyl, pyrrolyl, or imidazolyl) or heterocycloalkyl (morpholinyl or piperazinyl).
  • Halo can be F, I, Cl, or Br.
  • Mono-halo methyl can be -C(H 2 )F,
  • Di-halo methyl can be -C(H)F 2 , -C(H)I 2 , -C(H)C1 2 or -
  • Tri-halo methyl can be -CF3, -CI3, -CCI3 or -CBrs.
  • the Ci-Ce alkyl can be -CH3.
  • the Ci-Ce alkoxy can be -OCH3.
  • the compound can have the structure:
  • R 1 and R 2 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO 2 , a Ci-Ce alkyl, and a Ci-Ce alkoxy.
  • Halo can be F, I, Cl, or Br.
  • Mono-halo methyl can be -C(H 2 )F, -C(H 2 )I, - C(H 2 )C1 or -C(H 2 )Br.
  • Di-halo methyl can be -C(H)F 2 , -C(H)I 2 , -C(H)C1 2 or -C(H)Br 2 .
  • Tri- halo methyl can be -CF3, -CI3, -CCI3 or -CBrs.
  • the Ci-Ce alkyl can be -CH3.
  • the Ci-Ce alkoxy can be -OCH3. Still further provided is a compound of formula IV: Formula IV wherein:
  • R 3 and R 4 are each independently H, alkyl or acyl or, R 3 and R 4 , together with the atoms to which they are each attached, form an aryl or heteroaryl group;
  • R 5 and R 6 are each independently H or alkyl or, R 5 and R 6 , together with the atoms to which they are attached, form a heterocyclyl group;
  • R 7 and R 8 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy; or
  • R 7 and R 8 together with the atoms to which they are each attached, form an aryl or heteroaryl group
  • X 1 is NR 5 , O, S or CR 9 2, wherein each R 9 is independently, H, alkyl or acyl;
  • X 2 is O or S.
  • X 2 can be NR 13 , wherein R 13 is H or alkyl
  • halo for compounds of the formula IV are F, I, Cl, and Br.
  • monohalo methyl for compounds of the formula IV are -C(H2)F, -C(H2)I, -C(H2)C1, and -C(H2)Br.
  • di-halo methyl for compounds of the formula IV are -C(H)F2, -C(H)l2, - C(H)Ch, and -C(H)Br2.
  • tri-halo methyl for compounds of the formula IV are - CF3, -CI3, -CCI3, and -CBrs.
  • alkyl for any alkyl group for compounds of formula IV is Ci-Ce alkyl , which can be -CEE.
  • alkoxy for any alkyl group for compounds of formula IV is Ci-Ce alkoxy, which can be -OCH3.
  • R 3 and R 4 are each independently H, alkyl or acyl or, R 3 and R 4 , together with the atoms to which they are each attached, form an aryl or heteroaryl group;
  • R 5 and R 6 are each independently H or alkyl or, R 5 and R 6 , together with the atoms to which they are attached, form a heterocyclyl group;
  • R 7 and R 8 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy; or
  • R 7 and R 8 together with the atoms to which they are each attached, form an aryl or heteroaryl group
  • X 1 is NR 5 , O, S or CR 9 2, wherein each R 9 is independently, H, alkyl or acyl;
  • X 2 is O or S. In some embodiments, X 2 can be NR 13 , wherein R 13 is H or alkyl.
  • halo for compounds of the formula IV are F, I, Cl, and Br.
  • monohalo methyl for compounds of the formula IV are -C(H2)F, -C(H2)I, -C(H2)C1, and -C(H2)Br.
  • di-halo methyl for compounds of the formula IV are -C(H)F2, -C(H)l2, - C(H)Ch, and -C(H)Br2.
  • tri-halo methyl for compounds of the formula IV are - CF3, -CI3, -CCI3, and -CBrs.
  • alkyl for any alkyl group for compounds of formula IV is Ci-Ce alkyl , which can be -CEE.
  • alkoxy for any alkyl group for compounds of formula IV is Ci-Ce alkoxy, which can be -OCH3.
  • the compound of formula IV can be a compound of the formula:
  • the compound of the formula IV can be a compound of the formula:
  • the compound of formula IV can be a compound of the formula: such as compounds of the formula:
  • X 2 in the compounds of the formula IV can be S.
  • one R 9 in the compounds of the formula IV can be H.
  • both R 9 groups can be H.
  • R 11 is alkyl, cycloalkyl, aryl, arylalkyl, or arylacyl;
  • R 12 is cycloakyl, aryl, or heterocyclyl
  • R 5 and R 6 are each independently H or alkyl or, R 5 and R 6 , together with the atoms to which they are attached, form a heterocyclyl group;
  • X 2 is O or S.
  • X 2 can be NR 13 , wherein R 13 is H or alkyl.
  • aryl groups representing R 11 include napthyl (e.g., 1-, 2-, or 4-napthyl) and aryl groups substituted with one, two, or three F, Cl, I, acyl, alkyl, alkoxy, alkylthio, and amino.
  • alkyl groups representing R 11 include C1-C3 alkyl.
  • Examples of cycloalkyl groups representing R 11 include cyclopentyl and cyclohexyl.
  • arylacyl groups representing R 11 include groups of the formula:
  • arylalkyl groups representing R 11 include benzyl and phenethyl.
  • cycloalkyl groups representing R 12 include cyclopentyl and cyclohexyl.
  • aryl groups representing R 12 include fluorenyl (e.g., 3-fluorenyl) and aryl groups substituted with one or two haloalkyl (e.g., CF3), amino, morpholino, piperazinyl, and 2- thiazolyl.
  • heterocyclyl groups representing R 12 include 4-indolyl, 5-indolyl, and 6-indolyl.
  • R 11 and R 12 are not the same, such that the compounds of the formula V are assymetric.
  • a pharmaceutical composition comprising at least one compound of Formulae I-V and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising at least one compound of Formulae I-V can further comprise at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus and is selected from the group consisting of a sulfonylurea, a meglitinide, a biguanide, a thiazolidinedione (TZD), an a-glucosidase inhibitor, a dipeptidyl peptidase IV (DPP-4) inhibitor, a bile acid sequestrant, a dopamine agonist, a sodium-glucose transport protein 2 (SGLT2) inhibitor, and a glucagon like peptide 1 (GLP-1) receptor agonist.
  • a sulfonylurea a meglitinide, a biguanide, a thiazolidinedione (TZD), an a-glucos
  • the sulfonylurea can be at least one compound selected from the group consisting of chlorpropamide, tolazamide, tolbutamide, acetohexamide, glyburide, glipizide, glimepiride, and gliclazide.
  • the meglitinide can be repaglinide, netaglinide, or a combination thereof.
  • the biguanide can be metformin.
  • the TZD can be rosiglitazone, pioglitazone, or a combination thereof.
  • the a-glucosidase inhibitor can be at least one compound selected from the group consisting of acarbose, miglitol and voglibose.
  • the DPP-4 inhibitor can be at least one compound selected from the group consisting of alogliptin, linagliptin, sitagliptin, saxagliptin and vldagliptin.
  • the bile acid sequestrant can be colesevelam.
  • the dopamine agonist can be bromocriptine.
  • the SGLT2 inhibitor can be at least one compound selected from the group consisting of cangliflozin, dapagliflozin, empagliflozin and ertugliflozin.
  • the GLP-1 receptor agonist can be semaglutide.
  • a pharmaceutical composition comprising at least one compound of Formulae I-V can further comprise at least one other compound, which is a combination of compounds selected from (a) metformin and at least one of glyburide, glipizide, sitagliptin, saxagliptin, pioglitazone, repaglinide, rosiglitazone, linagliptin, alogliptin, canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin, (b) glimepiride and either or both of pioglitazone and rosiglitazone, (c) alogliptin and pioglitazone, (d) dapagliflozin and saxagliptin, (e) empagliflozin and linagliptin, (f) ertugliflozin and sitagliptin, (g) lixisenatide
  • a pharmaceutical composition comprising at least one compound of Formulae I-V can further comprise at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus and is selected from the group consisting of a GLP-1 receptor agonist and a dual GLP-1 receptor and GIP receptor agonist.
  • the GLP-1 receptor agonist can be exenatide, liraglutide, dulaglutide, lixisenatide or semaglutide.
  • the dual GLP-1 receptor and GIP receptor agonist can be tirzepatide.
  • the pharmaceutical composition can be formulated for administration by injection.
  • a pharmaceutical composition comprising at least one compound of Formula I can further comprise bexagliflozin.
  • the pharmaceutical composition can be formulated for oral administration.
  • a pharmaceutical composition comprising at least one compound of Formula II can further comprise bexagliflozin.
  • the pharmaceutical composition can be formulated for oral administration.
  • a pharmaceutical composition comprising at least one compound of Formula II can further comprise bexagliflozin.
  • the pharmaceutical composition can be formulated for oral administration.
  • a pharmaceutical composition comprising at least one compound of Formula IV can further comprise bexagliflozin.
  • the pharmaceutical composition can be formulated for oral administration.
  • a pharmaceutical composition comprising at least one compound of Formula V and a pharmaceutically acceptable carrier is also provided.
  • a method of inhibiting aggregation of misfolded islet amyloid protein (IAPP) in a human with, or at risk for, diabetes mellitus comprises administering to the human the compound of Formulae I-V, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit aggregation of misfolded IAPP, whereupon aggregation of misfolded IAPP is inhibited in the human with, or at risk for, diabetes mellitus.
  • IAPP misfolded islet amyloid protein
  • the method can further comprise administering, simultaneously or sequentially by the same or different routes, at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus and which is selected from the group consisting of a sulfonylurea, a meglitinide, a biguanide, a TZD, an a-glucosidase inhibitor, a DPP-4 inhibitor, a bile acid sequestrant, a dopamine agonist, a SGLT2 inhibitor, and a GLP-1 receptor agonist.
  • a sulfonylurea a meglitinide, a biguanide, a TZD, an a-glucosidase inhibitor, a DPP-4 inhibitor, a bile acid sequestrant, a dopamine agonist, a SGLT2 inhibitor, and a GLP-1 receptor agonist.
  • the sulfonylurea can be at least one compound selected from the group consisting of chlorpropamide, tolazamide, tolbutamide, acetohexamide, glyburide, glipizide, glimepiride, and gliclazide.
  • the meglitinide can be repaglinide, netaglinide, or a combination thereof.
  • the biguanide can be metformin.
  • the TZD can be rosiglitazone, pioglitazone, or a combination thereof.
  • the a-glucosidase inhibitor can be at least one compound selected from the group consisting of acarbose, miglitol and voglibose.
  • the DPP-4 inhibitor can be at least one compound selected from the group consisting of alogliptin, linagliptin, sitagliptin, saxagliptin and vldagliptin.
  • the bile acid sequestrant can be colesevelam.
  • the dopamine agonist can be bromocriptine.
  • the SGLT2 inhibitor can be at least one compound selected from the group consisting of cangliflozin, dapagliflozin, empagliflozin and ertugliflozin.
  • the GLP-1 receptor agonist can be semaglutide.
  • the at least one other compound can be a combination of compounds selected from (a) metformin and at least one of glyburide, glipizide, sitagliptin, saxagliptin, pioglitazone, repaglinide, rosiglitazone, linagliptin, alogliptin, canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin, (b) glimepiride and either or both of pioglitazone and rosiglitazone, (c) alogliptin and pioglitazone, (d) dapagliflozin and saxagliptin, (e) empagliflozin and linagliptin, (f) ertugliflozin and sitagliptin, (g) lixisenatide and glargine insulin, and (h) liraglutide
  • the method comprising administering to the human the compound of Formulae I-V, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit aggregation of misfolded IAPP can further comprise administering at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus, wherein the at least one other compound is selected from the group consisting of a GLP-1 receptor agonist and a dual GLP-1 receptor and GIP receptor agonist.
  • the GLP-1 receptor agonist can be exenatide, liraglutide, dulaglutide, lixisenatide or semaglutide.
  • the dual GLP-1 receptor and GIP receptor agonist can be tirzepatide.
  • the administering can be by injection.
  • a method of inhibiting aggregation of misfolded IAPP in a cat with, or at risk for, diabetes mellitus comprises administering to the cat the compound of Formulae I-V, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit aggregation of misfolded IAPP, whereupon aggregation of misfolded IAPP is inhibited in the cat with, or at risk for, diabetes mellitus.
  • the method can further comprise administering bexafliflozin.
  • the administering can be orally administering.
  • the method can further comprise administering insulin.
  • the administering of the compound of Formulae I-V can be orally, and the administering of the insulin can be by injection.
  • tubulin-associated unit (tau) protein aggregation in a subject having, or at risk for, tau protein aggregation.
  • the method comprises administering to the subject an above-described pharmaceutical composition in an amount effective to inhibit tau protein aggregation.
  • the subject can have, or be at risk for, Alzheimer’s disease.
  • a method of inhibiting alpha-synuclein (a-syn) protein aggregation in a subject having, or at risk for a-syn comprises administering to the subject an above-described pharmaceutical composition in an amount effective to inhibit a- syn protein aggregation.
  • the subject can have, or be at risk for, Parkinson’s disease.
  • Fig. 1 Amino acid sequences of human islet amyloid polypeptide (hlAPP; SEQ ID NO: 1
  • Fig- 3 Kinetics of amylin fibril formation with increasing doses of four synthesized compounds 14 (APS-22-96), 20 (APS-22-109), 26 (APS-22-110), and 27 (APS-22-107), assessed via thioflavin T (ThT) fluorescence assay.
  • the original stock of the compound was tested at 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 pM concentration. These compounds were tested in the presence of hlAPP and flAPP at 10 pM.
  • Fig. 5 TEM imaging of amylin fibrils incubated with four unique treatments. Samples of hlAPP and flAPP were solubilized at 10 pM in PBS buffer, after the end of kinetic of fibril formation. Samples were incubated with DMSO (0.25%), Compound 27 (100, Compound 20, or Compound 14. An image of hlAPP treated with Compound 14 was unable to be obtained. All images are displayed at 20k magnification.
  • ThT Thioflavin T assays performed with 10 pM of hlAPP and flAPP subjected to different treatments at pH 5.5 and 25 °C. Bars represent the average of maximum fluorescence values (triplicate) obtained at the plateau phase after 12 hours for each condition. ThT fluorescence intensities for the control DMSO (0.25%) indicated as hlAPP and flAPP were normalized to 100%. Error bars represent the S.E.M. A) ThT assays were performed with 10 pM of hlAPP in the presence of 0.25% DMSO (indicated as hlAPP) or different compounds 17-24 at a final concentration of 100 pM.
  • ThT assays were performed with 10 pM of flAPP in the presence of 0.25% DMSO (indicated as flAPP) or different compounds 17-24 at a final concentration of 100 pM.
  • C) ThT assays were performed with 10 pM of hlAPP in the presence of 0.25% DMSO (indicated as hlAPP) or compound 24 using different concentrations (25, 50, and 100 pM).
  • D) ThT assays were performed with 10 pM of flAPP in the presence of 0.25% DMSO (indicated as flAPP) or compound 24 using different concentrations (25, 50, and 100 pM).
  • Fig. 7 MIN-6 cell viability in the presence of feline islet amyloid polypeptide (flAPP) with DMSO (0.1%), resveratrol (12.5 pM), compound 24 (6.25 pM and 12.5 pM), and compound 12 (12.5 pM) and the absence of flAPP, as determined by resazurin-based assays.
  • flAPP was tested at 20 pM.
  • the peptide was pre-incubated for 48 hours at a concentration of 1 mM with a 4.8% DMSO in PBS (pH 7.4). The experiment lasted 24 hours for the treatment of the cell.
  • Fig- 9 The ThT fluorescence assay was employed to analyze the kinetic curves of compounds 14T and 6T at a concentration of 100 pM with: A. a-syn (6 pM); B. compound 14T dose-dependent inhibition with varying concentrations (3.125, 6.25, 12.5, 25, 50, 100 pM) against a-syn (6 pM) fibril formation. Data were collected in triplicate for each concentration at the plateau phase over the course of five consecutive time points. The error bars indicate the standard error of the mean (SEM) specific to each condition.
  • SEM standard error of the mean
  • Fig. 10 Thioflavin-T kinetic curve showing the effect of the best anti-fibrillary compound (14T and 6TB) when assessed with different tau isoforms.
  • the compounds were tested at a concentration of 100 pM along with 10 pM (10: 1) of A) tau 0N3R, B) tau 2N3R, C) tau 2N4R.
  • 10 pM (10: 1) of A) tau 0N3R, B) tau 2N3R, C) tau 2N4R Several compounds were tested for their anti-fibrillary effect on the tau 2N3R.
  • the kinetics were performed in the presence of 2.5 pM of heparin, 1 mM of dithiothreitol (DTT), 1 mM of 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, and 30 pM of Thioflavin- T (ThT) in a buffer solution containing 50 mM Tris, 25 mM NaCl, at pH 7.4.
  • the positive control involved tau isoform without compound treatment.
  • the background (BG) signal was obtained with all components in the absence of tau protein and compounds.
  • the depicted curves represent the average data obtained from three technical replicates.
  • Compound 14T mainly prevents a-syn inclusion formation.
  • FIG. 12 Compounds 6T and 14T reduce tau seeding activity in vitro.
  • A Schematic of experimental setup to test the effect of compounds on tau seeding activity.
  • hTauP301S plasmid was transfected to over-express tau in HEK293T cells and the cells were treated with the compounds 24 hours later. Cell viability and tau seeding activity were assessed 48 hr after treatment with compounds.
  • B Viability of HEK cells after treatment with the compounds.
  • C Representative images of FRET signal from biosensor cells after transfection with HEK cell lysates.
  • D The seeding activity of cells overexpressing hTauP301S and after treatment with the compounds.
  • IAPP islet amyloid protein
  • serum amyloid A serum amyloid A
  • alpha-synuclein alpha-synuclein
  • amyloid-beta proteins This new class of compounds possesses the necessary attributes for further drug development due to their selectivity for IAPP, with early in vitro studies suggesting them to be non-toxic.
  • two specific N-phenyl-N'- (2-ethyl)ureas were identified as inhibitors of hlAPP fibril formation, reducing its subsequent cytotoxicity.
  • Described herein is a novel class of small molecules, which contain a urea linker, are highly potent in the prevention of IAPP fibril formation and inhibit toxic IAPP oligomer accumulation.
  • These compounds were developed using a simple synthetic approach utilizing a diverse set of commercially available amines and isocyanates. Compounds from three series (2-aminofluorenes, 4-morpholino anilines, and 4-aminoindoles) were coupled with various substituted isocyanates to generate the urea derivatives. Synthesized compounds were evaluated for their effects on hlAPP and fLAPP fibril formation using Thioflavin T (ThT) to compare their selectivity and antiaggregation potential. The best-performing compounds were then assessed using transmission electron microscopy (TEM) to study their impact on the fibril structure and morphology.
  • TEM transmission electron microscopy
  • R 1 and R 2 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy.
  • Halo can be F, I, Cl, or Br.
  • Mono-halo methyl can be -C(H2)F, -C(H2)I, - C(H 2 )C1 or -C(H 2 )Br.
  • Di-halo methyl can be -C(H)F 2 , -C(H)I 2 , -C(H)C1 2 or -C(H)Br 2 .
  • Tri- halo methyl can be -CF3, -CI3, -CCI3 or -CBrs.
  • the Ci-Ce alkyl can be -CH3.
  • the Ci-Ce alkoxy can be -OCH3.
  • the compound can have the structure:
  • R 1 and R 2 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy; and R 10 is heteroaryl (pyridinyl, pyrrolyl, or imidazolyl) or heterocycloalkyl (morpholinyl or piperazinyl).
  • Halo can be F, I, Cl, or Br.
  • Mono-halo methyl can be -C(H2)F, - C(H 2 )I, -C(H 2 )C1 or -C(H 2 )Br.
  • Di-halo methyl can be -C(H)F 2 , -C(H)I 2 , -C(H)C1 2 or - C(H)Br2.
  • Tri-halo methyl can be -CF3, -CI3, -CCI3 or -CBrs.
  • the Ci-Ce alkyl can be -CH3.
  • the Ci-Ce alkoxy can be -OCH3.
  • the compound can have the structure:
  • R 1 and R 2 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy.
  • Halo can be F, I, Cl, or Br.
  • Mono-halo methyl can be -C(H2)F, -C(H2)I, - C(H 2 )C1 or -C(H 2 )Br.
  • Di-halo methyl can be -C(H)F 2 , -C(H)I 2 , -C(H)C1 2 or -C(H)Br 2 .
  • Tri- halo methyl can be -CF3, -CI3, -CCI3 or -CBr 3 .
  • the Ci-Ce alkyl can be -CH 3 .
  • the Ci-Ce alkoxy can be -OCH3.
  • R 3 and R 4 are each independently H, alkyl or acyl or, R 3 and R 4 , together with the atoms to which they are each attached, form an aryl or heteroaryl group;
  • R 5 and R 6 are each independently H or alkyl or, R 5 and R 6 , together with the atoms to which they are attached, form a heterocyclyl group;
  • R 7 and R 8 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy; or
  • R 7 and R 8 together with the atoms to which they are each attached, form an aryl or heteroaryl group
  • X 1 is NR 5 , O, S or CR 9 2, wherein each R 9 is independently, H, alkyl or acyl;
  • X 2 is O or S. In some embodiments, X 2 can be NR 13 , wherein R 13 is H or alkyl.
  • Examples of halo for compounds of the formula IV are F, I, Cl, and Br.
  • Examples of mono- halo methyl for compounds of the formula IV are -C(H2)F, -C(H2)I, -C(H2)C1, and -C(H2)Br.
  • Examples of di-halo methyl for compounds of the formula IV are -C(H)F2, -C(H)l2, - C(H)Ch, and -C(H)Br2.
  • Examples of tri-halo methyl for compounds of the formula IV are - CF3, -CI3, -CCI3, and -CBrs.
  • An example of alkyl for any alkyl group for compounds of formula IV is Ci-Ce alkyl , which can be -CEE.
  • An example of alkoxy for any alkyl group for compounds of formula IV is Ci-Ce alkoxy, which can be -OCH3.
  • the compound of formula IV can be a compound of the formula:
  • the compound of the formula IV can be a compound of the formula: such as compounds of the formula:
  • the compound of formula IV can be a compound of the formula:
  • X 2 in the compounds of the formula IV can be S.
  • one R 9 in the compounds of the formula IV can be H.
  • both R 9 groups can be H.
  • R 11 is alkyl, cycloalkyl, aryl, arylakyl, or arylacyl
  • R 12 is cycloakyl, aryl, or heterocyclyl
  • R 5 and R 6 are each independently H or alkyl or, R 5 and R 6 , together with the atoms to which they are attached, form a heterocyclyl group;
  • X 2 is O or S.
  • X 2 can be NR 13 , wherein R 13 is H or alkyl.
  • aryl groups representing R 11 include napthyl (e.g., 1-, 2-, or 4-napthyl) and aryl groups substituted with one, two, or three F, Cl, I, acyl, alkyl, alkoxy, alkylthio, and amino.
  • alkyl groups representing R 11 include Ci-Csalkyl.
  • cycloalkyl groups representing R 11 include cyclopentyl and cyclohexyl.
  • arylacyl groups representing R 11 include groups of the formula: .
  • Examples of arylalkyl groups representing R 11 include benzyl and phenethyl.
  • Examples of cycloalkyl groups representing R 12 include cyclopentyl and cyclohexyl.
  • Examples of aryl groups representing R 12 include fluorenyl (e.g., 3-fluorenyl) and aryl groups substituted with one or two haloalkyl (e.g., CF3), amino, morpholino, piperazinyl, and 2- thiazolyl.
  • Examples of heterocyclyl groups representing R 12 include 4-indolyl, 5-indolyl, and 6-indolyl.
  • R 11 and R 12 are not the same, such that the compounds of the formula V are assymetric.
  • the compounds can be used to inhibit aggregation of misfolded islet amyloid protein (IAPP) in a human with, or at risk for, diabetes mellitus.
  • the compounds also can be used to inhibit aggregation of misfolded IAPP in a feline with, or at risk for, diabetes mellitus.
  • the compounds can be formulated as pharmaceutical compositions comprising a pharmaceutically acceptable carrier using methods well-known in the art.
  • Carrier is used generically herein to refer to pharmaceutically acceptable carriers, diluents, adjuvants, and excipients. See, e.g., Remington: The Science and Practice of Pharmacy, 23 rd edition, October 30, 2020, Adeboye Adejare, ed. Accordingly, provided is a pharmaceutical composition comprising at least one compound of Formulae I-V and a pharmaceutically acceptable carrier.
  • a pharmaceutical composition comprising at least one compound of Formulae I-V can further comprise at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus and is selected from the group consisting of a sulfonylurea, a meglitinide, a biguanide, a thiazolidinedione (TZD), an a- glucosidase inhibitor, a dipeptidyl peptidase IV (DPP-4) inhibitor, a bile acid sequestrant, a dopamine agonist, a sodium-glucose transport protein 2 (SGLT2) inhibitor, and a glucagon like peptide 1 (GLP-1) receptor agonist.
  • a sulfonylurea a meglitinide, a biguanide, a thiazolidinedione (TZD), an a- glucosidase inhibitor, a dipeptidyl peptidase IV (DPP-4) inhibitor
  • the sulfonylurea can be at least one compound selected from the group consisting of chlorpropamide, tolazamide, tolbutamide, acetohexamide, glyburide, glipizide, glimepiride, and gliclazide.
  • the meglitinide can be repaglinide, netaglinide, or a combination thereof.
  • the biguanide can be metformin.
  • the TZD can be rosiglitazone, pioglitazone, or a combination thereof.
  • the a-glucosidase inhibitor can be at least one compound selected from the group consisting of acarbose, miglitol and voglibose.
  • the DPP-4 inhibitor can be at least one compound selected from the group consisting of alogliptin, linagliptin, sitagliptin, saxagliptin and vldagliptin.
  • the bile acid sequestrant can be colesevelam.
  • the dopamine agonist can be bromocriptine.
  • the SGLT2 inhibitor can be at least one compound selected from the group consisting of cangliflozin, dapagliflozin, empagliflozin and ertugliflozin.
  • the GLP-1 receptor agonist can be semaglutide.
  • a pharmaceutical composition comprising at least one compound of Formulae I-V can further comprise at least one other compound, which is a combination of compounds selected from (a) metformin and at least one of glyburide, glipizide, sitagliptin, saxagliptin, pioglitazone, repaglinide, rosiglitazone, linagliptin, alogliptin, canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin, (b) glimepiride and either or both of pioglitazone and rosiglitazone, (c) alogliptin and pioglitazone, (d) dapagliflozin and saxagliptin, (e) empagliflozin and linagliptin, (f) ertugliflozin and sitagliptin, (g) lixisenatide
  • a pharmaceutical composition comprising at least one compound of Formulae I-V can further comprise at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus and is selected from the group consisting of a GLP-1 receptor agonist and a dual GLP-1 receptor and GIP receptor agonist.
  • the GLP-1 receptor agonist can be exenatide, liraglutide, dulaglutide, lixisenatide or semaglutide.
  • the dual GLP-1 receptor and GIP receptor agonist can be tirzepatide.
  • the pharmaceutical composition can be formulated for administration by injection.
  • a pharmaceutical composition comprising at least one compound of Formula I can further comprise bexagliflozin.
  • the pharmaceutical composition can be formulated for oral administration.
  • a pharmaceutical composition comprising at least one compound of Formula II can further comprise bexagliflozin.
  • the pharmaceutical composition can be formulated for oral administration.
  • a pharmaceutical composition comprising at least one compound of Formula III can further comprise bexagliflozin.
  • the pharmaceutical composition can be formulated for oral administration.
  • a pharmaceutical composition comprising at least one compound of Formula IV can further comprise bexagliflozin.
  • the pharmaceutical composition can be formulated for oral administration.
  • a pharmaceutical composition comprising at least one compound of Formula V can further comprise bexagliflozin.
  • the pharmaceutical composition can be formulated for oral administration.
  • a method of inhibiting aggregation of misfolded islet amyloid protein (IAPP) in a human with, or at risk for, diabetes mellitus comprises administering to the human the compound of Formulae I-V, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit aggregation of misfolded IAPP, whereupon aggregation of misfolded IAPP is inhibited in the human with, or at risk for, diabetes mellitus.
  • IAPP misfolded islet amyloid protein
  • the method can further comprise administering, simultaneously or sequentially by the same or different routes, at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus and which is selected from the group consisting of a sulfonylurea, a meglitinide, a biguanide, a TZD, an a-glucosidase inhibitor, a DPP-4 inhibitor, a bile acid sequestrant, a dopamine agonist, a SGLT2 inhibitor, and a GLP-1 receptor agonist.
  • a sulfonylurea a meglitinide, a biguanide, a TZD, an a-glucosidase inhibitor, a DPP-4 inhibitor, a bile acid sequestrant, a dopamine agonist, a SGLT2 inhibitor, and a GLP-1 receptor agonist.
  • the sulfonylurea can be at least one compound selected from the group consisting of chlorpropamide, tolazamide, tolbutamide, acetohexamide, glyburide, glipizide, glimepiride, and gliclazide.
  • the meglitinide can be repaglinide, netaglinide, or a combination thereof.
  • the biguanide can be metformin.
  • the TZD can be rosiglitazone, pioglitazone, or a combination thereof.
  • the a-glucosidase inhibitor can be at least one compound selected from the group consisting of acarbose, miglitol and voglibose.
  • the DPP-4 inhibitor can be at least one compound selected from the group consisting of alogliptin, linagliptin, sitagliptin, saxagliptin and vldagliptin.
  • the bile acid sequestrant can be colesevelam.
  • the dopamine agonist can be bromocriptine.
  • the SGLT2 inhibitor can be at least one compound selected from the group consisting of cangliflozin, dapagliflozin, empagliflozin and ertugliflozin.
  • the GLP-1 receptor agonist can be semaglutide.
  • the at least one other compound can be a combination of compounds selected from (a) metformin and at least one of glyburide, glipizide, sitagliptin, saxagliptin, pioglitazone, repaglinide, rosiglitazone, linagliptin, alogliptin, canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin, (b) glimepiride and either or both of pioglitazone and rosiglitazone, (c) alogliptin and pioglitazone, (d) dapagliflozin and saxagliptin, (e) empagliflozin and linagliptin, (f) ertugliflozin and sitagliptin, (g) lixisenatide and glargine insulin, and (h) liraglutide
  • the method comprising administering to the human the compound of Formulae I-V, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit aggregation of misfolded IAPP can further comprise administering at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus, wherein the at least one other compound is selected from the group consisting of a GLP-1 receptor agonist and a dual GLP-1 receptor and GIP receptor agonist.
  • the GLP-1 receptor agonist can be exenatide, liraglutide, dulaglutide, lixisenatide or semaglutide.
  • the dual GLP-1 receptor and GIP receptor agonist can be tirzepatide.
  • the administering can be by injection.
  • a method of inhibiting aggregation of misfolded IAPP in a cat with, or at risk for, diabetes mellitus comprises administering to the cat the compound of Formulae I-V, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit aggregation of misfolded IAPP, whereupon aggregation of misfolded IAPP is inhibited in the cat with, or at risk for, diabetes mellitus.
  • the method can further comprise administering bexafliflozin.
  • the administering can be orally administering.
  • the method can further comprise administering insulin.
  • the administering of the compound of Formula I-V can be orally, and the administering of the insulin can be by injection.
  • tubulin-associated unit (tau) protein aggregation in a subject having, or at risk for, tau protein aggregation.
  • the method comprises administering to the subject an above-described pharmaceutical composition in an amount effective to inhibit tau protein aggregation.
  • the subject can have, or be at risk for, Alzheimer’s disease.
  • a method of inhibiting alpha-synuclein (a-syn) protein aggregation in a subject having, or at risk for a-syn comprises administering to the subject an above-described pharmaceutical composition in an amount effective to inhibit a- syn protein aggregation.
  • the subject can have, or be at risk for, Parkinson’s disease.
  • an effective amount can be determined by one of ordinary skill in the art using dosage range determining methods known in the art.
  • a physician or veterinarian for non-human subjects
  • the specific dose level and frequency of dosage for an individual may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, gender, diet, mode and time of administration, rate of excretion, other administered drugs, and the severity of the condition.
  • the above compounds include isotopic variants and compounds in which one or more hydrogen atoms have been substituted with deuterium.
  • the compounds may contain one or more chiral centers or may otherwise be capable of existing as multiple stereoisomers.
  • the compounds are not limited to any particular stereochemical requirement, and that the compounds, and compositions, methods, uses, and medicaments that include them may be optically pure, or may be any of a variety of stereoisomeric mixtures, including racemic and other mixtures of enantiomers, other mixtures of diastereomers, and the like.
  • Such mixtures of stereoisomers may include a single stereochemical configuration at one or more chiral centers, while including mixtures of stereochemical configuration at one or more other chiral centers.
  • the compounds may include geometric centers, such as cis, trans isomers, diastereomers, enantiomers, and E and Z double bonds.
  • the compounds are not limited to any particular geometric isomer requirement, and that the compounds, and compositions, methods, uses, and medicaments that include them may be pure, or may be any of a variety of geometric isomer mixtures.
  • Such mixtures of geometric isomers may include a single configuration at one or more double bonds and chiral carbons, while including mixtures of geometry at one or more other double bonds and chiral carbons.
  • salts and “pharmaceutically acceptable salts” refer to derivatives of the compounds wherein the parent compound is modified by making acid or base salts thereof.
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic groups such as amines; and alkali or organic salts of acidic groups such as carboxylic acids.
  • Pharmaceutically acceptable salts include the conventional nontoxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, and isethionic, and the like.
  • inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric
  • organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic,
  • salts can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods.
  • such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
  • Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, the disclosure of which is hereby incorporated by reference for its teachings regarding same.
  • solvate means a compound, or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. Where the solvent is water, the solvate is a hydrate.
  • any suitable route of administration can be used in the above methods. Examples include, but are not limited to, oral, parenteral, intravenous, intracranial, intracerebroventricular, and intracerebral.
  • An effective amount can be determined by one of ordinary skill in the art using dosage range determining methods known in the art. Typically, a physician (or veterinarian for non-human subjects) will determine the actual dosage, which will be most suitable for an individual subject.
  • the specific dose level and frequency of dosage for an individual may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, gender, diet, mode and time of administration, rate of excretion, other administered drugs, and the severity of the particular condition.
  • the compound/compositions described herein can be administered with other biologically active compounds as appropriate.
  • substituted refers to a group that can be or is substituted onto a molecule or onto another group (e.g., on an aryl or an alkyl group).
  • substituents include, but are not limited to, a halogen (e.g., F, Cl, Br, and I), OR, OC(O)N(R) 2 , CN, NO, NO2, ONO2, azido, CF 3 , OCF3, R, O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, -(CEhjo- 2 P(O)(OR)2, C(O)R, C(O)C(O)R, C(O)CH 2 C(O)R, C(S)R, C(O)OR, OC(O)R, C(0)N
  • a halogen e.g., F, Cl,
  • alkyl refers to substituted or unsubstituted straight chain and branched mono- or divalent alkyl groups and cycloalkyl groups having from 1 to 40 carbon atoms (C1-C40), 1 to about 20 carbon atoms (C1-C20), 1 to 12 carbons (C1-C12), 1 to 8 carbon atoms (Ci-Cs), or, in some embodiments, from 1 to 6 carbon atoms (Ci-Ce).
  • straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups.
  • branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups.
  • alkyl encompasses n-alkyl, isoalkyl, and c c-isoalkyl groups as well as other branched chain forms of alkyl.
  • Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.
  • alkenyl refers to substituted or unsubstituted straight chain and branched mono- or divalent alkenyl groups and cycloalkenyl groups having at least one double bond and having from 1 to 40 carbon atoms (C1-C40), 1 to about 20 carbon atoms (Ci- C20), 1 to 12 carbons (C1-C12), 1 to 8 carbon atoms (Ci-Cs), or, in some embodiments, from 1 to 6 carbon atoms (Ci-Ce).
  • Representative substituted alkenyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.
  • cycloalkyl refers to substituted or unsubstituted cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups.
  • the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7.
  • Cycloalkyl groups can have any number of carbon atoms, e.g., 3 to 8 carbon atoms (Cs-Cs), 3 to 6 carbon atoms (C3-C6), and 4 to 8 carbon atoms (C4-Cs). Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbomyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like.
  • cycloalkylalkyl refers to substituted or unsubstituted alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group as defined herein is replaced with a bond to a cycloalkyl group as defined herein.
  • Representative cycloalkylalkyl groups include, but are not limited to, cyclopentylalkyl.
  • alkylcycloalkyl refers to substituted or unsubstituted cycloalkyl groups as defined herein in which a hydrogen of a cycloalkyl group as defined herein is replaced with a bond to an alkyl group as defined herein.
  • Representative alkylcycloalkyl groups include, but are not limited to, alkylcyclopropyl.
  • acyl refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom.
  • the carbonyl carbon atom is also bonded to another carbon atom, which can be part of a substituted or unsubstituted alkyl, aryl, aralkyl cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl group or the like.
  • the group is a “formyl” group, an acyl group as the term is defined herein.
  • An acyl group can include 0 to about 12-40, 6-10, 1-5 or 2-5 additional carbon atoms bonded to the carbonyl group.
  • An acryloyl group is an example of an acyl group.
  • An acyl group can also include heteroatoms within the meaning here.
  • a nicotinoyl group (pyridyl-3 -carbonyl) is an example of an acyl group within the meaning herein.
  • Other examples include acetyl, benzoyl, phenylacetyl, pyridyl acetyl, cinnamoyl, and acryloyl groups and the like.
  • the group containing the carbon atom that is bonded to the carbonyl carbon atom contains a halogen, the group is termed a “haloacyl” group.
  • An example is a trifluoroacetyl group.
  • heterocyclylcarbonyl is an example of an acyl group that is bonded to a substituted or unsubstituted heterocyclyl group, as the term “heterocyclyl” is defined herein.
  • An example of a heterocyclylcarbonyl group is a prolyl group, wherein the prolyl group can be a D- or an L-prolyl group.
  • aryl refers to substituted or unsubstituted cyclic aromatic hydrocarbons that do not contain heteroatoms in the ring.
  • aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups.
  • aryl groups contain about 6 to about 14 carbons (Ce-Cu) or from 6 to 10 carbon atoms (Ce-Cio) in the ring portions of the groups.
  • Aryl groups can be unsubstituted or substituted, as defined herein.
  • “Aryl” and the phrase “aryl group” includes fused ring species including those that include fused aromatic and non-aromatic groups. Accordingly, “aryl” and the phrase “aryl group” include groups of the formula: , each of which can be substituted or unsubstituted, such as hydroxy substituted.
  • Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or 2-8 substituted naphthyl groups, which can be substituted with carbon or non-carbon groups such as those listed herein.
  • aralkyl and arylalkyl refer to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein.
  • Representative aralkyl groups include benzyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl.
  • Aralkenyl groups are alkenyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein.
  • heterocyclyl refers to substituted or unsubstituted aromatic and non-aromatic ring compounds containing 3 or more ring members, of which one or more (e.g., 1, 2 or 3) is a heteroatom such as, but not limited to, N, O, and S.
  • a heterocyclyl can be a cycloheteroalkyl or a heteroaryl or, if polycyclic, any combination thereof.
  • heterocyclyl groups include 3 to about 20 ring members, whereas other such groups have 3 to about 15 ring members.
  • heterocyclyl groups include heterocyclyl groups that include 3 to 8 carbon atoms (Cs-Cs), 3 to 6 carbon atoms (Cs-Ce), 3 to 5 carbon atoms (C3-C5) or 6 to 8 carbon atoms (Ce-Cs).
  • a heterocyclyl group designated as a C2-heterocyclyl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and fthe heteroatoms and so forth.
  • a C4-heterocyclyl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth.
  • a heterocyclyl ring can also include one or more double bonds, such as in the group 3,6-dihydro-2H-pyran and 3,4-dihydro-2H-pyran, having the formula: respectively, each of which can be substituted.
  • heteroaryl ring is an embodiment of a heterocyclyl group.
  • heterocyclyl group includes fused ring species including those that include fused aromatic and non- aromatic groups.
  • Representative heterocyclyl groups include, but are not limited to tetrahydro-2H-thiopyran- 1,1 -di oxide, having the formula:
  • isoindolinonyl groups include groups having the general formula: , wherein R is as defined herein.
  • benzoxazolinyl groups include groups having the general formula: , wherein R is as defined herein.
  • benzthiazolinyl groups include groups having the general formula: , wherein R is as defined herein.
  • the group R in benzoxazolinyl and benzthiazolinyl groups is an N(R) 2 group.
  • each R is hydrogen or alkyl, wherein the alkyl group is substituted or unsubstituted.
  • the alkyl group is substituted with a heterocyclyl group (e.g., with a pyrrolidinyl group).
  • heterocyclylalkyl refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group as defined herein is replaced with a bond to a heterocyclyl group as defined herein.
  • heterocyclylalkyl groups include, but are not limited to, furan-2-yl methyl, furan-3-yl methyl, pyridine-3-yl methyl, tetrahydrofuran-2-yl methyl, and indol-2-yl propyl.
  • heterocyclylalkoxy refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group as defined herein is replaced with a bond to a heterocyclyl group as defined herein and the alkyl group is attached to an oxygen.
  • Representative heterocyclylalkoxy groups include, but are not limited to, -O- (CH2)qheterocyclyl, wherein q is an integer from 1 to 5.
  • heterocyclylalkoxy groups include -O-(CH2) q morpholinyl such as -O-CH2CH2-morpholine.
  • heteroarylalkyl refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined herein.
  • alkoxy refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein.
  • linear alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like.
  • branched alkoxy include, but are not limited to, isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like.
  • cyclic alkoxy include, but are not limited to, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like.
  • An alkoxy group can include one to about 12-20 or about 12-40 carbon atoms bonded to the oxygen atom, can further include double or triple bonds, and can also include heteroatoms.
  • an allyloxy group is an alkoxy group within the meaning herein.
  • a methoxy ethoxy group is also an alkoxy group within the meaning herein, as is a methylenedi oxy group in a context where two adjacent atoms of a structure are substituted therewith.
  • amine refers to a substituent of the form -NH2, -NHR, -NR2, or -NRE, wherein each R is defined herein, and protonated forms of each, except for -NR3 , which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine.
  • An “amino group” within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group.
  • alkylamino group includes a monoalkylamino, dialkylamino, and trialkylamino group.
  • An example of a “alkylamino” is -NH-alkyl and -N(alkyl)2.
  • An example of a “cycloalkylamino” group is -NH-cycloalkyl and -N(cycloalkyl)2.
  • cycloalkyl heterocycloamino is -NH-(heterocyclo cycloalkyl), wherein the heterocyclo group is attached to the nitrogen and the cycloalkyl group is attached to the heterocyclo group.
  • heterocyclo cycloamino group is -NH-(cycloalkyl heterocycle), wherein the cycloalkyl group is attached to the nitrogen and the heterocyclo group is attached to the cycloalkyl group.
  • halo means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
  • haloalkyl group includes mono-halo alkyl groups, poly-halo alkyl groups, wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro.
  • haloalkyl include trifluoromethyl, 1,1 -di chloroethyl, 1,2-di chloroethyl, l,3-dibromo-3,3-difluoropropyl, perfluorobutyl, -CF(CH3)2 and the like.
  • treat is an approach for obtaining beneficial or desired results including and preferably clinical results and includes, but is not limited to, one or more of the following: improving a condition associated with a disease, curing a disease, lessening severity of a disease, delaying progression of a disease, alleviating one or more symptoms associated with a disease, increasing the quality of life of one suffering from a disease, prolonging survival and/or prophylactic or preventative treatment.
  • an “effective amount” refers to any amount that is sufficient to achieve a desired biological effect. Combined with the teachings provided herein, by choosing among the various active conjugates or compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial unwanted toxicity and yet is effective to treat the particular subject.
  • the effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular compound being administered, the size of the subject, or the severity of the disease or condition.
  • One of ordinary skill in the art can empirically determine the effective amount of a particular compound and/or other therapeutic agent without necessitating undue experimentation.
  • a maximum dose can be used, that is, the highest safe dose according to some medical judgment. Multiple doses per day can be used to achieve appropriate systemic levels of compounds. Appropriate systemic levels can be determined by, for example, measurement of the patient’s peak or sustained plasma level of the drug. “Dose” and “dosage” are used interchangeably herein.
  • daily oral doses of a compound are, for human subjects, from about 0.01 milligrams/kg per day to 1,000 milligrams/kg per day. Oral doses in the range of 0.5 to 50 milligrams/kg, in one or more administrations per day, can yield therapeutic results. Dosage can be adjusted appropriately to achieve desired drug levels, local or systemic, depending upon the mode of administration. For example, intravenous administration can vary from one order to several orders of magnitude lower dose per day. If the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) can be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of the compound.
  • a “therapeutically effective amount” (or “effective amount”) of a compound with respect to use in treatment refers to an amount of the compound in a preparation which, when administered as part of a desired dosage regimen (to a mammal, such as a human) alleviates a symptom, ameliorates a condition, or slows the onset of disease conditions according to clinically acceptable standards for the disorder or condition to be treated or the cosmetic purpose, e.g., at a reasonable benefit/risk ratio applicable to any medical treatment.
  • a therapeutically effective amount can be initially determined from animal models.
  • a therapeutically effective dose can also be determined from human data for compounds which have been tested in humans and for compounds which are known to exhibit similar pharmacological activities, such as other related active agents. Higher doses may be required for parenteral administration.
  • the applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan.
  • the formulations can be administered in pharmaceutically acceptable solutions, which can routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.
  • an effective amount of the compound can be administered to a subject by any mode that delivers the compound to the desired surface.
  • Administering a pharmaceutical composition can be accomplished by any means known to the skilled artisan. Routes of administration include, but are not limited to, intravenous, intramuscular, intraperitoneal, intravesical (urinary bladder), oral, subcutaneous, direct injection (for example, into a tumor or abscess), mucosal (e.g., topical to eye), inhalation, and topical.
  • a compound can be formulated as a lyophilized preparation, as a lyophilized preparation of liposome-intercalated or -encapsulated active compound, as a lipid complex in aqueous suspension, or as a salt complex.
  • Lyophilized formulations are generally reconstituted in suitable aqueous solution, e.g., in sterile water or saline, shortly prior to administration.
  • the compounds can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well-known in the art.
  • Such carriers enable the compounds to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated.
  • a pharmaceutical preparation for oral use can be obtained as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, and sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone (PVP).
  • disintegrating agents can be added, such as the cross-linked PVP, agar, or alginic acid or a salt thereof such as sodium alginate.
  • the oral formulations can also be formulated in saline or buffers, e.g., EDTA for neutralizing internal acid conditions, or can be administered without any carriers.
  • the compounds can be chemically modified so that oral delivery of the derivative is efficacious.
  • the chemical modification contemplated is the attachment of at least one moiety to the compound itself, where said moiety permits (a) inhibition of acid hydrolysis; and (b) uptake into the blood stream from the stomach or intestine.
  • the increase in overall stability of the compounds and increase in circulation time in the body examples include polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, PVP and polyproline.
  • the location of release of a compound hereof can be the stomach, the small intestine (e.g., the duodenum, the jejunum, or the ileum), or the large intestine.
  • the small intestine e.g., the duodenum, the jejunum, or the ileum
  • One skilled in the art has available formulations, which will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine.
  • the release can avoid the deleterious effects of the stomach environment, either by protection of the compound or by release of the compound beyond the stomach environment, such as in the intestine.
  • a coating impermeable to at least pH 5.0 is essential.
  • examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and shellac. These coatings can be used as mixed films.
  • a coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make the tablet easier to swallow.
  • Capsules can consist of a hard shell (such as gelatin) for delivery of dry therapeutic (e.g., powder); for liquid forms, a soft gelatin shell can be used.
  • the shell material of cachets could be thick starch or other edible paper. For pills, lozenges, molded tablets or tablet triturates, moist massing techniques can be used.
  • the compound can be included in the formulation as fine multi-particulates in the form of granules or pellets of particle size about 1 mm.
  • the formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets.
  • Therapeutic agent could be prepared by compression.
  • Colorants and flavoring agents may all be included.
  • the compound can be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.
  • diluents can include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch.
  • Certain inorganic salts also can be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride.
  • Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.
  • Disintegrants can be included in the formulation of therapeutic agent into a solid dosage form.
  • Materials used as disintegrates include, but are not limited to, starch, including the commercial disintegrant based on starch, Explotab.
  • Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used.
  • Another form of the disintegrant is the insoluble cationic exchange resin.
  • Powdered gums can be used as disintegrants and as binders, and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.
  • Binders can be used to hold the compound together to form a hard tablet and can include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). PVP and hydroxypropylmethyl cellulose (HPMC) can both be used in alcoholic solutions to granulate therapeutic agent.
  • MC methyl cellulose
  • EC ethyl cellulose
  • CMC carboxymethyl cellulose
  • HPMC hydroxypropylmethyl cellulose
  • Lubricants can be used as a layer between therapeutic agent and the die wall, and these can include, but are not limited to, stearic acid, including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants can also be used, such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.
  • Glidants which can improve the flow properties of the drug during formulation and aid rearrangement during compression, can be added.
  • the glidants can include starch, talc, pyrogenic silica and hydrated silicoaluminate.
  • surfactant can be added as a wetting agent.
  • Surfactants can include anionic detergents, such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
  • anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
  • Cationic detergents which can be used, include benzalkonium chloride and benzethonium chloride.
  • Nonionic detergents that can be included in the formulation as surfactants include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the compound or derivative thereof either alone or as a mixture in different ratios.
  • Pharmaceutical preparations, which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • suitable liquids such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers can be added.
  • Microspheres formulated for oral administration can also be used. Such microspheres have been well-defined in the art. All formulations for oral administration should be in dosages suitable for such administration.
  • compositions can take the form of tablets or lozenges formulated in conventional manner.
  • the compound can be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art.
  • Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal oral or pulmonary administration.
  • compounds can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, di chlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, di chlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit can be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.
  • the compound is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream.
  • inhaled molecules include Adjei et al., Pharm Res 7:565-569 (1990); Adjei et al., Int J Pharmaceutics 63: 135-144 (1990) (leuprolide acetate); Braquet et al., J Cardiovasc Pharmacol 13(suppl.
  • Contemplated for use are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products including, but not limited to, nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.
  • Nasal delivery of a pharmaceutical composition is also contemplated.
  • Nasal delivery allows the passage of a pharmaceutical composition to the blood stream directly after administering therapeutic product to the nose, without the necessity for deposition of the product in the lung.
  • Formulations for nasal delivery include those with dextran or cyclodextran.
  • the compounds when it is desirable to deliver them systemically, can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multidose containers, with an added preservative.
  • the compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension can also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the active compounds can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • the compounds can also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • a compound in addition to the formulations described above, can also be formulated as a depot preparation.
  • Such long-acting formulations can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • compositions also can comprise suitable solid or gel phase carriers or excipients.
  • suitable solid or gel phase carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosolized, pelleted for implantation into the skin, or dried onto a sharp object to be scratched into the skin.
  • the pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above.
  • the pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer R, Science 249: 1527-1533 (1990).
  • the compound and optionally one or more other therapeutic agents can be administered per se (neat) or in the form of a pharmaceutically acceptable salt.
  • the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof.
  • Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2- sulphonic, and benzene sulphonic.
  • such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
  • Suitable buffering agents include acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v).
  • Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v); and thimerosal (0.004-0.02% w/v).
  • compositions contain an effective amount of a compound as described herein and optionally one or more other therapeutic agents included in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier means one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to a human or other vertebrate animal.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • the components of the pharmaceutical compositions also can be commingled with the compounds, and with each other, in a manner such that there is no interaction, which would substantially impair the desired pharmaceutical efficiency.
  • Therapeutic agent(s), including specifically, but not limited to, a compound, can be provided in particles.
  • “Particles” means nanoparticles or microparticles (or in some instances larger particles) that can consist in whole or in part of the compound or the other therapeutic agent(s).
  • the particles can contain therapeutic agent(s) in a core surrounded by a coating, including, but not limited to, an enteric coating.
  • Therapeutic agent(s) also can be dispersed throughout the particles.
  • Therapeutic agent(s) also can be adsorbed into the particles.
  • the particles can be of any order release kinetics, including zero-order release, first-order release, second-order release, delayed release, sustained release, immediate release, and any combination thereof, etc.
  • the particle can include, in addition to therapeutic agent(s), any of those materials routinely used in the art of pharmacy and medicine, including, but not limited to, erodible, nonerodible, biodegradable, or nonbiodegradable material or combinations thereof.
  • the particles can be microcapsules which contain the compound in a solution or in a semi-solid state.
  • the particles can be of virtually any shape.
  • Both non-biodegradable and biodegradable polymeric materials can be used in the manufacture of particles for delivering therapeutic agent(s).
  • Such polymers can be natural or synthetic polymers. The polymer is selected based on the period of time over which release is desired.
  • Bioadhesive polymers of particular interest include bioerodible hydrogels described in Sawhney et al., Macromolecules 26:5823-2787 (1993), the teachings of which are specifically incorporated by reference herein.
  • polyhyaluronic acids casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly (isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).
  • Control release refers to any drug-containing formulation in which the manner and profile of drug release from the formulation are controlled. This refers to immediate as well as non-immediate release formulations, with non-immediate release formulations including, but not limited to, sustained release and delayed release formulations.
  • sustained release also referred to as “extended release” refers to a drug formulation that provides for gradual release of a drug over an extended period of time, and that can result in substantially constant blood levels of a drug over an extended time period.
  • delayed release refers to a drug formulation in which there is a time delay between administration of the formulation and the release of the drug therefrom. “Delayed release” may or may not involve gradual release of drug over an extended period of time, and thus may or may not be “sustained release.”
  • Long-term sustained-release implant can be particularly suitable for treatment of chronic conditions.
  • “Long-term” release means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days, and up to 30-60 days.
  • Long-term sustained-release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
  • prodrug means a derivative of a compound that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide an active compound, particularly a compound of the invention.
  • prodrugs include, but are not limited to, derivatives and metabolites of a compound of the invention that include biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues.
  • Specific prodrugs of compounds with carboxyl functional groups are the lower alkyl esters of the carboxylic acid.
  • the carboxylate esters are conveniently formed by esterifying any of the carboxylic acid moieties present on the molecule.
  • Prodrugs can typically be prepared using well-known methods, such as those described by Burger’s Medicinal Chemistry and Drug Discovery 6th ed. (Donald J. Abraham ed., 2001, Wiley) and Design and Application of Prodrugs (H. Bundgaard ed., 1985, Harwood Academic Publishers GmbH).
  • the formulae include and represent not only all pharmaceutically acceptable salts of the compounds, but also include any and all hydrates and/or solvates of the compound formulae or salts thereof. It is to be appreciated that certain functional groups, such as the hydroxy, amino, and like groups form complexes and/or coordination compounds with water and/or various solvents, in the various physical forms of the compounds. Accordingly, the above formulae are to be understood to include and represent those various hydrates and/or solvates. In each of the foregoing and following embodiments, it is also to be understood that the formulae include and represent each possible isomer, such as stereoisomers and geometric isomers, both individually and in any and all possible mixtures. In each of the foregoing and following embodiments, it is also to be understood that the formulae include and represent any and all crystalline forms, partially crystalline forms, and non-crystalline and/or amorphous forms of the compounds.
  • pharmaceutically acceptable carrier refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof.
  • a pharmaceutically acceptable material such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof.
  • Each carrier must be "acceptable” in the sense of being compatible with the subject composition and its components and not injurious to the patient.
  • materials which may serve as pharmaceutically acceptable carriers, include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydro
  • administering includes all means of introducing the compounds and compositions described herein to the patient, including, but are not limited to, oral (po), intravenous (iv), intramuscular (im), subcutaneous (sc), transdermal, inhalation, buccal, ocular, sublingual, vaginal, rectal, and the like.
  • the compounds and compositions may be administered in unit dosage forms and/or formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles.
  • Illustrative formats for oral administration include tablets, capsules, elixirs, syrups, and the like.
  • Illustrative routes for parenteral administration include intravenous, intraarterial, intraperitoneal, epidural, intraurethral, intrastemal, intramuscular and subcutaneous, as well as any other art recognized route of parenteral administration.
  • parenteral administration examples include needle (including microneedle) injectors, needle-free injectors and infusion techniques, as well as any other means of parenteral administration recognized in the art.
  • Parenteral formulations are typically aqueous solutions, which may contain excipients such as salts, carbohydrates and buffering agents (preferably at a pH in the range from about 3 to about 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.
  • a suitable vehicle such as sterile, pyrogen-free water.
  • the preparation of parenteral formulations under sterile conditions for example, by lyophilization, may readily be accomplished using standard pharmaceutical techniques well- known to those skilled in the art.
  • Parenteral administration of a compound is illustratively performed in the form of saline solutions or with the compound incorporated into liposomes.
  • a solubilizer such as ethanol can be applied.
  • each compound of the claimed combinations depends on several factors, including: the administration method, the condition to be treated, the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the person to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular patient may affect the dosage regimen used.
  • the individual components of a co-administration, or combination can be administered by any suitable means, contemporaneously, simultaneously, sequentially in either order, separately or in a single pharmaceutical formulation.
  • the number of dosages administered per day for each compound may be the same or different.
  • the compounds or compositions may be administered via the same or different routes of administration.
  • the compounds or compositions may be administered according to simultaneous or alternating regimens, at the same or different times during the course of the therapy, concurrently in divided or single forms.
  • therapeutically effective amount refers to that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated.
  • the therapeutically effective amount is that which may treat or alleviate the disease or symptoms of the disease at a reasonable benefit/risk ratio applicable to any medical treatment.
  • the total daily usage of the compounds and compositions described herein may be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular patient will depend upon a variety of factors, including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, gender and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidentally with the specific compound employed; and like factors well-known to the researcher, veterinarian, medical doctor or other clinician of ordinary skill.
  • a wide range of permissible dosages are contemplated, including doses falling in the range from about 1 pg/kg to about 1 g/kg.
  • the dosages may be single or divided and may administered according to a wide variety of protocols, including q.d. (once a day), b.i.d. (twice a day), t.i.d. (three times a day), or even every other day, once a week, once a month, once a quarter, and the like.
  • the described therapeutically effective amounts correspond to the instance of administration, or alternatively to the total daily, weekly, month, or quarterly dose, as determined by the dosing protocol.
  • an effective amount of any one or a mixture of the compounds can be determined by the attending diagnostician or physician by the use of known techniques and/or by observing results obtained under analogous circumstances.
  • determining the effective amount or dose a number of factors are considered by the attending diagnostician or physician, including, but not limited to, the species of mammal, including human, its size, age, and general health, the specific disease or disorder involved, the degree of or involvement or the severity of the disease or disorder, the response of the individual patient, the particular compound administered, the mode of administration, the bioavailability characteristics of the preparation administered, the dose regimen selected, the use of concomitant medication, and other relevant circumstances.
  • the term “patient” includes human and non-human animals such as companion animals (dogs and cats and the like) and livestock animals. Livestock animals are animals raised for food production.
  • the patient to be treated is preferably a mammal, in particular a human.
  • the disclosure relates to, among other things, the following enumerated Embodiments, which listing does not represent an order of importance:
  • R 3 and R 4 are each independently H, alkyl or acyl; R 3 and R 4 , together with the atoms to which they are each attached, form an aryl or heteroaryl group; or the group NR 5 is connected at R 3 or R 4 ;
  • R 5 and R 6 are each independently H or alkyl or, R 5 and R 6 , together with the atoms to which they are attached, form a heterocyclyl group;
  • R 7 and R 8 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy; or
  • R 7 and R 8 together with the atoms to which they are each attached, form an aryl or heteroaryl group
  • X 1 is NR 5 , O, S or CR 9 2, wherein each R 9 is independently, H, alkyl or acyl;
  • X 2 is O or S.
  • R 3 and R 4 are each independently H, alkyl or acyl or, R 3 and R 4 , together with the atoms to which they are each attached, form an aryl or heteroaryl group;
  • R 5 and R 6 are each independently H or alkyl or, R 5 and R 6 , together with the atoms to which they are attached, form a heterocyclyl group;
  • R 7 and R 8 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy; or
  • R 7 and R 8 together with the atoms to which they are each attached, form an aryl or heteroaryl group
  • X 1 is NR 5 , O, S or CR 9 2, wherein each R 9 is independently, H, alkyl or acyl; and X 2 is O or S.
  • R 1 and R 2 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy.
  • a compound of formula Ila Formula Ila wherein R 1 and R 2 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy; and R 10 is heteroaryl or heterocycloalkyl.
  • a compound of formula III Formula III wherein R 1 and R 2 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy.
  • R 11 is alkyl, cycloalkyl, aryl, arylakyl, or arylacyl
  • R 12 is cycloakyl, aryl, or heterocyclyl
  • R 5 and R 6 are each independently H or alkyl or, R 5 and R 6 , together with the atoms to which they are attached, form a heterocyclyl group; and X 2 is O or S.
  • a pharmaceutical composition comprising at least one compound of any one of Embodiments 1-38 and a pharmaceutically acceptable carrier.
  • Embodiment 40 which further comprises at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus, wherein the at least one other compound is selected from the group consisting of a sulfonylurea, a meglitinide, a biguanide, a thiazolidinedione (TZD), an a-glucosidase inhibitor, a dipeptidyl peptidase IV (DPP -4) inhibitor, a bile acid sequestrant, a dopamine agonist, a sodium-glucose transport protein 2 (SGLT2) inhibitor, and a glucagon like peptide 1 (GLP-1) receptor agonist.
  • a sulfonylurea a meglitinide, a biguanide, a thiazolidinedione (TZD), an a-glucosidase inhibitor, a dipeptidyl peptidase IV (DPP -4) inhibitor,
  • the sulfonylurea is at least one compound selected from the group consisting of chlorpropamide, tolazamide, tolbutamide, acetohexamide, glyburide, glipizide, glimepiride, and gliclazide,
  • the meglitinide is repaglinide, netaglinide, or a combination thereof,
  • the TZD is rosiglitazone, pioglitazone, or a combination thereof
  • the a-glucosidase inhibitor is at least one compound selected from the group consisting of acarbose, miglitol and voglibose,
  • the DPP-4 inhibitor is at least one compound selected from the group consisting of alogliptin, linagliptin, sitagliptin, saxagliptin and vldagliptin,
  • the SGLT2 inhibitor is at least one compound selected from the group consisting of cangliflozin, dapagliflozin, empagliflozin and ertugliflozin, and
  • Embodiment 43 The pharmaceutical composition of Embodiment 41, wherein the at least one other compound is a combination of compounds selected from: (a) metformin and at least one of glyburide, glipizide, sitagliptin, saxagliptin, pioglitazone, repaglinide, rosiglitazone, linagliptin, alogliptin, canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin,
  • Embodiment 40 which further comprises at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus, wherein the at least one other compound is selected from the group consisting of a GLP-1 receptor agonist and a dual GLP-1 receptor and gastric inhibitory polypeptide (GIP) receptor agonist.
  • GIP gastric inhibitory polypeptide
  • the GLP-1 receptor agonist is exenatide, liraglutide, dulaglutide, lixisenatide or semaglutide, and
  • Embodiment 40 which further comprises bexagliflozin.
  • Embodiment 48 which is formulated for oral administration. a pharmaceutically acceptable carrier.
  • a method of inhibiting aggregation of misfolded islet amyloid protein (IAPP) in a human with, or at risk for, diabetes mellitus which method comprises administering to the human the compound of any one of Embodiments 1-39, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit aggregation of misfolded IAPP, whereupon aggregation of misfolded IAPP is inhibited in the human with, or at risk for, diabetes mellitus.
  • IAPP misfolded islet amyloid protein
  • Embodiment 50 which further comprises administering, simultaneously or sequentially by the same or different routes, at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus and which is selected from the group consisting of a sulfonylurea, a meglitinide, a biguanide, a thiazolidinedione (TZD), an a-glucosidase inhibitor, a dipeptidyl peptidase IV (DPP-4) inhibitor, a bile acid sequestrant, a dopamine agonist, a sodium-glucose transport protein 2 (SGLT2) inhibitor, and a glucagon like peptide 1 (GLP-1) receptor agonist.
  • a sulfonylurea a meglitinide, a biguanide, a thiazolidinedione (TZD), an a-glucosidase inhibitor, a dipeptidyl peptidase IV (DPP
  • the sulfonylurea is at least one compound selected from the group consisting of chlorpropamide, tolazamide, tolbutamide, acetohexamide, glyburide, glipizide, glimepiride, and gliclazide,
  • the meglitinide is repaglinide, netaglinide, or a combination thereof,
  • the TZD is rosiglitazone, pioglitazone, or a combination thereof
  • the a-glucosidase inhibitor is at least one compound selected from the group consisting of acarbose, miglitol and voglibose,
  • the DPP-4 inhibitor is at least one compound selected from the group consisting of alogliptin, linagliptin, sitagliptin, saxagliptin and vldagliptin,
  • the SGLT2 inhibitor is at least one compound selected from the group consisting of cangliflozin, dapagliflozin, empagliflozin and ertugliflozin, and
  • the GLP-1 receptor agonist is semaglutide. 53.
  • the at least one other compound is a combination of compounds selected from:
  • Embodiment 50 which further comprises administering at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus, wherein the at least one other compound is selected from the group consisting of a GLP-1 receptor agonist and a dual GLP-1 receptor and gastric inhibitory polypeptide (GIP) receptor agonist.
  • at least one other compound which is prophylactically or therapeutically effective for the treatment of diabetes mellitus
  • the at least one other compound is selected from the group consisting of a GLP-1 receptor agonist and a dual GLP-1 receptor and gastric inhibitory polypeptide (GIP) receptor agonist.
  • GIP gastric inhibitory polypeptide
  • the GLP-1 receptor agonist is exenatide, liraglutide, dulaglutide, lixisenatide or semaglutide, and
  • a method of inhibiting aggregation of misfolded islet amyloid protein (IAPP) in a cat with, or at risk for, diabetes mellitus comprises administering to the cat the compound of any one of Embodiments 1-39, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit aggregation of misfolded IAPP, whereupon aggregation of misfolded IAPP is inhibited in the cat with, or at risk for, diabetes mellitus.
  • IAPP islet amyloid protein
  • Embodiment 58 which further comprises administering bexafliflozin.
  • Embodiment 60 The method of Embodiment 58 or 59, wherein administering is orally administering.
  • Embodiment 58 which further comprises administering insulin.
  • Embodiment 61 wherein the compound of any one of Embodiments 1-39, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, is administered orally and insulin is administered by injection.
  • a method of inhibiting tubulin-associated unit (tau) protein aggregation in a subject having, or at risk for, tau protein aggregation comprises administering to subject any one of Embodiments 39, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit tubulin-associated unit (tau) protein aggregation
  • Embodiment 64 The method of Embodiment 63, wherein the subject has, or is at risk for, Alzheimer’s disease.
  • a method of inhibiting alpha-synuclein (a-syn) protein aggregation in a subject having, or at risk for a-syn comprises administering to subject any one of Embodiments 39, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit alpha-synuclein (a-syn) protein aggregation.
  • Embodiment 65 The method of Embodiment 65, wherein the subject has, or is at risk for, Parkinson’s disease.
  • DMSO dimethylsulfoxide
  • HFIP Hexafluoroisopropanol
  • ThT thioflavin-T
  • hlAPP human islet amyloid protein
  • flAPP feline IAPP
  • Thioflavin T (ThT) fluorescence assay hlAPP and flAPP peptides from the stock solution of 1 mM were each added to 10 mM phosphate-buffered saline (PBS) buffer (pH 7.4) and transferred to a black 96-well microtiter plate with transparent bottom. Each well contained a final volume of 150 pL with a final peptide concentration of 10 pM. Experiments in the presence of hlAPP and flAPP were performed with different compounds at a final concentration of 100 pM. For the dose response, final drug concentrations ranged from 1.5 to 100 pM. The background signal consisted of 0.25% DMSO without peptide and compound. The fluorescence emission experiments were performed using methods known in the art. The maximum fluorescence intensity in percentage reported in Table 1 was calculated as previously described.
  • hlAPP and flAPP were each incubated in 10 mM PBS (pH 7.4, 25 °C) at 10 pM for one hour with different compounds (14, 20, or 27) at 100 pM.
  • Disaggregation experiments were performed by incubating either hlAPP or flAPP for 24 hours prior to drug treatment, followed by incubation for 24 hours with the pharmacologic agent. Samples were treated with one of four conditions: DMSO (control), Compound 14 (APS-22-96), Compound 20 (APS- 22-109), or Compound 27 (APS-22-107).
  • the Malvern Zetasizer machine was utilized to conduct DLS, in which the particle size of each sample is analyzed.
  • hlAPP and flAPP were each incubated in 10 mM PBS (pH 7.4, 25 °C) at 10 pM for one hour with different compounds (14, 20, or 27) at 100 pM. to attain a 1 : 10 molar ratio.
  • a volume of 50 pL was added to a low-volume quartz cuvette (QS high precision cell, 105-251-85-40, Hellma analytics) and read at 25°C using the fluorescence filter. The average of three repetitions of each sample were recorded.
  • Min-6 (mice insulinoma) cells were purchased from AddexBio (Cat No COO 18006, San Diego, CA, USA). Min-6 cells were grown in AddexBio Advanced Medium with glucose, L-glutamine, sodium pyruvate (AddexBio, Cat No C0003-04, San Diego, CA, USA) supplemented with 50 mM 2-mercaptoethanol, 100 U/mL streptomycin, 100 U/mL of penicillin G (1% of Penicillin-Streptomycine solution from Cytiva, Hyclone Laboratories, Logan, Utah, USA) and 15% fetal bovine serum (Hyclone Laboratories, cat No SH30088.03, Logan, Utah, USA). Cells were maintained in a moisture saturated atmosphere at 37 °C under 5% CO 2 .
  • Cells were maintained in a 37 °C, 5% CO2 incubator, in exponential growth, for the duration of the experimentation.
  • cells were seeded in 96-well microtiter plates at a density of 5 x 10 3 Min-6 cells per well for 24 h.
  • Compounds were freshly solubilized in DMSO were added subsequently after stimulation with 20 pM of flAPP.
  • flAPP was dissolved using 4.8% DMSO in 10 mM PBS (pH 7.4, 25 °C) and incubated for 48 hours before addition to cells in order to generate fibrils.
  • Compounds were tested at 6.25 to 12.5 pM.
  • DMSO concentration was maintained at 0.12% to avoid growth inhibition.
  • Negative control consisted of DMSO alone. Plates were incubated for 24h in the presence of compounds or vehicle (0.12% DMSO), and flAPP. Resazurin-based reduction assays were performed as previously described (Fortin and Benoit-Biancaman
  • the diaryl urea derivatives 1-27 were synthesized by treating commercially available aromatic amines, namely 2-aminofluorene (series 1), 4-morpholinoaniline (series 2), and 4- aminoindole (series 3), with substituted aromatic isocyanates in anhydrous dichloromethane at room temperature for 12 hours.
  • the general synthetic route for the preparation of the diaryl derivatives is shown in Scheme 1. All compounds were obtained in moderate to high yields.
  • the compounds were characterized using both proton ( 1 H) and carbon ( 13 C) spectroscopy and high-resolution mass spectrometry (TERMS). The melting point for each compound was also recorded.
  • R2 refers to one of the following substituents: o- methylphenyl, o-methoxy phenyl, o-fluorophenyl, o-nitrophenyl, p-(trifluoromethyl)phenyl, o,p-dimethoxylphenyl, o-methylchloro, m-(trifluoromethyl)phenyl, o,p-dimethoxy, o- chlorophenyl.
  • the diaryl urea derivatives 1-27 were tested on hlAPP and flPAA to determine their effect on IAPP fibril formation by measuring their fluorescence intensity (Table 1). In this test, lower fluorescence corresponds with a greater reduction of fibrils in the sample. Compounds with a measured fluorescence percentage less than 10% for either hlAPP or flAPP were selected as the compounds with greatest potential for reducing amylin fibril accumulation.
  • 4-aminoindolyl urea derivatives 27 (APS-22-107) and 20 (APS-22-109) were found to be the most potent compounds with fluorescence intensities of 7.5 ⁇ 0.4% and 8.5 ⁇ 1.2%, respectively, for hlAAP; 6.9 ⁇ 1.4 and 9.0 ⁇ 0.6%, respectively, for flAPP. Moreover, these compounds were highly selective for both hlAAP and flAPP proteins. In the 2-aminoflurorene series, compounds 1 (APS-21-07), 2 (APS-22- 99), 8 (APS-21-06), and 9 (APS-21-10) were selective for both hlAPP and flAPP.
  • Compounds 11 (APS-22-98), 14 (APS-22-96), and 15 (APS-22-92) were selective for both hlAPP and flAPP while compounds 10 (APS-22-91), 12 (APS-22-100), 16 (APS-22-95), 17 (APS-22-94), and 18 (APS-22-93) were more selective for hlAPP than flAPP.
  • Compound 14 (APS-22-96) was the most potent anti-fibrillary agent in this series with a fluorescence intensity of 9.8 ⁇ 0.3 and 15.4 ⁇ 0.4% on hlAPP and flAPP, respectively.
  • 4-aminoindole urea series all compounds except compound 23 (APS-22-106) were found to be selective for both hlAPP and flAPP.
  • the ThT fluorescence assay is a biophysical method employed to study the kinetics of fibril formation in prone-to-aggregate proteins.
  • the diaryl derivatives were added followed by the addition of ThT.
  • hlAPP or flAPP was mixed in the buffer.
  • the kinetics of fibril formation begin when the protein is dissolved in the buffer and introduced onto the plate.
  • the plateau phase represents the formation of mature fibrils, where an equilibrium is achieved between the aggregation and disaggregation process.
  • the anti-fibrilization property of novel compounds is determined at the plateau phase, at which fluorescence intensity (%) of the tested compounds is compared for different incubations.
  • the kinetic curves obtained for compounds 1-27 are depicted in Fig.
  • DLS was used to determine the size and distribution of amylin proteins after one hour of incubation with the three most promising compounds.
  • 10 pM of hlAPP and flAPP mix were incubated with 100 pM of one of four treatments: DMSO (control), Compound 14 (APS-22-96), Compound 20 (APS-22-109), or Compound 27 (APS-22-107).
  • the particle diameters were analyzed via DLS after 60 minutes. When measured at 0 minutes, the stock solution predominantly contained monomers, with some pre-fibrillar proteins present. The amylin misfolds quickly, with pre-fibrillar proteins forming within 15 minutes.
  • TEM is one of the leading microscopy methods for imaging small particles, including proteinaceous fibrils, as the image is generated through the bombardment of electrons on the sample deposited onto a copper grid.
  • Both the hlAPP and flAPP samples treated with DMSO exhibited large clusters of dense fibrils.
  • hlAPP fibrils were not significantly different from treatment with DMSO, but flAPP fibrils were narrow and isolated, refusing to accumulate in large clusters.
  • Compound 20 was the most effective compound on both hlAPP and flAPP samples.
  • hlAPP samples exposed to Compound 20 depicted a fracturing of the fibrils, with small clusters of proteins being spread out across the field of view.
  • flAPP treated with the same compound yielded small accumulations of thin, thread-like fibrils; a clear reduction from the dense accumulations visualized in the control.
  • Such data support the anti-fibrillization properties of Compound 20 for both hlAPP and flAPP.
  • flAPP samples treated with Compound 14 also exhibited a great reduction in the clustered accumulation of fibrils, as much of the field was empty except for occasional small, isolated packets.
  • Example 5 The anti-fibrillar activity of compounds from series 3 at low pH condition. ThT experiment has been performed with the best compounds, which are part of series 3 (4- aminoindole-linked urea derivatives) using a Tris buffer at 20 mM with pH adjusted at 5.5. The experimental conditions were similar as described in the materials and methods. The only difference is that the Tris buffer was utilized instead of PBS (at pH 7.4). This was achieved to represent physiological milieu with low pH. The anti-fibrillar effects of series 3 are represented in the histograms in Figure 6. The ThT maximum fluorescence activity was lower with all compound treatments at 100 pM in comparison with the control DMSO ( Figure 6-A hlAPP and Figure 6-B flAPP conditions).
  • Compound 24 is nontoxic to Min-6 cell.
  • the cytoprotective effect of compound 24 was evaluated using mouse insulinoma cell line (Min-6).
  • FlAPP fibrils were preformed 12 hours prior the experiment by incubating the peptide at a concentration of 1 mM in PBS (pH 7.4) with 4.8% DMSO for 48 hours.
  • FlAPP treated cells demonstrated a reduction of survival ( Figure 7) as assessed with the resazurin assay, which is metabolized by mitochondria resulting in a fluorescent byproduct and allows for detection of alive cells.
  • the fluorescence intensity of Min-6 cells alone was set arbitrarily at 100%. Compound 24 resulted in an increase of fluorescent signal compared to the flAPP treated cells.
  • Resveratrol (res, a positive control) resulted in comparative effect compared to compound 24 at equivalent concentration. As expected, compound 12 did not result in an increase in the cell survival. However, the one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test did not show significant differences with the flAPP data abpve. Compounds 24 and 12 were not toxic to Min-6 cells at a concentration of 12.5 pM.
  • the aromatic moiety was sometime distanced from the linker by a methyl, ethyl, or carbonyl.
  • different aromatic moieties were attached to the urea/thiourea linker to the other side (Py in Fig. 8) of the molecules; the moieties being incorporated mainly consisted of an indole, benzothiazole, aminofluorene, 4-morpholino, 7V,7V-dimethylphenyl, p-trifluoromethyphenyl, cyclohexyl.
  • the nitrogen present in the urea/thiourea linker has been coupled with different substituents including methyl, ethyl, and isopropyl in some instances (Table 2).
  • Table 2 structure-activity relationships for various compounds.
  • ThT anti-fibrillization kinetics The ThT assay serves as the first line of testing to evaluate the potential of synthesized compounds for their ant-fibril effect.
  • This assay employs thioflavin T, which is a fluorescent dye that recognizes and binds to the P-pleated sheet structures of misfolded protein aggregates. Proteins that are susceptible to misfolding, such as tau and a-syn, typically undergo a structural transformation from their native conformation to adopt a P-sheet structure when aggregated. We screened our compounds for their anti-fibril effect on a-syn, and three isoforms of tau (0N3R, 2N3R, and 2N4R).
  • the first of the tau series was the 0N3R isoform, where only compounds 6T and 14T were tested, and compound 14T was deemed the best, having a 66% reduction in fibril formation as against 7% for compound 6T after 18 h of incubation (Fig. 10A).
  • additional sets of compounds (38T and 18T) on tau 2N3R At the conclusion of the experiment, it was observed that virtually all the compounds effectively halted tau 2N3R fibrillization (Fig. 10B) The compounds demonstrated a significant reduction in tau 2N3R, with the following order of efficacy: 18T (97%), 6T (96%), 14T (79%), and 38T (70%).
  • tau 2N4R we observed a decrease of 34.2% and 67.1% for 6T and 14T respectively (Figure 2B).
  • the PICUP assay utilizes the action of Tris(2,2'-bipyridyl)ruthenium(II) chloride (Ru(bpy)3) and ammonium persulfate (APS), which are electron and radical -generating compounds respectively, to induce oligomer formation.
  • This photochemical assay has been utilized to examine the impact of compounds on the formation of oligomers, which are the initial events preceding fibril formation.
  • Compounds that showed an anti-fibrillization effect from the ThT experiment with a cut off below 15% (including the subtraction of the SEM) were selected for the PICUP assay, i.e., compounds 6T, 14U, 14T, 15U, and 16T.
  • TEM transmission electron microscope
  • the fibrils obtained from the kinetics are individualized and elongated. Similar trend was observed from the two compound treatments.
  • the compound 14T showed an effect that resulted in the formation of short and less dense 2N4R fibrils when compared to the control.
  • compound 6T failed to reduce the density of fibrils.
  • the multi-target effect of compounds 6T and 14T were further explored to determine if the compounds had a disaggregation potential on amyloid-beta (AP) plaques isolated from AD patients.
  • Samples were obtained from the brain of an Alzheimer’s disease patient and treated with the compounds 6T and 14T at 50 pM.
  • TEM micrographs for Ap plaques were taken at both 2500, 20k, and 40k magnifications.
  • the 2500 magnification demonstrates the plaque-like materials present after the incubation with different treatments.
  • the 20k magnification vividly shows the compact arrangement (density) of the formed fibrils, while the 40k magnification helps to distinguish the changes in the fibrillar structures postcompound treatment.
  • M17D neuroblastoma cells expressing the fusion protein S3K::YFP were utilized to assess the impact of compounds 6T and 14T on both cell viability and inclusion formation.
  • aS3K mutations make the protein, a- syn, readily prone to form round cytoplasmic inclusions in the cell system utilized, herein the M17D.
  • the neuroblastoma cells expressing the aS3K have delayed growth and present more cell stress and toxicity.
  • Several compounds were capable to reduce the formation of the aS inclusion formation and aS-induced cytotoxicity.
  • the neuroblastoma cells utilized in this assay expressed an S3K::YFP fusion protein in a doxycycline inducible-dependent manner.
  • the induction caused a pronounced round YFP-positive inclusions for the cell treated with the vehicle (DMSO) in the absence of compound.
  • Compounds 14U, 15U, and 16T exhibited anti-fibrillary activity below 15% but were unable to reduce the oligomer formation. These compounds were used as negative controls and did not exhibit a significant anti-inclusion activity below a concentration of 40 pM.
  • the cell-based experiment entails the production of tau seeds and treatment of biosensor cells with the tau seeds.
  • the production of tau seeds is achieved by expression of htau P301S in vitro.
  • Human embryonic kidney (HEK) 293T cells were transfected with the htauP301S plasmid to overexpress human P301S tau.
  • the compounds (14T and 6T) or vehicle (control, 0.01% DMSO) were applied to HEK 293T cells post 24 h of transfection. Following 48 h of exposure to the compounds at 5, and 20 pM doses, cell viability and tau seeding activity were evaluated (Fig. 12A).
  • HEK 293T cell viability was not impacted by treatment with vehicle (0.01% DMSO) or compounds 14T and 6T (Fig. 12B).
  • Cell lysates from P301S tau overexpressing cells treated with compounds 14T and 6T exhibited reduced seeding activity when compared to lysates from cells treated with the vehicle (0.01% DMSO) alone (Figs. 12C-D).
  • the control (imipramine) has a retention capacity of 44% of its initial amount after 30 minutes.
  • compound 6T demonstrates a higher value of 74% indicating potentially higher stability while compound 14T exhibits a lower percentage remaining at 25%, suggesting it might be more prone to degradation.
  • PK pharmacokinetics
  • the pharmacokinetics (PK) study for the best drug candidate was carried out using CD1 male mouse and single intravenous administration. The PK studies were performed to determine the potential of the best compounds to cross the blood-brain barrier.
  • compound 19U a previously characterized in vitro, was included for the PK study due to its excellent a-syn anti-oligomer, anti-fibril, anti-inclusion activities.
  • Compounds 6T, 14T, and 19U resulted in a plasma: brain ratio of about 1.
  • the term “substantially” can allow for a degree of variability in a value or range, for example, within 90%, within 95%, or within 99% of a stated value or of a stated limit of a range.

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Abstract

Diaryl urea-based compounds, compositions comprising same, and methods of use in the prophylactic and therapeutic treatment of diabetes mellitus in humans and felines.

Description

DIARYL UREA-BASED COMPOUNDS AND RELATED COMPOSITIONS AND
METHODS OF USE IN THE TREATMENT OF DIABETES MELLITUS AND
NEURODEGENERATIVE DISEASE
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. Provisional Appl. No. 63/463,470, filed May 2, 2023, which is incorporated by reference as if fully set forth herein.
STATEMENT OF GOVERNMENT SUPPORT
This invention was made with government support under contracts AG070447 and AG071985 awarded by the National Institutes of Health. The government has certain rights in the invention.
STATEMENT OF GRANT SUPPORT
The work disclosed herein was supported in part by a grant (W20-020) awarded by the EveryCat Health Foundation.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
This application contains a Sequence Listing which has been submitted electronically in ST26 format and hereby incorporated by reference in its entirety. Said ST26 file, created on May 2, 2024, is name 1165181W01.xml and is 2,983bytes in size.
TECHNICAL FIELD
The present disclosure relates to diaryl urea-based compounds, pharmaceutical compositions comprising same, their use in the prophylactic and therapeutic treatment of diabetes mellitus in humans and felines and their use in the treatment of neurodegenerative disease, such as diseases involving tubulin-associated unit and alpha-synuclein protein aggregation.
BACKGROUND
Amyloidosis is a term encompassing a wide range of protein misfolding disorders characterized by the accumulation of toxic protein aggregates. Notable examples include Alzheimer’s disease, Parkinson’s disease, and type 2 diabetes. Diabetes mellitus, also known as type 2 diabetes (T2D), is one disorder in which about 70% of cases involve the accumulation of misfolded islet amyloid protein (IAPP) affecting both humans and other species, including the cat. As of 2021, 537 million people worldwide are living with T2D, with this number expected to rise to 783 million by the year 2045. Around 0.5% of domestic cats are diagnosed with diabetes mellitus, with Burmese cats and cats older than six years of age having the greatest predisposition [6], Risk factors for T2D that are shared between cats and humans include obesity and the consumption of processed foods. Further, pancreatic aggregates of IAPP are reported in 70% of tested feline and human T2D patients.
IAPP, also known as amylin, is normally co-secreted with insulin by pancreatic P- cells. Normal IAPP is a hormone that signals satiety, with its main function being insulin and glucagon inhibition. However, when IAPP misfolds and aggregates in the islets of Langerhans, P-cell mass and function both decrease, suggesting intermediate IAPP species (oligomers) are cytotoxic toward pancreatic P-cells. IAPP is a prone-to-aggregate protein comprised of 37 amino acid residues. Of these, the hydrophobic core spanning residues 20-29 has been suggested as the region most likely to promote aggregation, especially in humans. IAPP has been identified in a multitude of mammals other than humans and cats, such as the Chinese hamster, cow, hare, degu, tamarin, mouse, raccoon, and baboon. Differences in the genetic sequences encoding IAPP correlate with its ability to form fibrils. For instance, rodent IAPP does not have a propensity to aggregate because its 20-29 residue core contains more proline, which can disassemble beta sheet conformations. Human IAPP (hlAPP) and feline IAPP (flAPP) share the most similarities in IAPP sequence (Fig. 1) and a high propensity for aggregation, oftentimes leading to T2D in both species.
As of now, there is no cure for T2D in humans or felines. Further, there are no current treatments for pancreatic amyloidosis. However, various therapeutics are currently under investigation. One group of researchers investigated the anti-amyloidogenic properties of flavonoid compounds derived from Scutellaria baicalensis. a popular Chinese herb with antioxidant potential. A multitude of these derivatives was able to prevent hlAPP from misfolding, presumably due to an ortho-hydroxybenzene structure present in many of the derivatives. Another team of researchers in Vancouver, Canada, was able to identify a small molecule that reduces hlAPP oligomer accumulation in mice by clearance through autophagy [26], Finally, Garcia- Vinuales et al. investigated the anti-aggregation potential of Silybin A and Silybin B, two components derived from the milk thistle plant. Silybin B was shown to be the more potent of the two compounds, interacting heavily with the hlAPP core and reducing its toxicity. It is an object of the present disclosure to provide urea-based inhibitors of IAPP fibril formation. Neurodegenerative diseases, such as Alzheimer’s (AD) and Parkinson’s (PD) can trace their origin to protein fibrillization, the process by which a misfolded protein develops fibrils (Mahmoudi et al., Nanoscale 5(7): 2570-2588 (2013)). Two major neuropathological hallmarks characterize typical AD, the accumulation of extracellular P-amyloid (AP) plaques and neurofibrillary tangles (NFTs) in the brain (Jagtap et al., Bioorg Chem 95: 103135 (2020); and Yan et al., Eur J Med Chem 135: 462-475 (2017)). NFTs contain misfolded and hyperphosphorylated tubulin-associated unit (tau) protein (Jagtap et al., supra, and Yan et al., supra). Hypothetically, Ap plaques accumulate in the brain and consequently activate microglia - neuroimmune cells involved in sensing, housekeeping, and defense of the central nervous system (CNS) (Hickman et al., Nat Neurosci 21(10): 1359-1369 (2018). These microglial cells then induce a pre-inflammatory state, which contributes to the hyperphosphorylation of tau (p-tau). The insolubility of tau protein fibrils leads to NFTs, which result in neuron toxicity and an acceleration of AD neurodegeneration (Ashrafian et al., Int J Biol Macromol 183: 1939-1947 (2021); and Bucciantini et al., Nature 461(6880): 507-511 (2002)). The spatiotemporal distribution of NFTs have been shown to coincide with neurodegeneration and cognitive impairment (Hickman et al. supra).
There are six naturally occurring isoforms of the tau protein in the human brain. The isoforms vary by the presence or absence of inserts encoded by the Tau gene (Zhong et al., J. Biol. Chem. 287(24): 20711-20719 (2012)). The six isoforms (0N3R, 0N4R, 1N3R, 1N4R, 2N3R, 2N4R) are transcribed in the human brain by alternative splicing and are expressed in different ratios during neurodegeneration resulting in tau pathological effects (Boyarko et al., Front Neurosci 15: 702788 (2021)). Each isoform is distinguished by two criteria: the absence, presence, or partial presence of two 29-amino acid inserts encoded by exons 2 and 3 resulting in ON (no insert), IN (one insert), or 2N (two inserts) tau; and the presence (4R) or absence (3R) of the R2 domain of the microtubule binding domain region (MTBR) in addition to the already present Rl, R3, and R4 domains (Boyarko et al., supra). Tau isoforms are differentially expressed across the hippocampus during various stages of development; in fetal stages only the 0N3R isoform is expressed, while all six isoforms are expressed in adulthood (Sergeant et al., Biochim Biophy Acta 1739(2-3): 179-197 (2005)). In healthy adults the ratio of 3R to 4R isoforms is approximately 1 (Boyarko et al., supra, and Bachmann et al., Front Neurosci 15: 643115 (2021)). In AD brains, this ratio becomes skewed with the 3R isoform becoming preferentially added to the tau fibrils as the disease progresses (Cherry et al., Acta Neuropathol Comm 9(1): 86 (2021)). Isoform 4R on the other hand is more efficient at promoting microtubule assembly compared to 3R, with the R1-R2 region being unique to 4R-Tau; when the ratio of 4R to 3R drastically increases in the human brain this leads to Argyrophilic Grain Disease (AGD), a form of dementia (Sergeant et al., supra, Tolnay et al., Neuropathol Appl Neurobiol 25(3): 171-187 (1999): and Togo et al., Acta Neuropathol 104(4): 398-402 (2002)). The 1 : 1 ratio of 3R and 4R isoforms is important to maintain as seen by the progression of neurodegenerative diseases when this ratio is altered towards the 3R or 4R direction.
An antibody, which apparently stimulates the clearance of amyloid plaques has been approved by the Food and Drug Administration for the treatment of AD. However, it is the formation of the neurofibrillary tangles and the spatiotemporal distribute of the tangles that correlate with the loss of cognition. In view of the foregoing, there is an unmet need for a small molecule that can inhibit the formation of oligomers, i.e., early-stage aggregation. Accordingly, it is an object of the present disclosure to provide such small molecules and related compositions, which can be used to inhibit tau protein aggregation and alpha- synuclein (a-syn) protein aggregation (e.g., Lewy bodies and Lewy neurites).
Other objects, as well as inventive features, will be apparent from the detailed description provided herein.
SUMMARY
Provided is a compound of formula I:
Figure imgf000005_0001
Formula I wherein R1 and R2 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy. Halo can be F, I, Cl, or Br. Mono-halo methyl can be -C(H2)F, -C(H2)I, - C(H2)C1 or -C(H2)Br. Di-halo methyl can be -C(H)F2, -C(H)I2, -C(H)C12 or -C(H)Br2. Tri- halo methyl can be -CF3, -CI3, -CCI3 or -CBrs. The Ci-Ce alkyl can be -CH3. The Ci-Ce alkoxy can be -OCH3. The compound can have the structure:
Figure imgf000005_0002
. The compound can have the structure:
Figure imgf000006_0001
Further provided is a compound of formula II or Ila:
Figure imgf000006_0004
wherein R1 and R2 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy; and R10 is heteroaryl (pyridinyl, pyrrolyl, or imidazolyl) or heterocycloalkyl (morpholinyl or piperazinyl). Halo can be F, I, Cl, or Br. Mono-halo methyl can be -C(H2)F,
-C(H2)I, -C(H2)C1 or -C(H2)Br. Di-halo methyl can be -C(H)F2, -C(H)I2, -C(H)C12 or -
C(H)Br2. Tri-halo methyl can be -CF3, -CI3, -CCI3 or -CBrs. The Ci-Ce alkyl can be -CH3.
The Ci-Ce alkoxy can be -OCH3. The compound can have the structure:
Figure imgf000006_0002
Still further provided is a compound of formula III:
Figure imgf000006_0003
Formula III wherein R1 and R2 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy. Halo can be F, I, Cl, or Br. Mono-halo methyl can be -C(H2)F, -C(H2)I, - C(H2)C1 or -C(H2)Br. Di-halo methyl can be -C(H)F2, -C(H)I2, -C(H)C12 or -C(H)Br2. Tri- halo methyl can be -CF3, -CI3, -CCI3 or -CBrs. The Ci-Ce alkyl can be -CH3. The Ci-Ce alkoxy can be -OCH3. Still further provided is a compound of formula IV:
Figure imgf000007_0001
Formula IV wherein:
R3 and R4 are each independently H, alkyl or acyl or, R3 and R4, together with the atoms to which they are each attached, form an aryl or heteroaryl group;
R5 and R6 are each independently H or alkyl or, R5 and R6, together with the atoms to which they are attached, form a heterocyclyl group;
R7 and R8 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy; or
R7 and R8, together with the atoms to which they are each attached, form an aryl or heteroaryl group;
X1 is NR5, O, S or CR92, wherein each R9 is independently, H, alkyl or acyl; and
X2 is O or S. In some embodiments, X2 can be NR13, wherein R13 is H or alkyl Examples of halo for compounds of the formula IV are F, I, Cl, and Br. Examples of monohalo methyl for compounds of the formula IV are -C(H2)F, -C(H2)I, -C(H2)C1, and -C(H2)Br. Examples of di-halo methyl for compounds of the formula IV are -C(H)F2, -C(H)l2, - C(H)Ch, and -C(H)Br2. Examples of tri-halo methyl for compounds of the formula IV are - CF3, -CI3, -CCI3, and -CBrs. An example of alkyl for any alkyl group for compounds of formula IV is Ci-Ce alkyl , which can be -CEE. An example of alkoxy for any alkyl group for compounds of formula IV is Ci-Ce alkoxy, which can be -OCH3.
Still further provided is a compound of formula IV:
Figure imgf000007_0002
Formula IV wherein:
R3 and R4 are each independently H, alkyl or acyl or, R3 and R4, together with the atoms to which they are each attached, form an aryl or heteroaryl group; R5 and R6 are each independently H or alkyl or, R5 and R6, together with the atoms to which they are attached, form a heterocyclyl group;
R7 and R8 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy; or
R7 and R8, together with the atoms to which they are each attached, form an aryl or heteroaryl group;
X1 is NR5, O, S or CR92, wherein each R9 is independently, H, alkyl or acyl; and
X2 is O or S. In some embodiments, X2 can be NR13, wherein R13 is H or alkyl. Examples of halo for compounds of the formula IV are F, I, Cl, and Br. Examples of monohalo methyl for compounds of the formula IV are -C(H2)F, -C(H2)I, -C(H2)C1, and -C(H2)Br. Examples of di-halo methyl for compounds of the formula IV are -C(H)F2, -C(H)l2, - C(H)Ch, and -C(H)Br2. Examples of tri-halo methyl for compounds of the formula IV are - CF3, -CI3, -CCI3, and -CBrs. An example of alkyl for any alkyl group for compounds of formula IV is Ci-Ce alkyl , which can be -CEE. An example of alkoxy for any alkyl group for compounds of formula IV is Ci-Ce alkoxy, which can be -OCH3.
In embodiments, the compound of formula IV can be a compound of the formula:
Figure imgf000008_0001
In embodiments, the compound of the formula IV can be a compound of the formula:
Figure imgf000009_0001
such as compounds of the formula:
Figure imgf000009_0002
including compounds of the formula:
Figure imgf000010_0001
In embodiments, the compound of formula IV can be a compound of the formula:
Figure imgf000010_0002
such as compounds of the formula:
Figure imgf000011_0001
including compounds of the formula:
Figure imgf000011_0002
In any of the foregoing, X2 in the compounds of the formula IV can be S. In any of the foregoing, in the compounds of the formula IV, one R9 can be H. In embodiments, both R9 groups can be H.
Still further provided is a compound of formula V:
Figure imgf000012_0001
Formula V wherein:
R11 is alkyl, cycloalkyl, aryl, arylalkyl, or arylacyl;
R12 is cycloakyl, aryl, or heterocyclyl;
R5 and R6 are each independently H or alkyl or, R5 and R6, together with the atoms to which they are attached, form a heterocyclyl group; and
X2 is O or S. In some embodiments, X2 can be NR13, wherein R13 is H or alkyl. Examples of aryl groups representing R11 include napthyl (e.g., 1-, 2-, or 4-napthyl) and aryl groups substituted with one, two, or three F, Cl, I, acyl, alkyl, alkoxy, alkylthio, and amino. Examples of alkyl groups representing R11 include C1-C3 alkyl. Examples of cycloalkyl groups representing R11 include cyclopentyl and cyclohexyl. Examples of arylacyl groups representing R11 include groups of the formula:
O
QA . Examples of arylalkyl groups representing R11 include benzyl and phenethyl. Examples of cycloalkyl groups representing R12 include cyclopentyl and cyclohexyl. Examples of aryl groups representing R12 include fluorenyl (e.g., 3-fluorenyl) and aryl groups substituted with one or two haloalkyl (e.g., CF3), amino, morpholino, piperazinyl, and 2- thiazolyl. Examples of heterocyclyl groups representing R12 include 4-indolyl, 5-indolyl, and 6-indolyl. In embodiments, R11 and R12 are not the same, such that the compounds of the formula V are assymetric.
In view of the above, provided is a pharmaceutical composition comprising at least one compound of Formulae I-V and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising at least one compound of Formulae I-V can further comprise at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus and is selected from the group consisting of a sulfonylurea, a meglitinide, a biguanide, a thiazolidinedione (TZD), an a-glucosidase inhibitor, a dipeptidyl peptidase IV (DPP-4) inhibitor, a bile acid sequestrant, a dopamine agonist, a sodium-glucose transport protein 2 (SGLT2) inhibitor, and a glucagon like peptide 1 (GLP-1) receptor agonist. The sulfonylurea can be at least one compound selected from the group consisting of chlorpropamide, tolazamide, tolbutamide, acetohexamide, glyburide, glipizide, glimepiride, and gliclazide. The meglitinide can be repaglinide, netaglinide, or a combination thereof. The biguanide can be metformin. The TZD can be rosiglitazone, pioglitazone, or a combination thereof. The a-glucosidase inhibitor can be at least one compound selected from the group consisting of acarbose, miglitol and voglibose. The DPP-4 inhibitor can be at least one compound selected from the group consisting of alogliptin, linagliptin, sitagliptin, saxagliptin and vldagliptin. The bile acid sequestrant can be colesevelam. The dopamine agonist can be bromocriptine. The SGLT2 inhibitor can be at least one compound selected from the group consisting of cangliflozin, dapagliflozin, empagliflozin and ertugliflozin. The GLP-1 receptor agonist can be semaglutide. A pharmaceutical composition comprising at least one compound of Formulae I-V can further comprise at least one other compound, which is a combination of compounds selected from (a) metformin and at least one of glyburide, glipizide, sitagliptin, saxagliptin, pioglitazone, repaglinide, rosiglitazone, linagliptin, alogliptin, canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin, (b) glimepiride and either or both of pioglitazone and rosiglitazone, (c) alogliptin and pioglitazone, (d) dapagliflozin and saxagliptin, (e) empagliflozin and linagliptin, (f) ertugliflozin and sitagliptin, (g) lixisenatide and glargine insulin, and (h) liraglutide and degludec insulin. The pharmaceutical composition can be formulated for oral administration.
A pharmaceutical composition comprising at least one compound of Formulae I-V can further comprise at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus and is selected from the group consisting of a GLP-1 receptor agonist and a dual GLP-1 receptor and GIP receptor agonist. The GLP-1 receptor agonist can be exenatide, liraglutide, dulaglutide, lixisenatide or semaglutide. The dual GLP-1 receptor and GIP receptor agonist can be tirzepatide. The pharmaceutical composition can be formulated for administration by injection.
A pharmaceutical composition comprising at least one compound of Formula I can further comprise bexagliflozin. The pharmaceutical composition can be formulated for oral administration.
A pharmaceutical composition comprising at least one compound of Formula II can further comprise bexagliflozin. The pharmaceutical composition can be formulated for oral administration.
A pharmaceutical composition comprising at least one compound of Formula II can further comprise bexagliflozin. The pharmaceutical composition can be formulated for oral administration. A pharmaceutical composition comprising at least one compound of Formula IV can further comprise bexagliflozin. The pharmaceutical composition can be formulated for oral administration.
A pharmaceutical composition comprising at least one compound of Formula V and a pharmaceutically acceptable carrier is also provided.
Further in view of the above, a method of inhibiting aggregation of misfolded islet amyloid protein (IAPP) in a human with, or at risk for, diabetes mellitus is provided. The method comprises administering to the human the compound of Formulae I-V, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit aggregation of misfolded IAPP, whereupon aggregation of misfolded IAPP is inhibited in the human with, or at risk for, diabetes mellitus. The method can further comprise administering, simultaneously or sequentially by the same or different routes, at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus and which is selected from the group consisting of a sulfonylurea, a meglitinide, a biguanide, a TZD, an a-glucosidase inhibitor, a DPP-4 inhibitor, a bile acid sequestrant, a dopamine agonist, a SGLT2 inhibitor, and a GLP-1 receptor agonist. The sulfonylurea can be at least one compound selected from the group consisting of chlorpropamide, tolazamide, tolbutamide, acetohexamide, glyburide, glipizide, glimepiride, and gliclazide. The meglitinide can be repaglinide, netaglinide, or a combination thereof. The biguanide can be metformin. The TZD can be rosiglitazone, pioglitazone, or a combination thereof. The a-glucosidase inhibitor can be at least one compound selected from the group consisting of acarbose, miglitol and voglibose. The DPP-4 inhibitor can be at least one compound selected from the group consisting of alogliptin, linagliptin, sitagliptin, saxagliptin and vldagliptin. The bile acid sequestrant can be colesevelam. The dopamine agonist can be bromocriptine. The SGLT2 inhibitor can be at least one compound selected from the group consisting of cangliflozin, dapagliflozin, empagliflozin and ertugliflozin. The GLP-1 receptor agonist can be semaglutide. The at least one other compound can be a combination of compounds selected from (a) metformin and at least one of glyburide, glipizide, sitagliptin, saxagliptin, pioglitazone, repaglinide, rosiglitazone, linagliptin, alogliptin, canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin, (b) glimepiride and either or both of pioglitazone and rosiglitazone, (c) alogliptin and pioglitazone, (d) dapagliflozin and saxagliptin, (e) empagliflozin and linagliptin, (f) ertugliflozin and sitagliptin, (g) lixisenatide and glargine insulin, and (h) liraglutide and degludec insulin. The compounds in the combination can be administered simultaneously or sequentially by the same or different routes. Administering can be orally administering.
The method comprising administering to the human the compound of Formulae I-V, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit aggregation of misfolded IAPP can further comprise administering at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus, wherein the at least one other compound is selected from the group consisting of a GLP-1 receptor agonist and a dual GLP-1 receptor and GIP receptor agonist. The GLP-1 receptor agonist can be exenatide, liraglutide, dulaglutide, lixisenatide or semaglutide. The dual GLP-1 receptor and GIP receptor agonist can be tirzepatide. The administering can be by injection.
Still further in view of the above, a method of inhibiting aggregation of misfolded IAPP in a cat with, or at risk for, diabetes mellitus is provided. The method comprises administering to the cat the compound of Formulae I-V, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit aggregation of misfolded IAPP, whereupon aggregation of misfolded IAPP is inhibited in the cat with, or at risk for, diabetes mellitus. The method can further comprise administering bexafliflozin. The administering can be orally administering. Alternatively, the method can further comprise administering insulin. The administering of the compound of Formulae I-V can be orally, and the administering of the insulin can be by injection.
Still further provided is a method of inhibiting tubulin-associated unit (tau) protein aggregation in a subject having, or at risk for, tau protein aggregation. The method comprises administering to the subject an above-described pharmaceutical composition in an amount effective to inhibit tau protein aggregation. The subject can have, or be at risk for, Alzheimer’s disease.
A method of inhibiting alpha-synuclein (a-syn) protein aggregation in a subject having, or at risk for a-syn is also provided. The method comprises administering to the subject an above-described pharmaceutical composition in an amount effective to inhibit a- syn protein aggregation. The subject can have, or be at risk for, Parkinson’s disease.
DESCRIPTION OF THE FIGURES
Fig. 1. Amino acid sequences of human islet amyloid polypeptide (hlAPP; SEQ ID
NO: 1) and feline IAPP (flAPP; SEQ ID NO: 2). The four amino acids in flAPP that differ from those in hlAPP are shown in red ink. Fig- 2. Kinetics of IAPP fibril formation obtained with different diaryl derivatives of urea monitored with the thioflavin T (ThT) fluorescence assays. In this first-tier assay, compounds were tested at a final concentration of 100 pM in the presence of hlAPP and flAPP at 10 pM. Molar ratio peptide: compound is 1 : 10. Series 1, 2, and 3 represent the 2- aminofluorene, morpholino, and 4-aminoindole, respectively.
Fig- 3. Kinetics of amylin fibril formation with increasing doses of four synthesized compounds 14 (APS-22-96), 20 (APS-22-109), 26 (APS-22-110), and 27 (APS-22-107), assessed via thioflavin T (ThT) fluorescence assay. The original stock of the compound was tested at 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 pM concentration. These compounds were tested in the presence of hlAPP and flAPP at 10 pM.
Fig- 4. Size of amylin-based protein based on light scattering. Control at 60 min showed one major fibril peak in the 1000 nm range. The three tested compounds (14, 20, and 27) were individually added to aliquots of amylin and incubated for 60 min before analysis to determine the size of the proteins contained within the sample.
Fig. 5. TEM imaging of amylin fibrils incubated with four unique treatments. Samples of hlAPP and flAPP were solubilized at 10 pM in PBS buffer, after the end of kinetic of fibril formation. Samples were incubated with DMSO (0.25%), Compound 27 (100, Compound 20, or Compound 14. An image of hlAPP treated with Compound 14 was unable to be obtained. All images are displayed at 20k magnification.
Fig. 6. Thioflavin T (ThT) assays performed with 10 pM of hlAPP and flAPP subjected to different treatments at pH 5.5 and 25 °C. Bars represent the average of maximum fluorescence values (triplicate) obtained at the plateau phase after 12 hours for each condition. ThT fluorescence intensities for the control DMSO (0.25%) indicated as hlAPP and flAPP were normalized to 100%. Error bars represent the S.E.M. A) ThT assays were performed with 10 pM of hlAPP in the presence of 0.25% DMSO (indicated as hlAPP) or different compounds 17-24 at a final concentration of 100 pM. B) ThT assays were performed with 10 pM of flAPP in the presence of 0.25% DMSO (indicated as flAPP) or different compounds 17-24 at a final concentration of 100 pM. C) ThT assays were performed with 10 pM of hlAPP in the presence of 0.25% DMSO (indicated as hlAPP) or compound 24 using different concentrations (25, 50, and 100 pM). D) ThT assays were performed with 10 pM of flAPP in the presence of 0.25% DMSO (indicated as flAPP) or compound 24 using different concentrations (25, 50, and 100 pM).
Fig. 7. MIN-6 cell viability in the presence of feline islet amyloid polypeptide (flAPP) with DMSO (0.1%), resveratrol (12.5 pM), compound 24 (6.25 pM and 12.5 pM), and compound 12 (12.5 pM) and the absence of flAPP, as determined by resazurin-based assays. flAPP was tested at 20 pM. The peptide was pre-incubated for 48 hours at a concentration of 1 mM with a 4.8% DMSO in PBS (pH 7.4). The experiment lasted 24 hours for the treatment of the cell.
Fig. 8. Synthesis of urea (U) and thiourea (T) analogs using respective isocyanate and isothiocyanate reacting with amines. (Ri = H, Me, Et, or i-Pr and R2 = H).
Fig- 9. The ThT fluorescence assay was employed to analyze the kinetic curves of compounds 14T and 6T at a concentration of 100 pM with: A. a-syn (6 pM); B. compound 14T dose-dependent inhibition with varying concentrations (3.125, 6.25, 12.5, 25, 50, 100 pM) against a-syn (6 pM) fibril formation. Data were collected in triplicate for each concentration at the plateau phase over the course of five consecutive time points. The error bars indicate the standard error of the mean (SEM) specific to each condition.
Fig. 10. Thioflavin-T kinetic curve showing the effect of the best anti-fibrillary compound (14T and 6TB) when assessed with different tau isoforms. The compounds were tested at a concentration of 100 pM along with 10 pM (10: 1) of A) tau 0N3R, B) tau 2N3R, C) tau 2N4R. Several compounds were tested for their anti-fibrillary effect on the tau 2N3R. The best anti-fibrillary compound (14T and 6TB) when assessed with tau 0N3R and 2N4R. The kinetics were performed in the presence of 2.5 pM of heparin, 1 mM of dithiothreitol (DTT), 1 mM of 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, and 30 pM of Thioflavin- T (ThT) in a buffer solution containing 50 mM Tris, 25 mM NaCl, at pH 7.4. The positive control involved tau isoform without compound treatment. The background (BG) signal was obtained with all components in the absence of tau protein and compounds. The depicted curves represent the average data obtained from three technical replicates.
Fig. 11. Compound 14T mainly prevents a-syn inclusion formation. M17D cells expressing the inclusion-prone aS-3K::YFP fusion protein (dox-inducible) were treated with 0.1% DMSO (vehicle; “0 pM”) as well as 1.25, 2.5, 5, 10, 20 and 40 pM of compounds 6T and 14T at t = 24 h after plating. Cells were induced with doxycycline at t = 48 h. A) Incucyte- based analysis of punctate YFP signals relative to 0.1% DMSO was done at t = 96 h in N = 4 (compound 6T) or N =3 (compound 14T) independent experiments, n = 9-24 individual wells total. For compound 6T: 0 pM, n = 18; 40, 20 and 10 pM, n = 9; all other concentrations, n = 18. For compound 14T: 0 pM, n = 24; 40, 20 and 10 pM, n = 11; all other concentrations, n = 24. B) Same as panel A, but confluence fold changes relative to DMSO vehicle (0 pM) were plotted. All data are presented as fold-changes relative to DMSO control + I- standard deviation. Kruskal-Wallis tests plus Dunn’s multiple comparisons test; *, p < 0.05; ***, p
0.001, ****, p < 0.0001, ns = non-significant.
Fig. 12. Compounds 6T and 14T reduce tau seeding activity in vitro. (A) Schematic of experimental setup to test the effect of compounds on tau seeding activity. hTauP301S plasmid was transfected to over-express tau in HEK293T cells and the cells were treated with the compounds 24 hours later. Cell viability and tau seeding activity were assessed 48 hr after treatment with compounds. (B) Viability of HEK cells after treatment with the compounds. (C) Representative images of FRET signal from biosensor cells after transfection with HEK cell lysates. (D) The seeding activity of cells overexpressing hTauP301S and after treatment with the compounds.
DETAILED DESCRIPTION
The potential of urea and urea derivatives in reducing misfolded protein accumulations has been the focus of recent research. Urea has been suggested to weaken hydrophobic interactions and disaggregate fibril structures of islet amyloid protein (IAPP), serum amyloid A, alpha-synuclein, and amyloid-beta proteins. This new class of compounds possesses the necessary attributes for further drug development due to their selectivity for IAPP, with early in vitro studies suggesting them to be non-toxic. In a previous investigation from Fortin et al., two specific N-phenyl-N'- (2-ethyl)ureas were identified as inhibitors of hlAPP fibril formation, reducing its subsequent cytotoxicity. Described herein is a novel class of small molecules, which contain a urea linker, are highly potent in the prevention of IAPP fibril formation and inhibit toxic IAPP oligomer accumulation. These compounds were developed using a simple synthetic approach utilizing a diverse set of commercially available amines and isocyanates. Compounds from three series (2-aminofluorenes, 4-morpholino anilines, and 4-aminoindoles) were coupled with various substituted isocyanates to generate the urea derivatives. Synthesized compounds were evaluated for their effects on hlAPP and fLAPP fibril formation using Thioflavin T (ThT) to compare their selectivity and antiaggregation potential. The best-performing compounds were then assessed using transmission electron microscopy (TEM) to study their impact on the fibril structure and morphology.
In view of the above, provided is a compound of formula I:
Figure imgf000019_0001
wherein R1 and R2 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy. Halo can be F, I, Cl, or Br. Mono-halo methyl can be -C(H2)F, -C(H2)I, - C(H2)C1 or -C(H2)Br. Di-halo methyl can be -C(H)F2, -C(H)I2, -C(H)C12 or -C(H)Br2. Tri- halo methyl can be -CF3, -CI3, -CCI3 or -CBrs. The Ci-Ce alkyl can be -CH3. The Ci-Ce alkoxy can be -OCH3. The compound can have the structure:
Figure imgf000019_0002
Further provided is a compound of formula II or Ila:
Figure imgf000019_0003
wherein R1 and R2 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy; and R10 is heteroaryl (pyridinyl, pyrrolyl, or imidazolyl) or heterocycloalkyl (morpholinyl or piperazinyl). Halo can be F, I, Cl, or Br. Mono-halo methyl can be -C(H2)F, - C(H2)I, -C(H2)C1 or -C(H2)Br. Di-halo methyl can be -C(H)F2, -C(H)I2, -C(H)C12 or - C(H)Br2. Tri-halo methyl can be -CF3, -CI3, -CCI3 or -CBrs. The Ci-Ce alkyl can be -CH3. The Ci-Ce alkoxy can be -OCH3. The compound can have the structure:
Figure imgf000020_0001
Still further provided is a compound of formula III:
Figure imgf000020_0002
Formula III wherein R1 and R2 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy. Halo can be F, I, Cl, or Br. Mono-halo methyl can be -C(H2)F, -C(H2)I, - C(H2)C1 or -C(H2)Br. Di-halo methyl can be -C(H)F2, -C(H)I2, -C(H)C12 or -C(H)Br2. Tri- halo methyl can be -CF3, -CI3, -CCI3 or -CBr3. The Ci-Ce alkyl can be -CH3. The Ci-Ce alkoxy can be -OCH3.
Still further provided is a compound of formula IV:
Figure imgf000020_0003
Formula IV wherein:
R3 and R4 are each independently H, alkyl or acyl or, R3 and R4, together with the atoms to which they are each attached, form an aryl or heteroaryl group;
R5 and R6 are each independently H or alkyl or, R5 and R6, together with the atoms to which they are attached, form a heterocyclyl group;
R7 and R8 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy; or
R7 and R8, together with the atoms to which they are each attached, form an aryl or heteroaryl group;
X1 is NR5, O, S or CR92, wherein each R9 is independently, H, alkyl or acyl; and
X2 is O or S. In some embodiments, X2 can be NR13, wherein R13 is H or alkyl.
Examples of halo for compounds of the formula IV are F, I, Cl, and Br. Examples of mono- halo methyl for compounds of the formula IV are -C(H2)F, -C(H2)I, -C(H2)C1, and -C(H2)Br. Examples of di-halo methyl for compounds of the formula IV are -C(H)F2, -C(H)l2, - C(H)Ch, and -C(H)Br2. Examples of tri-halo methyl for compounds of the formula IV are - CF3, -CI3, -CCI3, and -CBrs. An example of alkyl for any alkyl group for compounds of formula IV is Ci-Ce alkyl , which can be -CEE. An example of alkoxy for any alkyl group for compounds of formula IV is Ci-Ce alkoxy, which can be -OCH3.
In embodiments, the compound of formula IV can be a compound of the formula:
Figure imgf000021_0001
In embodiments, the compound of the formula IV can be a compound of the formula:
Figure imgf000021_0002
such as compounds of the formula:
Figure imgf000022_0001
In embodiments, the compound of formula IV can be a compound of the formula:
Figure imgf000023_0001
such as compounds of the formula:
Figure imgf000023_0002
including compounds of the formula:
Figure imgf000024_0001
In any of the foregoing, X2 in the compounds of the formula IV can be S. In any of the foregoing, in the compounds of the formula IV, one R9 can be H. In embodiments, both R9 groups can be H.
Still further provided is a compound of formula V:
X2 R11 JL R12
R5 R6 Formula V wherein:
R11 is alkyl, cycloalkyl, aryl, arylakyl, or arylacyl;
R12 is cycloakyl, aryl, or heterocyclyl;
R5 and R6 are each independently H or alkyl or, R5 and R6, together with the atoms to which they are attached, form a heterocyclyl group; and
X2 is O or S. In some embodiments, X2 can be NR13, wherein R13 is H or alkyl. Examples of aryl groups representing R11 include napthyl (e.g., 1-, 2-, or 4-napthyl) and aryl groups substituted with one, two, or three F, Cl, I, acyl, alkyl, alkoxy, alkylthio, and amino. Examples of alkyl groups representing R11 include Ci-Csalkyl. Examples of cycloalkyl groups representing R11 include cyclopentyl and cyclohexyl. Examples of arylacyl groups representing R11 include groups of the formula:
Figure imgf000025_0001
. Examples of arylalkyl groups representing R11 include benzyl and phenethyl. Examples of cycloalkyl groups representing R12 include cyclopentyl and cyclohexyl. Examples of aryl groups representing R12 include fluorenyl (e.g., 3-fluorenyl) and aryl groups substituted with one or two haloalkyl (e.g., CF3), amino, morpholino, piperazinyl, and 2- thiazolyl. Examples of heterocyclyl groups representing R12 include 4-indolyl, 5-indolyl, and 6-indolyl. In embodiments, R11 and R12 are not the same, such that the compounds of the formula V are assymetric.
The compounds can be used to inhibit aggregation of misfolded islet amyloid protein (IAPP) in a human with, or at risk for, diabetes mellitus. The compounds also can be used to inhibit aggregation of misfolded IAPP in a feline with, or at risk for, diabetes mellitus.
The compounds can be formulated as pharmaceutical compositions comprising a pharmaceutically acceptable carrier using methods well-known in the art. “Carrier” is used generically herein to refer to pharmaceutically acceptable carriers, diluents, adjuvants, and excipients. See, e.g., Remington: The Science and Practice of Pharmacy, 23rd edition, October 30, 2020, Adeboye Adejare, ed. Accordingly, provided is a pharmaceutical composition comprising at least one compound of Formulae I-V and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising at least one compound of Formulae I-V can further comprise at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus and is selected from the group consisting of a sulfonylurea, a meglitinide, a biguanide, a thiazolidinedione (TZD), an a- glucosidase inhibitor, a dipeptidyl peptidase IV (DPP-4) inhibitor, a bile acid sequestrant, a dopamine agonist, a sodium-glucose transport protein 2 (SGLT2) inhibitor, and a glucagon like peptide 1 (GLP-1) receptor agonist. The sulfonylurea can be at least one compound selected from the group consisting of chlorpropamide, tolazamide, tolbutamide, acetohexamide, glyburide, glipizide, glimepiride, and gliclazide. The meglitinide can be repaglinide, netaglinide, or a combination thereof. The biguanide can be metformin. The TZD can be rosiglitazone, pioglitazone, or a combination thereof. The a-glucosidase inhibitor can be at least one compound selected from the group consisting of acarbose, miglitol and voglibose. The DPP-4 inhibitor can be at least one compound selected from the group consisting of alogliptin, linagliptin, sitagliptin, saxagliptin and vldagliptin. The bile acid sequestrant can be colesevelam. The dopamine agonist can be bromocriptine. The SGLT2 inhibitor can be at least one compound selected from the group consisting of cangliflozin, dapagliflozin, empagliflozin and ertugliflozin. The GLP-1 receptor agonist can be semaglutide. A pharmaceutical composition comprising at least one compound of Formulae I-V can further comprise at least one other compound, which is a combination of compounds selected from (a) metformin and at least one of glyburide, glipizide, sitagliptin, saxagliptin, pioglitazone, repaglinide, rosiglitazone, linagliptin, alogliptin, canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin, (b) glimepiride and either or both of pioglitazone and rosiglitazone, (c) alogliptin and pioglitazone, (d) dapagliflozin and saxagliptin, (e) empagliflozin and linagliptin, (f) ertugliflozin and sitagliptin, (g) lixisenatide and glargine insulin, and (h) liraglutide and degludec insulin. The pharmaceutical composition can be formulated for oral administration.
A pharmaceutical composition comprising at least one compound of Formulae I-V can further comprise at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus and is selected from the group consisting of a GLP-1 receptor agonist and a dual GLP-1 receptor and GIP receptor agonist. The GLP-1 receptor agonist can be exenatide, liraglutide, dulaglutide, lixisenatide or semaglutide. The dual GLP-1 receptor and GIP receptor agonist can be tirzepatide. The pharmaceutical composition can be formulated for administration by injection.
A pharmaceutical composition comprising at least one compound of Formula I can further comprise bexagliflozin. The pharmaceutical composition can be formulated for oral administration.
A pharmaceutical composition comprising at least one compound of Formula II can further comprise bexagliflozin. The pharmaceutical composition can be formulated for oral administration.
A pharmaceutical composition comprising at least one compound of Formula III can further comprise bexagliflozin. The pharmaceutical composition can be formulated for oral administration.
A pharmaceutical composition comprising at least one compound of Formula IV can further comprise bexagliflozin. The pharmaceutical composition can be formulated for oral administration.
A pharmaceutical composition comprising at least one compound of Formula V can further comprise bexagliflozin. The pharmaceutical composition can be formulated for oral administration.
Further in view of the above, a method of inhibiting aggregation of misfolded islet amyloid protein (IAPP) in a human with, or at risk for, diabetes mellitus is provided. The method comprises administering to the human the compound of Formulae I-V, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit aggregation of misfolded IAPP, whereupon aggregation of misfolded IAPP is inhibited in the human with, or at risk for, diabetes mellitus. The method can further comprise administering, simultaneously or sequentially by the same or different routes, at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus and which is selected from the group consisting of a sulfonylurea, a meglitinide, a biguanide, a TZD, an a-glucosidase inhibitor, a DPP-4 inhibitor, a bile acid sequestrant, a dopamine agonist, a SGLT2 inhibitor, and a GLP-1 receptor agonist. The sulfonylurea can be at least one compound selected from the group consisting of chlorpropamide, tolazamide, tolbutamide, acetohexamide, glyburide, glipizide, glimepiride, and gliclazide. The meglitinide can be repaglinide, netaglinide, or a combination thereof. The biguanide can be metformin. The TZD can be rosiglitazone, pioglitazone, or a combination thereof. The a-glucosidase inhibitor can be at least one compound selected from the group consisting of acarbose, miglitol and voglibose. The DPP-4 inhibitor can be at least one compound selected from the group consisting of alogliptin, linagliptin, sitagliptin, saxagliptin and vldagliptin. The bile acid sequestrant can be colesevelam. The dopamine agonist can be bromocriptine. The SGLT2 inhibitor can be at least one compound selected from the group consisting of cangliflozin, dapagliflozin, empagliflozin and ertugliflozin. The GLP-1 receptor agonist can be semaglutide. The at least one other compound can be a combination of compounds selected from (a) metformin and at least one of glyburide, glipizide, sitagliptin, saxagliptin, pioglitazone, repaglinide, rosiglitazone, linagliptin, alogliptin, canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin, (b) glimepiride and either or both of pioglitazone and rosiglitazone, (c) alogliptin and pioglitazone, (d) dapagliflozin and saxagliptin, (e) empagliflozin and linagliptin, (f) ertugliflozin and sitagliptin, (g) lixisenatide and glargine insulin, and (h) liraglutide and degludec insulin. The compounds in the combination can be administered simultaneously or sequentially by the same or different routes. Administering can be orally administering.
The method comprising administering to the human the compound of Formulae I-V, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit aggregation of misfolded IAPP can further comprise administering at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus, wherein the at least one other compound is selected from the group consisting of a GLP-1 receptor agonist and a dual GLP-1 receptor and GIP receptor agonist. The GLP-1 receptor agonist can be exenatide, liraglutide, dulaglutide, lixisenatide or semaglutide. The dual GLP-1 receptor and GIP receptor agonist can be tirzepatide. The administering can be by injection.
Still further in view of the above, a method of inhibiting aggregation of misfolded IAPP in a cat with, or at risk for, diabetes mellitus is provided. The method comprises administering to the cat the compound of Formulae I-V, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit aggregation of misfolded IAPP, whereupon aggregation of misfolded IAPP is inhibited in the cat with, or at risk for, diabetes mellitus. The method can further comprise administering bexafliflozin. The administering can be orally administering. Alternatively, the method can further comprise administering insulin. The administering of the compound of Formula I-V can be orally, and the administering of the insulin can be by injection.
Still further provided is a method of inhibiting tubulin-associated unit (tau) protein aggregation in a subject having, or at risk for, tau protein aggregation. The method comprises administering to the subject an above-described pharmaceutical composition in an amount effective to inhibit tau protein aggregation. The subject can have, or be at risk for, Alzheimer’s disease.
A method of inhibiting alpha-synuclein (a-syn) protein aggregation in a subject having, or at risk for a-syn is also provided. The method comprises administering to the subject an above-described pharmaceutical composition in an amount effective to inhibit a- syn protein aggregation. The subject can have, or be at risk for, Parkinson’s disease.
With regard to the above, methods an effective amount can be determined by one of ordinary skill in the art using dosage range determining methods known in the art. Typically, a physician (or veterinarian for non-human subjects) will determine the actual dosage, which will be most suitable for an individual subject (e.g., human or feline). The specific dose level and frequency of dosage for an individual may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, gender, diet, mode and time of administration, rate of excretion, other administered drugs, and the severity of the condition.
The above compounds include isotopic variants and compounds in which one or more hydrogen atoms have been substituted with deuterium. The compounds may contain one or more chiral centers or may otherwise be capable of existing as multiple stereoisomers. In one embodiment, the compounds are not limited to any particular stereochemical requirement, and that the compounds, and compositions, methods, uses, and medicaments that include them may be optically pure, or may be any of a variety of stereoisomeric mixtures, including racemic and other mixtures of enantiomers, other mixtures of diastereomers, and the like. Such mixtures of stereoisomers may include a single stereochemical configuration at one or more chiral centers, while including mixtures of stereochemical configuration at one or more other chiral centers.
Similarly, the compounds may include geometric centers, such as cis, trans isomers, diastereomers, enantiomers, and E and Z double bonds. In another embodiment, the compounds are not limited to any particular geometric isomer requirement, and that the compounds, and compositions, methods, uses, and medicaments that include them may be pure, or may be any of a variety of geometric isomer mixtures. Such mixtures of geometric isomers may include a single configuration at one or more double bonds and chiral carbons, while including mixtures of geometry at one or more other double bonds and chiral carbons.
The above compounds, and pharmaceutically acceptable salts and solvates thereof, can be synthesized in accordance with methods known in the art and exemplified herein. See, e.g., Example 1.
The terms “salts” and “pharmaceutically acceptable salts” refer to derivatives of the compounds wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic groups such as amines; and alkali or organic salts of acidic groups such as carboxylic acids. Pharmaceutically acceptable salts include the conventional nontoxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, and isethionic, and the like.
Pharmaceutically acceptable salts can be synthesized from the parent compound, which contains a basic or acidic moiety, by conventional chemical methods. In some instances, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, the disclosure of which is hereby incorporated by reference for its teachings regarding same.
The term “solvate” means a compound, or a salt thereof, that further includes a stoichiometric or non-stoichiometric amount of solvent bound by non-covalent intermolecular forces. Where the solvent is water, the solvate is a hydrate.
Any suitable route of administration can be used in the above methods. Examples include, but are not limited to, oral, parenteral, intravenous, intracranial, intracerebroventricular, and intracerebral. An effective amount can be determined by one of ordinary skill in the art using dosage range determining methods known in the art. Typically, a physician (or veterinarian for non-human subjects) will determine the actual dosage, which will be most suitable for an individual subject. The specific dose level and frequency of dosage for an individual may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, gender, diet, mode and time of administration, rate of excretion, other administered drugs, and the severity of the particular condition. The compound/compositions described herein can be administered with other biologically active compounds as appropriate.
The terms “substituted,” “substituent,” and “functional group” refer to a group that can be or is substituted onto a molecule or onto another group (e.g., on an aryl or an alkyl group). Examples of substituents include, but are not limited to, a halogen (e.g., F, Cl, Br, and I), OR, OC(O)N(R)2, CN, NO, NO2, ONO2, azido, CF3, OCF3, R, O (oxo), S (thiono), C(O), S(O), methylenedioxy, ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, -(CEhjo- 2P(O)(OR)2, C(O)R, C(O)C(O)R, C(O)CH2C(O)R, C(S)R, C(O)OR, OC(O)R, C(0)N(R)2, 0C(0)N(R)2, C(S)N(R)2, (CH2)O-2N(R)C(0)R, (CH2)O-2N(R)C(0)OR, (CH2)O-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, or C(=NOR)R wherein each R can be, independently, hydrogen, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl, wherein any alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl or two R groups bonded to a nitrogen atom or to adjacent nitrogen atoms can together with the nitrogen atom or atoms form a heterocyclyl, which can be mono- or independently multi-substituted. The term “alkyl” as used herein refers to substituted or unsubstituted straight chain and branched mono- or divalent alkyl groups and cycloalkyl groups having from 1 to 40 carbon atoms (C1-C40), 1 to about 20 carbon atoms (C1-C20), 1 to 12 carbons (C1-C12), 1 to 8 carbon atoms (Ci-Cs), or, in some embodiments, from 1 to 6 carbon atoms (Ci-Ce). Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. As used herein, the term “alkyl” encompasses n-alkyl, isoalkyl, and c c-isoalkyl groups as well as other branched chain forms of alkyl. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.
The term “alkenyl” as used herein refers to substituted or unsubstituted straight chain and branched mono- or divalent alkenyl groups and cycloalkenyl groups having at least one double bond and having from 1 to 40 carbon atoms (C1-C40), 1 to about 20 carbon atoms (Ci- C20), 1 to 12 carbons (C1-C12), 1 to 8 carbon atoms (Ci-Cs), or, in some embodiments, from 1 to 6 carbon atoms (Ci-Ce). Examples of straight chain alkenyl groups include those with from 1 to 8 carbon atoms such as -CH=CH-, -CH=CHCH3, and -CH2CH=CHCH2- groups, wherein the double bonds can have an E- or Z-configuration. And when there are multiple bonds, each double bond can, independently, have an E- or a Z-configuration. Examples of branched alkenyl groups include, but are not limited to, -CH=C(CH3)- and CH2C=CH(CH3) groups. Representative substituted alkenyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.
The term “cycloalkyl” as used herein refers to substituted or unsubstituted cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7. Cycloalkyl groups can have any number of carbon atoms, e.g., 3 to 8 carbon atoms (Cs-Cs), 3 to 6 carbon atoms (C3-C6), and 4 to 8 carbon atoms (C4-Cs). Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbomyl, adamantyl, bornyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. The term “cycloalkylalkyl” as used herein refers to substituted or unsubstituted alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group as defined herein is replaced with a bond to a cycloalkyl group as defined herein. Representative cycloalkylalkyl groups include, but are not limited to, cyclopentylalkyl.
The term “alkylcycloalkyl” as used herein refers to substituted or unsubstituted cycloalkyl groups as defined herein in which a hydrogen of a cycloalkyl group as defined herein is replaced with a bond to an alkyl group as defined herein. Representative alkylcycloalkyl groups include, but are not limited to, alkylcyclopropyl.
The term “acyl” as used herein refers to a group containing a carbonyl moiety wherein the group is bonded via the carbonyl carbon atom. The carbonyl carbon atom is also bonded to another carbon atom, which can be part of a substituted or unsubstituted alkyl, aryl, aralkyl cycloalkyl, cycloalkylalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, heteroarylalkyl group or the like. In the special case wherein the carbonyl carbon atom is bonded to a hydrogen, the group is a “formyl” group, an acyl group as the term is defined herein. An acyl group can include 0 to about 12-40, 6-10, 1-5 or 2-5 additional carbon atoms bonded to the carbonyl group. An acryloyl group is an example of an acyl group. An acyl group can also include heteroatoms within the meaning here. A nicotinoyl group (pyridyl-3 -carbonyl) is an example of an acyl group within the meaning herein. Other examples include acetyl, benzoyl, phenylacetyl, pyridyl acetyl, cinnamoyl, and acryloyl groups and the like. When the group containing the carbon atom that is bonded to the carbonyl carbon atom contains a halogen, the group is termed a “haloacyl” group. An example is a trifluoroacetyl group.
The term “heterocyclylcarbonyl” is an example of an acyl group that is bonded to a substituted or unsubstituted heterocyclyl group, as the term “heterocyclyl” is defined herein. An example of a heterocyclylcarbonyl group is a prolyl group, wherein the prolyl group can be a D- or an L-prolyl group.
The term “aryl” as used herein refers to substituted or unsubstituted cyclic aromatic hydrocarbons that do not contain heteroatoms in the ring. Thus, aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, aryl groups contain about 6 to about 14 carbons (Ce-Cu) or from 6 to 10 carbon atoms (Ce-Cio) in the ring portions of the groups. Aryl groups can be unsubstituted or substituted, as defined herein. “Aryl” and the phrase “aryl group” includes fused ring species including those that include fused aromatic and non-aromatic groups. Accordingly, “aryl” and the phrase “aryl group” include groups of the formula:
Figure imgf000033_0001
, each of which can be substituted or unsubstituted, such as hydroxy substituted.
Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2-, 3-, 4-, 5-, or 6-substituted phenyl or 2-8 substituted naphthyl groups, which can be substituted with carbon or non-carbon groups such as those listed herein.
The terms “aralkyl” and “arylalkyl” refer to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein. Representative aralkyl groups include benzyl and phenylethyl groups and fused (cycloalkylaryl)alkyl groups such as 4-ethyl-indanyl. Aralkenyl groups are alkenyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to an aryl group as defined herein.
The term “heterocyclyl” or “heterocyclo” refers to substituted or unsubstituted aromatic and non-aromatic ring compounds containing 3 or more ring members, of which one or more (e.g., 1, 2 or 3) is a heteroatom such as, but not limited to, N, O, and S. Thus, a heterocyclyl can be a cycloheteroalkyl or a heteroaryl or, if polycyclic, any combination thereof. In some embodiments, heterocyclyl groups include 3 to about 20 ring members, whereas other such groups have 3 to about 15 ring members. In some embodiments, heterocyclyl groups include heterocyclyl groups that include 3 to 8 carbon atoms (Cs-Cs), 3 to 6 carbon atoms (Cs-Ce), 3 to 5 carbon atoms (C3-C5) or 6 to 8 carbon atoms (Ce-Cs). A heterocyclyl group designated as a C2-heterocyclyl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and fthe heteroatoms and so forth. Likewise, a C4-heterocyclyl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. A heterocyclyl ring can also include one or more double bonds, such as in the group 3,6-dihydro-2H-pyran and 3,4-dihydro-2H-pyran, having the formula:
Figure imgf000033_0002
respectively, each of which can be substituted.
A heteroaryl ring is an embodiment of a heterocyclyl group. The phrase “heterocyclyl group” includes fused ring species including those that include fused aromatic and non- aromatic groups. Representative heterocyclyl groups include, but are not limited to tetrahydro-2H-thiopyran- 1,1 -di oxide, having the formula:
O
Figure imgf000034_0001
, which can be substituted, 4a,5,6,7-tetrahydro-4H-pyrrolo[l,2- d][l,3,4]oxadiazinyl, having the formula:
Figure imgf000034_0002
, which can be substituted, pyrrolidinyl, pyrrolidinone (e.g., pyrrolidin-2-one), azetidinyl, piperidynyl, piperazinyl, morpholinyl, chromanyl, indolinonyl, isoindolinonyl, furanyl, pyrrolidinyl, pyridinyl, pyrazinyl, pyrimidinyl, triazinyl, thiophenyl, tetrahydrofuranyl, pyrrolyl, oxazolyl, oxadiazolyl, imidazolyl, imidazo[l,2-a]pyridinyl, having the formula:
Figure imgf000034_0003
, which can be substituted, triazyolyl, tetrazolyl, benzoxazolinyl, thiazolyl, benzthiazolinyl, and benzimidazolinyl groups. Examples of indolinonyl groups include groups having the general formula:
Figure imgf000034_0004
, wherein R is as defined herein.
Examples of isoindolinonyl groups include groups having the general formula:
Figure imgf000034_0005
, wherein R is as defined herein.
Examples of benzoxazolinyl groups include groups having the general formula:
Figure imgf000034_0006
, wherein R is as defined herein.
Examples of benzthiazolinyl groups include groups having the general formula:
Figure imgf000034_0007
, wherein R is as defined herein.
In some embodiments, the group R in benzoxazolinyl and benzthiazolinyl groups is an N(R)2 group. In some embodiments, each R is hydrogen or alkyl, wherein the alkyl group is substituted or unsubstituted. In some embodiments, the alkyl group is substituted with a heterocyclyl group (e.g., with a pyrrolidinyl group). The term “heterocyclylalkyl” refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group as defined herein is replaced with a bond to a heterocyclyl group as defined herein. Representative heterocyclylalkyl groups include, but are not limited to, furan-2-yl methyl, furan-3-yl methyl, pyridine-3-yl methyl, tetrahydrofuran-2-yl methyl, and indol-2-yl propyl.
The term “heterocyclylalkoxy” refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group as defined herein is replaced with a bond to a heterocyclyl group as defined herein and the alkyl group is attached to an oxygen. Representative heterocyclylalkoxy groups include, but are not limited to, -O- (CH2)qheterocyclyl, wherein q is an integer from 1 to 5. In some embodiments, heterocyclylalkoxy groups include -O-(CH2)qmorpholinyl such as -O-CH2CH2-morpholine.
The term “heteroarylalkyl” refers to alkyl groups as defined herein in which a hydrogen or carbon bond of an alkyl group is replaced with a bond to a heteroaryl group as defined herein.
The term “alkoxy” refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein. Examples of linear alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, pentyloxy, hexyloxy, and the like. Examples of branched alkoxy include, but are not limited to, isopropoxy, sec-butoxy, tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy include, but are not limited to, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the like. An alkoxy group can include one to about 12-20 or about 12-40 carbon atoms bonded to the oxygen atom, can further include double or triple bonds, and can also include heteroatoms. For example, an allyloxy group is an alkoxy group within the meaning herein. A methoxy ethoxy group is also an alkoxy group within the meaning herein, as is a methylenedi oxy group in a context where two adjacent atoms of a structure are substituted therewith.
The terms “amine,” “amine group,” “amino,” and “amino group” refer to a substituent of the form -NH2, -NHR, -NR2, or -NRE, wherein each R is defined herein, and protonated forms of each, except for -NR3 , which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine. An “amino group” within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group.
An “alkylamino” group includes a monoalkylamino, dialkylamino, and trialkylamino group. An example of a “alkylamino” is -NH-alkyl and -N(alkyl)2. An example of a “cycloalkylamino” group is -NH-cycloalkyl and -N(cycloalkyl)2.
An example of a “cycloalkyl heterocycloamino” group is -NH-(heterocyclo cycloalkyl), wherein the heterocyclo group is attached to the nitrogen and the cycloalkyl group is attached to the heterocyclo group.
An example of a “heterocyclo cycloamino” group is -NH-(cycloalkyl heterocycle), wherein the cycloalkyl group is attached to the nitrogen and the heterocyclo group is attached to the cycloalkyl group.
The term “amido” refers to a group of the formula -C(0)NR2, wherein R is defined herein.
The terms “halo,” “halogen,” and “halide” group, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.
The term “haloalkyl” group includes mono-halo alkyl groups, poly-halo alkyl groups, wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkyl include trifluoromethyl, 1,1 -di chloroethyl, 1,2-di chloroethyl, l,3-dibromo-3,3-difluoropropyl, perfluorobutyl, -CF(CH3)2 and the like.
The terms “treat,” “treating,” “treated,” or “treatment” (with respect to a disease or condition) is an approach for obtaining beneficial or desired results including and preferably clinical results and includes, but is not limited to, one or more of the following: improving a condition associated with a disease, curing a disease, lessening severity of a disease, delaying progression of a disease, alleviating one or more symptoms associated with a disease, increasing the quality of life of one suffering from a disease, prolonging survival and/or prophylactic or preventative treatment.
An “effective amount” refers to any amount that is sufficient to achieve a desired biological effect. Combined with the teachings provided herein, by choosing among the various active conjugates or compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial unwanted toxicity and yet is effective to treat the particular subject. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular compound being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular compound and/or other therapeutic agent without necessitating undue experimentation. A maximum dose can be used, that is, the highest safe dose according to some medical judgment. Multiple doses per day can be used to achieve appropriate systemic levels of compounds. Appropriate systemic levels can be determined by, for example, measurement of the patient’s peak or sustained plasma level of the drug. “Dose” and “dosage” are used interchangeably herein.
Generally, daily oral doses of a compound are, for human subjects, from about 0.01 milligrams/kg per day to 1,000 milligrams/kg per day. Oral doses in the range of 0.5 to 50 milligrams/kg, in one or more administrations per day, can yield therapeutic results. Dosage can be adjusted appropriately to achieve desired drug levels, local or systemic, depending upon the mode of administration. For example, intravenous administration can vary from one order to several orders of magnitude lower dose per day. If the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) can be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of the compound.
A “therapeutically effective amount” (or “effective amount”) of a compound with respect to use in treatment, refers to an amount of the compound in a preparation which, when administered as part of a desired dosage regimen (to a mammal, such as a human) alleviates a symptom, ameliorates a condition, or slows the onset of disease conditions according to clinically acceptable standards for the disorder or condition to be treated or the cosmetic purpose, e.g., at a reasonable benefit/risk ratio applicable to any medical treatment.
For any compound a therapeutically effective amount can be initially determined from animal models. A therapeutically effective dose can also be determined from human data for compounds which have been tested in humans and for compounds which are known to exhibit similar pharmacological activities, such as other related active agents. Higher doses may be required for parenteral administration. The applied dose can be adjusted based on the relative bioavailability and potency of the administered compound. Adjusting the dose to achieve maximal efficacy based on the methods described above and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan.
The formulations can be administered in pharmaceutically acceptable solutions, which can routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients. For use in therapy, an effective amount of the compound can be administered to a subject by any mode that delivers the compound to the desired surface. Administering a pharmaceutical composition can be accomplished by any means known to the skilled artisan. Routes of administration include, but are not limited to, intravenous, intramuscular, intraperitoneal, intravesical (urinary bladder), oral, subcutaneous, direct injection (for example, into a tumor or abscess), mucosal (e.g., topical to eye), inhalation, and topical.
For intravenous and other parenteral routes of administration, a compound can be formulated as a lyophilized preparation, as a lyophilized preparation of liposome-intercalated or -encapsulated active compound, as a lipid complex in aqueous suspension, or as a salt complex. Lyophilized formulations are generally reconstituted in suitable aqueous solution, e.g., in sterile water or saline, shortly prior to administration.
For oral administration, the compounds can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well-known in the art. Such carriers enable the compounds to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. A pharmaceutical preparation for oral use can be obtained as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, and sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone (PVP). If desired, disintegrating agents can be added, such as the cross-linked PVP, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations can also be formulated in saline or buffers, e.g., EDTA for neutralizing internal acid conditions, or can be administered without any carriers.
Also contemplated are oral dosage forms of the compounds. The compounds can be chemically modified so that oral delivery of the derivative is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the compound itself, where said moiety permits (a) inhibition of acid hydrolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the compounds and increase in circulation time in the body. Examples of such moieties include polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, PVP and polyproline. Abuchowski and Davis, “Soluble Polymer-Enzyme Adducts,” In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., pp. 367-383 (1981); Newmark et al., J Appl Biochem 4: 185-189 (1982). Other polymers that could be used are poly-1, 3-dioxolane and poly-1, 3, 6-tioxocane. For pharmaceutical usage, as indicated above, polyethylene glycol moieties are suitable.
The location of release of a compound hereof can be the stomach, the small intestine (e.g., the duodenum, the jejunum, or the ileum), or the large intestine. One skilled in the art has available formulations, which will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine. The release can avoid the deleterious effects of the stomach environment, either by protection of the compound or by release of the compound beyond the stomach environment, such as in the intestine.
To ensure full gastric resistance a coating impermeable to at least pH 5.0 is essential. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and shellac. These coatings can be used as mixed films.
A coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make the tablet easier to swallow. Capsules can consist of a hard shell (such as gelatin) for delivery of dry therapeutic (e.g., powder); for liquid forms, a soft gelatin shell can be used. The shell material of cachets could be thick starch or other edible paper. For pills, lozenges, molded tablets or tablet triturates, moist massing techniques can be used.
The compound can be included in the formulation as fine multi-particulates in the form of granules or pellets of particle size about 1 mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. Therapeutic agent could be prepared by compression.
Colorants and flavoring agents may all be included. For example, the compound can be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.
One may dilute or increase the volume of the compound with an inert material. These diluents can include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts also can be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.
Disintegrants can be included in the formulation of therapeutic agent into a solid dosage form. Materials used as disintegrates include, but are not limited to, starch, including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used. Another form of the disintegrant is the insoluble cationic exchange resin. Powdered gums can be used as disintegrants and as binders, and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.
Binders can be used to hold the compound together to form a hard tablet and can include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). PVP and hydroxypropylmethyl cellulose (HPMC) can both be used in alcoholic solutions to granulate therapeutic agent.
An anti-frictional agent can be included in the formulation of therapeutic to prevent sticking during the formulation process. Lubricants can be used as a layer between therapeutic agent and the die wall, and these can include, but are not limited to, stearic acid, including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants can also be used, such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.
Glidants, which can improve the flow properties of the drug during formulation and aid rearrangement during compression, can be added. The glidants can include starch, talc, pyrogenic silica and hydrated silicoaluminate.
To aid dissolution of therapeutic agent into the aqueous environment a surfactant can be added as a wetting agent. Surfactants can include anionic detergents, such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents, which can be used, include benzalkonium chloride and benzethonium chloride. Potential nonionic detergents that can be included in the formulation as surfactants include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the compound or derivative thereof either alone or as a mixture in different ratios. Pharmaceutical preparations, which can be used orally, include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers can be added. Microspheres formulated for oral administration can also be used. Such microspheres have been well-defined in the art. All formulations for oral administration should be in dosages suitable for such administration.
For buccal administration, the compositions can take the form of tablets or lozenges formulated in conventional manner.
For topical administration, the compound can be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art. Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal oral or pulmonary administration.
For administration by inhalation, compounds can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, di chlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator, can be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.
Also contemplated is pulmonary delivery of the compounds (or salts thereof). The compound is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. Other reports of inhaled molecules include Adjei et al., Pharm Res 7:565-569 (1990); Adjei et al., Int J Pharmaceutics 63: 135-144 (1990) (leuprolide acetate); Braquet et al., J Cardiovasc Pharmacol 13(suppl. 5): 143-146 (1989) (endothelin-1); Hubbard et al., Annal Int Med 3:206-212 (1989) (al -antitrypsin); Smith et al., 1989, J Clin Invest 84: 1145-1146 (a- 1 -proteinase); Oswein et al., 1990, "Aerosolization of Proteins," Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (recombinant hepatocyte growth hormone); Debs et al., 1988, J Immunol 140:3482-3488 (interferon-gamma and tumor necrosis factor alpha) and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor; incorporated herein by reference). A method and composition for pulmonary delivery of drugs for systemic effect is described in U.S. Pat. No. 5,451,569 (specifically incorporated herein by reference for its disclosure regarding same), issued Sep. 19, 1995, to Wong et al.
Contemplated for use are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products including, but not limited to, nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.
Nasal delivery of a pharmaceutical composition is also contemplated. Nasal delivery allows the passage of a pharmaceutical composition to the blood stream directly after administering therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran.
The compounds, when it is desirable to deliver them systemically, can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multidose containers, with an added preservative. The compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds can be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions can contain substances which increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension can also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
Alternatively, the active compounds can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds can also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described above, a compound can also be formulated as a depot preparation. Such long-acting formulations can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
The pharmaceutical compositions also can comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosolized, pelleted for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer R, Science 249: 1527-1533 (1990).
The compound and optionally one or more other therapeutic agents can be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2- sulphonic, and benzene sulphonic. Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
Suitable buffering agents include acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v); and thimerosal (0.004-0.02% w/v).
Pharmaceutical compositions contain an effective amount of a compound as described herein and optionally one or more other therapeutic agents included in a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” means one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to a human or other vertebrate animal. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also can be commingled with the compounds, and with each other, in a manner such that there is no interaction, which would substantially impair the desired pharmaceutical efficiency.
Therapeutic agent(s), including specifically, but not limited to, a compound, can be provided in particles. “Particles” means nanoparticles or microparticles (or in some instances larger particles) that can consist in whole or in part of the compound or the other therapeutic agent(s). The particles can contain therapeutic agent(s) in a core surrounded by a coating, including, but not limited to, an enteric coating. Therapeutic agent(s) also can be dispersed throughout the particles. Therapeutic agent(s) also can be adsorbed into the particles. The particles can be of any order release kinetics, including zero-order release, first-order release, second-order release, delayed release, sustained release, immediate release, and any combination thereof, etc. The particle can include, in addition to therapeutic agent(s), any of those materials routinely used in the art of pharmacy and medicine, including, but not limited to, erodible, nonerodible, biodegradable, or nonbiodegradable material or combinations thereof. The particles can be microcapsules which contain the compound in a solution or in a semi-solid state. The particles can be of virtually any shape.
Both non-biodegradable and biodegradable polymeric materials can be used in the manufacture of particles for delivering therapeutic agent(s). Such polymers can be natural or synthetic polymers. The polymer is selected based on the period of time over which release is desired. Bioadhesive polymers of particular interest include bioerodible hydrogels described in Sawhney et al., Macromolecules 26:5823-2787 (1993), the teachings of which are specifically incorporated by reference herein. These include polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly (isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).
Therapeutic agent(s) can be contained in controlled-release systems. The term “controlled release” refers to any drug-containing formulation in which the manner and profile of drug release from the formulation are controlled. This refers to immediate as well as non-immediate release formulations, with non-immediate release formulations including, but not limited to, sustained release and delayed release formulations. The term “sustained release” (also referred to as “extended release”) refers to a drug formulation that provides for gradual release of a drug over an extended period of time, and that can result in substantially constant blood levels of a drug over an extended time period. The term “delayed release” refers to a drug formulation in which there is a time delay between administration of the formulation and the release of the drug therefrom. “Delayed release” may or may not involve gradual release of drug over an extended period of time, and thus may or may not be “sustained release.”
Use of a long-term, sustained-release implant can be particularly suitable for treatment of chronic conditions. “Long-term” release means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days, and up to 30-60 days. Long-term sustained-release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
The term “prodrug” means a derivative of a compound that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide an active compound, particularly a compound of the invention. Examples of prodrugs include, but are not limited to, derivatives and metabolites of a compound of the invention that include biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Specific prodrugs of compounds with carboxyl functional groups are the lower alkyl esters of the carboxylic acid. The carboxylate esters are conveniently formed by esterifying any of the carboxylic acid moieties present on the molecule. Prodrugs can typically be prepared using well-known methods, such as those described by Burger’s Medicinal Chemistry and Drug Discovery 6th ed. (Donald J. Abraham ed., 2001, Wiley) and Design and Application of Prodrugs (H. Bundgaard ed., 1985, Harwood Academic Publishers GmbH).
Further, in each of the foregoing and following embodiments, it is to be understood that the formulae include and represent not only all pharmaceutically acceptable salts of the compounds, but also include any and all hydrates and/or solvates of the compound formulae or salts thereof. It is to be appreciated that certain functional groups, such as the hydroxy, amino, and like groups form complexes and/or coordination compounds with water and/or various solvents, in the various physical forms of the compounds. Accordingly, the above formulae are to be understood to include and represent those various hydrates and/or solvates. In each of the foregoing and following embodiments, it is also to be understood that the formulae include and represent each possible isomer, such as stereoisomers and geometric isomers, both individually and in any and all possible mixtures. In each of the foregoing and following embodiments, it is also to be understood that the formulae include and represent any and all crystalline forms, partially crystalline forms, and non-crystalline and/or amorphous forms of the compounds.
The term “pharmaceutically acceptable carrier” is art-recognized and refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any subject composition or component thereof. Each carrier must be "acceptable" in the sense of being compatible with the subject composition and its components and not injurious to the patient. Some examples of materials, which may serve as pharmaceutically acceptable carriers, include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.
The term “administering” includes all means of introducing the compounds and compositions described herein to the patient, including, but are not limited to, oral (po), intravenous (iv), intramuscular (im), subcutaneous (sc), transdermal, inhalation, buccal, ocular, sublingual, vaginal, rectal, and the like. The compounds and compositions may be administered in unit dosage forms and/or formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles.
Illustrative formats for oral administration include tablets, capsules, elixirs, syrups, and the like. Illustrative routes for parenteral administration include intravenous, intraarterial, intraperitoneal, epidural, intraurethral, intrastemal, intramuscular and subcutaneous, as well as any other art recognized route of parenteral administration.
Illustrative means of parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques, as well as any other means of parenteral administration recognized in the art. Parenteral formulations are typically aqueous solutions, which may contain excipients such as salts, carbohydrates and buffering agents (preferably at a pH in the range from about 3 to about 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water. The preparation of parenteral formulations under sterile conditions, for example, by lyophilization, may readily be accomplished using standard pharmaceutical techniques well- known to those skilled in the art. Parenteral administration of a compound is illustratively performed in the form of saline solutions or with the compound incorporated into liposomes. In cases where the compound, itself, is not sufficiently soluble to be dissolved, a solubilizer such as ethanol can be applied.
The dosage of each compound of the claimed combinations depends on several factors, including: the administration method, the condition to be treated, the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the person to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular patient may affect the dosage regimen used.
In the methods the individual components of a co-administration, or combination, can be administered by any suitable means, contemporaneously, simultaneously, sequentially in either order, separately or in a single pharmaceutical formulation. Where the co-administered compounds or compositions are administered in separate dosage forms, the number of dosages administered per day for each compound may be the same or different. The compounds or compositions may be administered via the same or different routes of administration. The compounds or compositions may be administered according to simultaneous or alternating regimens, at the same or different times during the course of the therapy, concurrently in divided or single forms.
The term “therapeutically effective amount” refers to that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes alleviation of the symptoms of the disease or disorder being treated. In one aspect, the therapeutically effective amount is that which may treat or alleviate the disease or symptoms of the disease at a reasonable benefit/risk ratio applicable to any medical treatment. However, it is to be understood that the total daily usage of the compounds and compositions described herein may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors, including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, gender and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidentally with the specific compound employed; and like factors well-known to the researcher, veterinarian, medical doctor or other clinician of ordinary skill.
Depending upon the route of administration, a wide range of permissible dosages are contemplated, including doses falling in the range from about 1 pg/kg to about 1 g/kg. The dosages may be single or divided and may administered according to a wide variety of protocols, including q.d. (once a day), b.i.d. (twice a day), t.i.d. (three times a day), or even every other day, once a week, once a month, once a quarter, and the like. In each of these cases the described therapeutically effective amounts correspond to the instance of administration, or alternatively to the total daily, weekly, month, or quarterly dose, as determined by the dosing protocol.
An effective amount of any one or a mixture of the compounds can be determined by the attending diagnostician or physician by the use of known techniques and/or by observing results obtained under analogous circumstances. In determining the effective amount or dose, a number of factors are considered by the attending diagnostician or physician, including, but not limited to, the species of mammal, including human, its size, age, and general health, the specific disease or disorder involved, the degree of or involvement or the severity of the disease or disorder, the response of the individual patient, the particular compound administered, the mode of administration, the bioavailability characteristics of the preparation administered, the dose regimen selected, the use of concomitant medication, and other relevant circumstances.
The term “patient” includes human and non-human animals such as companion animals (dogs and cats and the like) and livestock animals. Livestock animals are animals raised for food production. The patient to be treated is preferably a mammal, in particular a human.
The disclosure relates to, among other things, the following enumerated Embodiments, which listing does not represent an order of importance:
1. A compound of formula IV:
Figure imgf000049_0001
Formula IV wherein:
R3 and R4 are each independently H, alkyl or acyl; R3 and R4, together with the atoms to which they are each attached, form an aryl or heteroaryl group; or the group NR5 is connected at R3 or R4;
R5 and R6 are each independently H or alkyl or, R5 and R6, together with the atoms to which they are attached, form a heterocyclyl group;
R7 and R8 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy; or
R7 and R8, together with the atoms to which they are each attached, form an aryl or heteroaryl group;
X1 is NR5, O, S or CR92, wherein each R9 is independently, H, alkyl or acyl; and
X2 is O or S.
2. A compound of the formula:
Figure imgf000049_0002
Formula IV wherein:
R3 and R4 are each independently H, alkyl or acyl or, R3 and R4, together with the atoms to which they are each attached, form an aryl or heteroaryl group;
R5 and R6 are each independently H or alkyl or, R5 and R6, together with the atoms to which they are attached, form a heterocyclyl group;
R7 and R8 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy; or
R7 and R8, together with the atoms to which they are each attached, form an aryl or heteroaryl group;
X1 is NR5, O, S or CR92, wherein each R9 is independently, H, alkyl or acyl; and X2 is O or S.
3. The compound of Embodiment 1, wherein the compound of formula IV is a compound of the formula:
Figure imgf000050_0001
4. The compound of Embodiment 1, wherein the compound of the formula IV is a compound of the formula:
Figure imgf000050_0002
5. The compound of Embodiment 1, wherein the compound is of the formula:
Figure imgf000051_0001
6. The compound of Embodiment 1, wherein the compound is of the formula:
Figure imgf000051_0002
7. The compound of Embodiment 1, wherein the compound of formula IV is a compound of the formula:
Figure imgf000052_0001
8. The compound of Embodiment 1, wherein the compound is of the formula:
Figure imgf000052_0002
9. The compound of Embodiment 1, wherein the compound is of the formula:
Figure imgf000053_0001
10. The compound of Embodiments 1-9, wherein X2 is S.
11. The compound of any of Embodiments 7-9, wherein one R9 is H.
12. The compound of any of Embodiments 7-11, wherein both R9 groups are H.
13. A compound of formula I:
Figure imgf000053_0002
F , ,
Formula 1 wherein R1 and R2 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy.
14. The compound of Embodiment 13, wherein halo is F, I, Cl, or Br.
15. The compound of Embodiment 14, wherein mono-halo methyl is -C(H2)F, - C(H2)I, -C(H2)C1 or -C(H2)Br. 16. The compound of Embodiment 14, wherein di-halo methyl is -C(H)F2, -C(H)l2, - C(H)Ch or -C(H)Br2.
17. The compound of Embodiment 14, wherein tri-halo methyl is -CF3, -CI3, -CCI3 or -CBr3.
18. The compound of Embodiment 13, wherein the Ci-Ce alkyl is -CH3.
19. The compound of Embodiment 13, wherein the Ci-Ce alkoxy is -OCH3.
20. The compound of Embodiment 13, which has the structure:
Figure imgf000054_0001
21. The compound of Embodiment 13, which has the structure:
Figure imgf000054_0002
22. A compound of formula Ila:
Figure imgf000054_0003
Formula Ila wherein R1 and R2 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy; and R10 is heteroaryl or heterocycloalkyl.
23. The compound of Embodiment 22, wherein the compound is of the formula:
Figure imgf000054_0004
24. The compound of Embodiment 22, wherein the compound is of the formula:
Figure imgf000055_0001
25. The compound of any one of Embodiments 22-24, wherein halo is F, I, Cl, or Br.
26. The compound of any one of Embodiments 22-24, wherein mono-halo methyl is - C(H2)F, -C(H2)I, -C(H2)C1 or -C(H2)Br.
27. The compound any one of Embodiments 22-24, wherein di-halo methyl is - C(H)F2, -C(H)I2, -C(H)C12 or -C(H)Br2.
28. The compound any one of Embodiments 22-24, wherein tri-halo methyl is -CF3, - CI3, -CCI3 or -CBr3.
29. The compound of any one of Embodiments 22-24, wherein the Ci-Ce alkyl is -
CH3.
30. The compound of any one of Embodiments 22-24, wherein the Ci-Ce alkoxy is -
OCH3.
31. The compound of any one of Embodiments 22-24, which has the structure:
Figure imgf000055_0002
32. A compound of formula III:
Figure imgf000056_0001
Formula III wherein R1 and R2 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy.
33. The compound of Embodiment 32, wherein halo is F, I, Cl, or Br.
34. The compound of Embodiment 33, wherein mono-halo methyl is -C(H2)F, - C(H2)I,
-C(H2)C1 or -C(H2)Br.
35. The compound of Embodiment 33, wherein di-halo methyl is -C(H)F2, -C(H)l2, -C(H)C12 or -C(H)Br2.
36. The compound of Embodiment 34, wherein tri-halo methyl is -CF3, -CI3, -CCI3 or
-CBr3.
37. The compound of Embodiment 33, wherein the Ci-Ce alkyl is -CH3.
38. The compound of Embodiment 32, wherein the Ci-Ce alkoxy is -OCH3.
39. A compound of formula V:
X2
R11 JL R12
R5 R6 Formula V wherein:
R11 is alkyl, cycloalkyl, aryl, arylakyl, or arylacyl;
R12 is cycloakyl, aryl, or heterocyclyl;
R5 and R6 are each independently H or alkyl or, R5 and R6, together with the atoms to which they are attached, form a heterocyclyl group; and X2 is O or S.
40. A pharmaceutical composition comprising at least one compound of any one of Embodiments 1-38 and a pharmaceutically acceptable carrier.
41. The pharmaceutical composition of Embodiment 40, which further comprises at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus, wherein the at least one other compound is selected from the group consisting of a sulfonylurea, a meglitinide, a biguanide, a thiazolidinedione (TZD), an a-glucosidase inhibitor, a dipeptidyl peptidase IV (DPP -4) inhibitor, a bile acid sequestrant, a dopamine agonist, a sodium-glucose transport protein 2 (SGLT2) inhibitor, and a glucagon like peptide 1 (GLP-1) receptor agonist.
42. The pharmaceutical composition of Embodiment 41, wherein:
(a) the sulfonylurea is at least one compound selected from the group consisting of chlorpropamide, tolazamide, tolbutamide, acetohexamide, glyburide, glipizide, glimepiride, and gliclazide,
(b) the meglitinide is repaglinide, netaglinide, or a combination thereof,
(c) the biguanide is metformin,
(d) the TZD is rosiglitazone, pioglitazone, or a combination thereof,
(e) the a-glucosidase inhibitor is at least one compound selected from the group consisting of acarbose, miglitol and voglibose,
(f) the DPP-4 inhibitor is at least one compound selected from the group consisting of alogliptin, linagliptin, sitagliptin, saxagliptin and vldagliptin,
(g) the bile acid sequestrant is colesevelam,
(h) the dopamine agonist is bromocriptine,
(i) the SGLT2 inhibitor is at least one compound selected from the group consisting of cangliflozin, dapagliflozin, empagliflozin and ertugliflozin, and
(j) the GLP-1 receptor agonist is semaglutide.
43. The pharmaceutical composition of Embodiment 41, wherein the at least one other compound is a combination of compounds selected from: (a) metformin and at least one of glyburide, glipizide, sitagliptin, saxagliptin, pioglitazone, repaglinide, rosiglitazone, linagliptin, alogliptin, canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin,
(b) glimepiride and either or both of pioglitazone and rosiglitazone,
(c) alogliptin and pioglitazone,
(d) dapagliflozin and saxagliptin,
(e) empagliflozin and linagliptin,
(f) ertugliflozin and sitagliptin,
(g) lixisenatide and glargine insulin, and
(h) liraglutide and degludec insulin.
44. The pharmaceutical composition of any one of Embodiments 40-44, which is formulated for oral administration.
45. The pharmaceutical composition of Embodiment 40, which further comprises at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus, wherein the at least one other compound is selected from the group consisting of a GLP-1 receptor agonist and a dual GLP-1 receptor and gastric inhibitory polypeptide (GIP) receptor agonist.
46. The pharmaceutical composition of Embodiment 45, wherein:
(a) the GLP-1 receptor agonist is exenatide, liraglutide, dulaglutide, lixisenatide or semaglutide, and
(b) the dual GLP-1 receptor and GIP receptor agonist is tirzepatide.
47. The pharmaceutical composition of any one of Embodiments 45 or 46, which is formulated for administration by injection.
48. The pharmaceutical composition of Embodiment 40, which further comprises bexagliflozin.
49. The pharmaceutical composition of Embodiment 48, which is formulated for oral administration. a pharmaceutically acceptable carrier. 50. A method of inhibiting aggregation of misfolded islet amyloid protein (IAPP) in a human with, or at risk for, diabetes mellitus, which method comprises administering to the human the compound of any one of Embodiments 1-39, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit aggregation of misfolded IAPP, whereupon aggregation of misfolded IAPP is inhibited in the human with, or at risk for, diabetes mellitus.
51. The method of Embodiment 50, which further comprises administering, simultaneously or sequentially by the same or different routes, at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus and which is selected from the group consisting of a sulfonylurea, a meglitinide, a biguanide, a thiazolidinedione (TZD), an a-glucosidase inhibitor, a dipeptidyl peptidase IV (DPP-4) inhibitor, a bile acid sequestrant, a dopamine agonist, a sodium-glucose transport protein 2 (SGLT2) inhibitor, and a glucagon like peptide 1 (GLP-1) receptor agonist.
52. The method of Embodiment 51, wherein:
(a) the sulfonylurea is at least one compound selected from the group consisting of chlorpropamide, tolazamide, tolbutamide, acetohexamide, glyburide, glipizide, glimepiride, and gliclazide,
(b) the meglitinide is repaglinide, netaglinide, or a combination thereof,
(c) the biguanide is metformin,
(d) the TZD is rosiglitazone, pioglitazone, or a combination thereof,
(e) the a-glucosidase inhibitor is at least one compound selected from the group consisting of acarbose, miglitol and voglibose,
(f) the DPP-4 inhibitor is at least one compound selected from the group consisting of alogliptin, linagliptin, sitagliptin, saxagliptin and vldagliptin,
(g) the bile acid sequestrant is colesevelam,
(h) the dopamine agonist is bromocriptine,
(i) the SGLT2 inhibitor is at least one compound selected from the group consisting of cangliflozin, dapagliflozin, empagliflozin and ertugliflozin, and
(j) the GLP-1 receptor agonist is semaglutide. 53. The method of Embodiment 51, wherein the at least one other compound is a combination of compounds selected from:
(a) metformin and at least one of glyburide, glipizide, sitagliptin, saxagliptin, pioglitazone, repaglinide, rosiglitazone, linagliptin, alogliptin, canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin,
(b) glimepiride and either or both of pioglitazone and rosiglitazone,
(c) alogliptin and pioglitazone,
(d) dapagliflozin and saxagliptin,
(e) empagliflozin and linagliptin,
(f) ertugliflozin and sitagliptin,
(g) lixisenatide and glargine insulin, and
(h) liraglutide and degludec insulin, wherein the compounds in the combination can be administered simultaneously or sequentially by the same or different routes.
54. The method of any one of Embodiments 50-53, wherein administering is orally administering.
55. The method of Embodiment 50, which further comprises administering at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus, wherein the at least one other compound is selected from the group consisting of a GLP-1 receptor agonist and a dual GLP-1 receptor and gastric inhibitory polypeptide (GIP) receptor agonist.
56. The method of Embodiment 55, wherein:
(a) the GLP-1 receptor agonist is exenatide, liraglutide, dulaglutide, lixisenatide or semaglutide, and
(b) the dual GLP-1 receptor and GIP receptor agonist is tirzepatide.
57. The method of 55 or 56, wherein administering is by injection.
58. A method of inhibiting aggregation of misfolded islet amyloid protein (IAPP) in a cat with, or at risk for, diabetes mellitus, which method comprises administering to the cat the compound of any one of Embodiments 1-39, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit aggregation of misfolded IAPP, whereupon aggregation of misfolded IAPP is inhibited in the cat with, or at risk for, diabetes mellitus.
59. The method of Embodiment 58, which further comprises administering bexafliflozin.
60. The method of Embodiment 58 or 59, wherein administering is orally administering.
61. The method of Embodiment 58, which further comprises administering insulin.
62. The method of Embodiment 61, wherein the compound of any one of Embodiments 1-39, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, is administered orally and insulin is administered by injection.
63. A method of inhibiting tubulin-associated unit (tau) protein aggregation in a subject having, or at risk for, tau protein aggregation, which method comprises administering to subject any one of Embodiments 39, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit tubulin-associated unit (tau) protein aggregation
64. The method of Embodiment 63, wherein the subject has, or is at risk for, Alzheimer’s disease.
65. A method of inhibiting alpha-synuclein (a-syn) protein aggregation in a subject having, or at risk for a-syn, which method comprises administering to subject any one of Embodiments 39, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit alpha-synuclein (a-syn) protein aggregation.
66. The method of Embodiment 65, wherein the subject has, or is at risk for, Parkinson’s disease. EXAMPLES
The following examples serve to illustrate the present disclosure. The examples are not intended to limit the scope of the claimed invention in any way.
Materials and methods
Chemicals
All drugs were dissolved in dimethylsulfoxide (DMSO) at 40 mM. Hexafluoroisopropanol (HFIP), DMSO, resazurin and thioflavin-T (ThT) were obtained from Alfa Aesar (Ward Hill, MA).
Peptide synthesis
Synthetic human islet amyloid protein (hlAPP) and feline IAPP (flAPP) (1-37) were obtained from AnaSpec (Fremont, CA, USA).
Thioflavin T (ThT) fluorescence assay hlAPP and flAPP peptides from the stock solution of 1 mM were each added to 10 mM phosphate-buffered saline (PBS) buffer (pH 7.4) and transferred to a black 96-well microtiter plate with transparent bottom. Each well contained a final volume of 150 pL with a final peptide concentration of 10 pM. Experiments in the presence of hlAPP and flAPP were performed with different compounds at a final concentration of 100 pM. For the dose response, final drug concentrations ranged from 1.5 to 100 pM. The background signal consisted of 0.25% DMSO without peptide and compound. The fluorescence emission experiments were performed using methods known in the art. The maximum fluorescence intensity in percentage reported in Table 1 was calculated as previously described.
Transmission electron microscopy (TEM) hlAPP and flAPP were each incubated in 10 mM PBS (pH 7.4, 25 °C) at 10 pM for one hour with different compounds (14, 20, or 27) at 100 pM. Disaggregation experiments were performed by incubating either hlAPP or flAPP for 24 hours prior to drug treatment, followed by incubation for 24 hours with the pharmacologic agent. Samples were treated with one of four conditions: DMSO (control), Compound 14 (APS-22-96), Compound 20 (APS- 22-109), or Compound 27 (APS-22-107). Following ThT kinetics, a volume of 10 pL was applied on a 400-mesh Formvar- carbon-coated copper grid (Electron Microscopy Sciences, Hatfield, PA). Grids were incubated for one minute and washed once with distilled water. After air-drying, grids were incubated for one minute in a fresh solution of 1% uranyl acetate. Grids were air-dried again and imaged via TEM (JEOL 1400 Flash, Japan). Pictures were acquired at an accelerating voltage of 100 kV and magnifications of 20k and 40k.
Dynamic Light Scattering (DLS)
The Malvern Zetasizer machine was utilized to conduct DLS, in which the particle size of each sample is analyzed. In microcentrifuge tubes, hlAPP and flAPP were each incubated in 10 mM PBS (pH 7.4, 25 °C) at 10 pM for one hour with different compounds (14, 20, or 27) at 100 pM. to attain a 1 : 10 molar ratio. From each sample, a volume of 50 pL was added to a low-volume quartz cuvette (QS high precision cell, 105-251-85-40, Hellma analytics) and read at 25°C using the fluorescence filter. The average of three repetitions of each sample were recorded.
Cell line and culture
Min-6 (mice insulinoma) cells were purchased from AddexBio (Cat No COO 18006, San Diego, CA, USA). Min-6 cells were grown in AddexBio Advanced Medium with glucose, L-glutamine, sodium pyruvate (AddexBio, Cat No C0003-04, San Diego, CA, USA) supplemented with 50 mM 2-mercaptoethanol, 100 U/mL streptomycin, 100 U/mL of penicillin G (1% of Penicillin-Streptomycine solution from Cytiva, Hyclone Laboratories, Logan, Utah, USA) and 15% fetal bovine serum (Hyclone Laboratories, cat No SH30088.03, Logan, Utah, USA). Cells were maintained in a moisture saturated atmosphere at 37 °C under 5% CO2.
Cell viability assay
Cells were maintained in a 37 °C, 5% CO2 incubator, in exponential growth, for the duration of the experimentation. For this assay, cells were seeded in 96-well microtiter plates at a density of 5 x 103 Min-6 cells per well for 24 h. Compounds were freshly solubilized in DMSO were added subsequently after stimulation with 20 pM of flAPP. flAPP was dissolved using 4.8% DMSO in 10 mM PBS (pH 7.4, 25 °C) and incubated for 48 hours before addition to cells in order to generate fibrils. Compounds were tested at 6.25 to 12.5 pM. DMSO concentration was maintained at 0.12% to avoid growth inhibition. Negative control consisted of DMSO alone. Plates were incubated for 24h in the presence of compounds or vehicle (0.12% DMSO), and flAPP. Resazurin-based reduction assays were performed as previously described (Fortin and Benoit-Biancamano 2015).
Example 1
Synthesis
The diaryl urea derivatives 1-27 were synthesized by treating commercially available aromatic amines, namely 2-aminofluorene (series 1), 4-morpholinoaniline (series 2), and 4- aminoindole (series 3), with substituted aromatic isocyanates in anhydrous dichloromethane at room temperature for 12 hours. The general synthetic route for the preparation of the diaryl derivatives is shown in Scheme 1. All compounds were obtained in moderate to high yields. The compounds were characterized using both proton (1H) and carbon (13C) spectroscopy and high-resolution mass spectrometry (TERMS). The melting point for each compound was also recorded.
Figure imgf000064_0001
Scheme 1: Preparation of diaryl urea derivatives; di chloromethane (DCM), room temperature (rt), 8-12 hours. An (Ri) is a placeholder for one of three series: 2-aminofluorene, 4- morpholinoaniline, or 4-aminoindole. An (R2) refers to one of the following substituents: o- methylphenyl, o-methoxy phenyl, o-fluorophenyl, o-nitrophenyl, p-(trifluoromethyl)phenyl, o,p-dimethoxylphenyl, o-methylchloro, m-(trifluoromethyl)phenyl, o,p-dimethoxy, o- chlorophenyl.
NMR Spectral Data
2-AMINOFLUORENE SERIES
Compound 1 APS-21-07: ’H NMR (500 MHz, DMSO) 5 9.16 (s, 1H), 8.57 (s, 1H), 8.18 (t, J = 8.2 Hz, 1H), 7.78 (d, J= 9.3 Hz, 3H), 7.52 (d, J= 7.5 Hz, 1H), 7.44 - 7.29 (m, 2H), 7.23 (t, J= 7.9 Hz, 2H), 7.14 (t, J= 7.9 Hz, 1H), 6.99 (d, J= 6.8 Hz, 1H), 3.89 (s, 2H). 13C NMR (126 MHz, DMSO) 5 153.4, 152.7, 151.5, 144.5, 143.1, 141.6, 139.0, 135.9, 128.1, 128.1, 127.2, 126.3, 125.5, 125.0, 122.9, 121.0, 120.8, 119.8, 117.5, 115.5, 115.4, 37.0. Compound 2 APS-22-99: ’H NMR (500 MHz, DMSO) 5 9.34 (s, 1H), 8.22 (s, 1H), 7.91 - 7.86 (m, 1H), 7.83 - 7.76 (m, 3H), 7.52 (d, J= 7.5 Hz, 1H), 7.41 (ddd, J= 13.7, 7.9, 1.8 Hz, 2H), 7.35 - 7.30 (m, 2H), 7.25 - 7.21 (m, 1H), 7.06 (td, J= 7.5, 1.2 Hz, 1H), 4.84 (s, 2H), 3.88 (s, 2H). (Xxs 2 Ar + 2 Aliph). 13C NMR (126 MHz, DMSO) 5 153.2, 144.5, 143.1,
141.7, 139.3, 138.0, 135.7, 131.1, 129.8, 128.1, 127.2, 126.3, 125.4, 123.6, 123.0, 120.8,
119.7, 117.5, 115.4, 44.0, 37.0.
Compound 3 APS-22-87: ’H NMR (500 MHz, DMSO) 5 9.06 (s, 1H), 8.87 (s, 1H), 8.05 (s, 1H), 7.83 - 7.74 (m, 3H), 7.60 - 7.48 (m, 3H), 7.40 (dd, J= 8.2, 2.0 Hz, 1H), 7.36 - 7.27 (m, 2H), 7.23 (td, J= 7.4, 1.2 Hz, 1H), 3.88 (s, 2H). 13C NMR (126 MHz, DMSO) 5 153.0, 144.4, 143.1, 141.6, 141.1, 138.9, 135.9, 130.4, 127.2, 126.4, 125.5, 122.3, 120.7,
119.8, 118.5, 117.8, 115.8, 114.6, 37.0.
Compound 4 APS-21-09: ’H NMR (500 MHz, DMSO) 5 9.11 (s, 1H), 8.88 (s, 1H), 7.78 (d, J= 7.2 Hz, 3H), 7.72 - 7.58 (m, 4H), 7.52 (d, J= 7.4 Hz, 1H), 7.45 - 7.37 (m, 1H), 7.33 (t, J= 7.4 Hz, 1H), 7.23 (t, J= 7.4 Hz, 1H), 3.89 (s, 2H). 13C NMR (126 MHz, DMSO) 5 152.8, 144.5, 144.0, 143.1, 141.6, 138.9, 136.0, 127.2, 126.6, 126.4, 125.5, 120.7, 119.8, 118.3, 117.8, 115.8, 37.0.
Compound 5 APS-21-08: ’H NMR (500 MHz, DMSO) 5 9.92 (s, 1H), 9.62 (s, 1H), 8.31 (d, J= 8.5 Hz, 1H), 8.13 - 8.03 (m, 1H), 7.80 (dd, J= 14.6, 7.0 Hz, 3H), 7.74 - 7.64 (m, 1H), 7.52 (d, J= 7.5 Hz, 1H), 7.46 - 7.38 (m, 1H), 7.33 (t, J= 7.4 Hz, 1H), 7.21 (dt, J= 23.0, 7.6 Hz, 2H), 3.89 (s, 2H). 13C NMR (126 MHz, DMSO) 5 152.3, 144.5, 143.2, 141.5, 138.8,
138.1, 136.2, 135.5, 127.2, 126.4, 125.9, 125.5, 123.0, 122.7, 120.8, 119.8, 117.9, 115.9, 37.0.
Compound 6 APS-21-15: ’H NMR (500 MHz, DMSO) 5 9.27 (s, 1H), 8.15 (s, 1H),
7.89 (d, J= 8.2 Hz, 1H), 7.86 - 7.72 (m, 3H), 7.52 (d, J= 7.4 Hz, 1H), 7.43 (d, J= 7.4 Hz, 2H), 7.33 (t, J= 7.6 Hz, 2H), 7.23 (t, J= 13 Hz, 1H), 7.06 (t, J= 7.5 Hz, 1H), 4.83 (s, 2H),
3.89 (s, 2H). 13C NMR (126 MHz, DMSO) 5 153.2, 144.5, 143.1, 141.7, 139.3, 138.0, 135.7,
131.1, 129.8, 128.1, 127.2, 126.3, 125.45, 123.7, 123.0, 120.8, 119.7, 117.5, 115.5, 44.0, 37.0.
Compound 7 APS-21-05: ’H NMR (500 MHz, DMSO) 5 9.11 (s, 1H), 7.93 (s, 1H), 7.87 - 7.70 (m, 4H), 7.52 (d, J= 7.4 Hz, 1H), 7.41 - 7.27 (m, 2H), 7.26 - 7.19 (m, 1H), 7.19 - 7.08 (m, 2H), 6.99 - 6.86 (m, 1H), 3.88 (s, 2H), 2.24 (s, 3H). 13C NMR (126 MHz, DMSO) 5 153.2, 144.5, 143.0, 141.7, 139.5, 137.9, 135.5, 130.7, 128.0, 127.2, 126.7, 126.3, 125.4,
123.1, 121.5, 120.8, 119.7, 117.3, 115.3, 37.0, 18.4. Compound 8 APS-21-06: ’H NMR (500 MHz, DMSO) 5 9.40 (s, 1H), 8.25 (s, 1H), 8.15 (dd, J= 7.8, 1.8 Hz, 1H), 7.84 - 7.73 (m, 3H), 7.52 (d, J= 7.4 Hz, 1H), 7.41 - 7.29 (m, 2H), 7.22 (td, J = 7.4, 1.1 Hz, 1H), 7.01 (dd, J= 8.0, 1.6 Hz, 1H), 6.91 (dtd, J= 22.8, 7.5, 1.6 Hz, 2H), 3.87 (s, 5H). 13C NMR (126 MHz, DMSO) 5 152.9, 148.1, 144.5, 143.0, 141.7,
139.5, 135.5, 129.2, 127.2, 126.2, 125.4, 122.3, 121.1, 120.7, 119.7, 118.7, 117.3, 115.2, 111.2, 56.3, 37.0.
Compound 9 APS-21-10: ’H NMR (500 MHz, DMSO) 5 9.24 (s, 1H), 8.01 (s, 1H), 7.95 (d, J= 8.8 Hz, 1H), 7.81 - 7.70 (m, 3H), 7.51 (d, J= 7.4 Hz, 1H), 7.33 (ddd, J= 16.8, 7.9, 1.6 Hz, 2H), 7.22 (td, J= 7.4, 1.2 Hz, 1H), 6.61 (d, J= 2.7 Hz, 1H), 6.48 (dd, J= 8.9, 2.7 Hz, 1H), 3.86 (d, J= 7.8 Hz, 5H), 3.72 (s, 3H). 13C NMR (126 MHz, DMSO) 5 155.4, 153.1,
149.6, 144.5, 143.0, 141.7, 139.7, 135.3, 127.2, 126.2, 125.4, 122.4, 120.7, 120.1, 119.6,
117.1, 115.1, 104.6, 99.3, 56.3, 55.8, 37.0.
4-MORPHOLINO AN ALINE SERIES
Compound 10 APS-22-91: ’H NMR (500 MHz, DMSO) 5 8.81 (s, 1H), 8.42 (s, 1H), 8.20 - 8.09 (m, 1H), 7.30 (d, J= 9.0 Hz, 2H), 7.20 (ddd, J= 11.7, 8.1, 1.5 Hz, 1H), 7.10 (td, J = 7.9, 1.5 Hz, 1H), 7.00 - 6.92 (m, 1H), 6.88 (d, J= 9.0 Hz, 2H), 3.71 (dd, J= 5.8, 3.7 Hz, 4H), 3.00 (s, 4H). 13C NMR (126 MHz, DMSO) 5 153.3, 152.8, 151.4, 147.1, 132.2, 128.3 (2C), 124.9, 122.6, 122.5, 120.8, 120.0, 116.4, 115.4, 115.3, 66.6, 49.7.
Compound 11 APS-22-98: ’H NMR (500 MHz, DMSO) 5 9.07 (s, 1H), 8.15 (s, 1H), 7.95 - 7.76 (m, 1H), 7.51 - 7.13 (m, 4H), 7.02 (td, J= 7.5, 1.3 Hz, 1H), 6.92 (d, J= 8.5 Hz, 2H), 3.73 (t, J= 4.6 Hz, 4H), 3.04 (s, 4H). 13C NMR (126 MHz, DMSO) 5 153.2, 138.2,
131.1, 129.7, 127.7, 123.3, 122.6, 119.9, 116.7, 66.5, 50.0, 44.0.
Compound 12 APS-22-100: ’H NMR (500 MHz, DMSO) 5 8.91 (s, 1H), 8.50 (s, 1H), 7.99 (s, 1H), 7.59 - 7.41 (m, 2H), 7.28 (dd, J= 22.2, 8.1 Hz, 3H), 6.87 (d, J= 8.6 Hz, 2H), 3.71 (t, J= 4.7 Hz, 4H), 3.01 (d, J= 4.8 Hz, 4H). 13C NMR (126 MHz, DMSO) 5 153.1,
147.2, 141.3, 132.0, 130.3, 122.1, 120.4, 118.2, 116.3, 114.4, 66.6, 49.6.
Compound 13 APS-22-101: ’H NMR (500 MHz, DMSO) 5 8.99 (s, 1H), 8.53 (s, 1H), 7.61 (m, 4H), 7.34 - 7.28 (m, 2H), 6.90 - 6.85 (m, 2H), 3.74 - 3.69 (m, 4H), 3.03 - 2.98 (m, 4H). 13C NMR (126 MHz, DMSO) 5 152.9, 147.2, 144.2, 132.0, 126.5, 124.0, 122.0, 121.8, 120.4, 118.1, 116.3, 66.6, 49.6.
Compound 14 APS-22-96: ’H NMR (500 MHz, DMSO) 5 9.61 (s, 1H), 9.54 (s, 1H), 8.31 (dt, J= 8.6, 1.0 Hz, 1H), 8.07 (dd, J= 8.4, 1.6 Hz, 1H), 7.66 (ddd, J = 8.7, 7.2, 1.6 Hz, 1H), 7.38 - 7.28 (m, 2H), 7.16 (ddd, J= 8.5, 7.2, 1.3 Hz, 1H), 6.94 - 6.81 (m, 2H), 3.75 - 3.67 (m, 4H), 3.05 - 2.97 (m, 4H). 13C NMR (126 MHz, DMSO) 5 152.3, 147.4, 137.8, 135.8, 135.5, 131.8, 125.9, 122.8, 122.4, 120.5, 116.3, 66.6, 49.5.
Compound 15 APS-22-92: ’H NMR (500 MHz, DMSO) 5 8.98 (s, 1H), 8.05 (s, 1H), 7.87 (dd, J= 8.2, 1.2 Hz, 1H), 7.39 (dd, J= 7.2, 1.2 Hz, 1H), 7.33 (dd, J= 9.3, 7.2 Hz, 2H), 7.12 - 6.77 (m, 3H), 4.80 (s, 2H), 3.72 (t, J= 4.8 Hz, 4H), 3.03 (t, J= 4.8 Hz, 4H). 13C NMR (126 MHz, DMSO) 5 153.2, 138.2, 131.1, 129.7, 127.7, 123.3, 122.6, 119.9, 116.6, 66.5,
49.9, 44.0.
Compound 16 APS-22-95: ’H NMR (500 MHz, DMSO) 5 8.75 (s, 1H), 7.83 (dd, J= 8.1, 1.3 Hz, 1H), 7.78 (s, 1H), 7.31 (d, J= 9.0 Hz, 2H), 7.18 - 7.05 (m, 2H), 6.96 - 6.77 (m, 3H), 3.83 - 3.60 (m, 4H), 3.08 - 2.89 (m, 4H), 2.21 (s, 3H). 13C NMR (126 MHz, DMSO) 5
153.3, 146.8, 138.1, 132.7, 130.6, 127.6, 126.6, 122.8, 121.2, 119.8, 116.4, 66.6, 49.7, 18.4.
Compound 17 APS-22-94: ’H NMR (500 MHz, DMSO) 5 9.05 (s, 1H), 8.15 - 8.05 (m, 2H), 7.34 - 7.23 (m, 2H), 6.98 (dd, J= 8.0, 1.6 Hz, 1H), 6.94 - 6.80 (m, 4H), 3.85 (s, 3H), 3.74 - 3.67 (m, 4H), 3.02 - 2.97 (m, 4H). 13C NMR (126 MHz, DMSO) 5 153.0, 148.0,
146.9, 132.7, 129.4, 122.0, 121.0, 119.7, 118.6, 116.4, 111.1, 66.6, 56.2, 49.7.
Compound 18 APS-22-93: ’H NMR (500 MHz, DMSO) 5 8.89 (s, 1H), 7.90 (d, J=
8.8 Hz, 1H), 7.85 (s, 1H), 7.28 (d, J= 9.0 Hz, 2H), 6.85 (d, J= 9.1 Hz, 2H), 6.59 (d, J= 2.7 Hz, 1H), 6.45 (dd, J= 8.9, 2.7 Hz, 1H), 3.83 (s, 3H), 3.71 (s, 7H), 2.99 (dd, J= 5.8, 3.8 Hz, 4H). 13C NMR (126 MHz, DMSO) 5 155.2, 153.2, 149.5, 146.7, 132.9, 122.6, 120.0, 119.6,
116.4, 104.6, 99.2, 66.6, 56.3, 55.7, 49.8.
4-AMINOINDOLE SERIES
Compound 19 APS-22-108: ’H NMR (500 MHz, DMSO) 5 11.13 (s, 1H), 8.90 (m, 2H), 8.30 - 8.15 (m, 1H), 7.68 (d, J= 7.6 Hz, 1H), 7.30 (t, J = 2.7 Hz, 1H), 7.27 - 7.17 (m, 1H), 7.13 (t, J= 7.8 Hz, 1H), 7.06 (d, J= 7.8 Hz, 1H), 6.99 (m, 2H), 6.58 (t, J= 2.5 Hz, 1H). 13C NMR (126 MHz, DMSO) 5 152.7, 151.4, 137.0, 131.8, 128.3, 125.0, 124.6, 122.6, 122.0, 120.8, 119.4, 115.5, 107.9, 106.5, 98.3.
Compound 20 APS-22-109: ’H NMR (500 MHz, DMSO) 5 11.46 (s, 1H), 11.13 (s, 1H), 10.31 (s, 1H), 7.89 (d, J= 7.9 Hz, 1H), 7.55 (dd, J= 18.7, 7.7 Hz, 2H), 7.45 - 7.31 (m, 3H), 7.21 (t, J= 2.9 Hz, 1H), 7.09 (t, J= 8.0 Hz, 2H), 6.95 (d, J= 7.5 Hz, 1H), 6.88 (t, J=
7.9 Hz, 1H), 6.80 (d, J= 7.7 Hz, 1H), 6.76 - 6.57 (m, 2H). 13C NMR (126 MHz, DMSO) 5 137.44, 134.74, 132.23, 131.82, 127.90, 126.39, 125.14, 121.65, 121.47, 111.71, 109.16, 99.91, 98.99, 40.14, 39.97, 39.81. Compound 21 APS-22-115: *H NMR (500 MHz, DMSO) 5 11.14 (s, 1H), 9.24 (s, 1H), 8.58 (s, 1H), 8.07 (d, J= 2.0 Hz, 1H), 7.63 (dd, J= 7.6, 0.9 Hz, 1H), 7.59 - 7.43 (m, 2H), 7.34 - 7.23 (m, 2H), 7.07 (dt, J= 8.1, 1.0 Hz, 1H), 7.00 (t, J= 7.9 Hz, 1H), 6.59 - 6.51 (m, 1H). 13C NMR (126 MHz, DMSO) 5 153.0, 141.2, 137.0, 131.5, 130.5, 124.7, 122.0, 119.6, 118.4, 114.3, 108.3, 106.7, 98.2.
Compound 22 APS-22-114: 3H NMR (500 MHz, DMSO) 5 11.14 (s, 1H), 9.31 (s, 1H), 8.61 (s, 1H), 7.69 (d, J= 8.5 Hz, 2H), 7.67 - 7.59 (m, 3H), 7.31 (t, J= 2.8 Hz, 1H), 7.08 (dt, J= 8.1, 1.0 Hz, 1H), 7.01 (t, .7= 7.9 Hz, 1H), 6.55 (td, J= 2.2, 1.2 Hz, 1H). 13C NMR (126 MHz, DMSO) 5 152.73, 144.05, 136.97, 131.50, 126.64, 126.61, 124.67, 121.99, 119.56, 118.11, 108.22, 106.75, 98.20, 40.13, 39.96, 39.79.
Compound 23 APS-22-106: ’H NMR (500 MHz, DMSO) 5 11.13 (s, 1H), 9.68 (s, 1H), 9.47 (s, 1H), 8.24 (d, J= 8.5 Hz, 1H), 8.15 - 7.97 (m, 1H), 7.75 - 7.60 (m, 1H), 7.54 (d, J= 7.7 Hz, 1H), 7.24 - 7.15 (m, 1H), 7.11 (d, J = 8.0 Hz, 1H), 7.01 (t, J= 7.9 Hz, 1H), 6.71 - 6.61 (m, 1H). 13C NMR (126 MHz, DMSO) 5 152.5, 138.7, 137.1, 135.2, 135.2, 131.2, 125.8, 124.7, 123.7, 122.8, 121.9, 120.3, 109.5, 107.3, 99.0.
Compound 24 APS-22-113: ’H NMR (500 MHz, DMSO) 5 11.11 (s, 1H), 9.05 (s, 1H), 8.66 (s, 1H), 7.89 (dd, J= 8.2, 1.2 Hz, 1H), 7.64 (d, J= 7.6 Hz, 1H), 7.42 (dd, J= 7.6,
1.6 Hz, 1H), 7.33 - 7.28 (m, 2H), 7.07 - 7.03 (m, 2H), 6.98 (d, J= 7.9 Hz, 1H), 6.71 (t, J=
2.6 Hz, 1H). 13C NMR (126 MHz, DMSO) 5 153.36, 138.13, 136.99, 132.01, 131.15, 129.68, 128.20, 124.37, 123.51, 123.10, 121.98, 119.62, 108.29, 106.41, 98.83, 44.07, 40.47, 40.40, 40.31, 40.23, 40.14, 39.97, 39.81, 39.64, 39.47.
Compound 25 APS-22-111 : ’H NMR (500 MHz, DMSO) 5 11.10 (s, 1H), 8.80 (s, 1H), 8.18 (s, 1H), 7.87 (dd, J= 8.2, 1.3 Hz, 1H), 7.65 (dd, J= 7.6, 1.0 Hz, 1H), 7.30 (t, J=
2.7 Hz, 1H), 7.19 - 7.11 (m, 2H), 7.04 (dt, J= 8.1, 1.0 Hz, 1H), 6.99 (t, J= 7.8 Hz, 1H), 6.93 (td, J= 7.4, 1.3 Hz, 1H), 6.64 - 6.54 (m, 1H), 2.28 (s, 3H). 13C NMR (126 MHz, DMSO) 5 153.24, 138.02, 136.97, 132.10, 130.67, 127.89, 126.61, 124.40, 123.01, 122.02, 121.62, 119.48, 108.16, 106.28, 98.44, 40.46, 40.30, 40.22, 40.13, 39.96, 39.79, 39.63, 39.46, 18.54.
Compound 26 APS-22-110: ’H NMR (500 MHz, DMSO) 5 11.08 (s, 1H), 9.03 (s, 1H), 8.60 (s, 1H), 8.16 (d, J= 7.8 Hz, 1H), 7.68 (d, J= 7.5 Hz, 1H), 7.27 (t, J= 2.7 Hz, 1H), 7.06 - 6.96 (m, 3H), 6.91 (dt, J= 22.5, 7.5 Hz, 2H), 6.65 (s, 1H), 3.89 (s, 3H). 13C NMR (126 MHz, DMSO) 5 153.07, 148.25, 136.95, 132.16, 129.33, 124.26, 122.13, 121.98, 121.00, 119.46, 119.05, 111.19, 107.98, 106.22, 98.68, 56.21, 40.48, 40.40, 40.31, 40.24, 40.14, 39.97, 39.81, 39.64, 39.47. Compound 27 APS-22-107: ’H NMR (500 MHz, DMSO) 5 11.07 (s, 1H), 8.88 (s, 1H), 8.37 (s, 1H), 7.97 (d, J= 92 Hz, 1H), 7.67 (d, J= 7.6 Hz, 1H), 7.26 (t, J= 2.5 Hz, 1H), 6.99 (m, 2H), 6.63 (dd, J= 9.2, 2.4 Hz, 2H), 6.48 (dd, J= 8.9, 2.7 Hz, 1H), 3.87 (s, 3H), 3.72 (s, 3H). 13C NMR (126 MHz, DMSO) 5 155.3, 153.2, 149.7, 137.0, 132.4, 124.2, 122.5, 122.0, 120.4, 119.3, 107.7, 106.0, 104.6, 99.2, 98.6, 56.3, 55.7.
Table 1: Structure- Activity Relationships
2-aminofluorene series
Figure imgf000070_0001
4-morpholino alinine series
SNO Structure CP
Figure imgf000070_0002
Figure imgf000071_0001
-aminoindole series
SNO
Figure imgf000071_0002
Figure imgf000072_0001
Example 2 Inhibition of IAPP fibril formation
The diaryl urea derivatives 1-27 were tested on hlAPP and flPAA to determine their effect on IAPP fibril formation by measuring their fluorescence intensity (Table 1). In this test, lower fluorescence corresponds with a greater reduction of fibrils in the sample. Compounds with a measured fluorescence percentage less than 10% for either hlAPP or flAPP were selected as the compounds with greatest potential for reducing amylin fibril accumulation. Based on the data obtained, 4-aminoindolyl urea derivatives 27 (APS-22-107) and 20 (APS-22-109) were found to be the most potent compounds with fluorescence intensities of 7.5 ± 0.4% and 8.5 ± 1.2%, respectively, for hlAAP; 6.9 ± 1.4 and 9.0 ± 0.6%, respectively, for flAPP. Moreover, these compounds were highly selective for both hlAAP and flAPP proteins. In the 2-aminoflurorene series, compounds 1 (APS-21-07), 2 (APS-22- 99), 8 (APS-21-06), and 9 (APS-21-10) were selective for both hlAPP and flAPP. Compound 7 (APS-21-05) was two-fold more selective for hlAPP over flAPP while compound 6 (APS-21-15) was found to be more selective for flAPP over hlAPP. Substitution on the electron-withdrawing group of the phenyl ring, such as chlorine, led to a reduction in fluorescence intensity when compared to substitutions with electron-donating groups, such as methoxy and methyl, at the same position. In the 4-morpholino urea series, a similar trend was observed. Compounds 11 (APS-22-98), 14 (APS-22-96), and 15 (APS-22-92) were selective for both hlAPP and flAPP while compounds 10 (APS-22-91), 12 (APS-22-100), 16 (APS-22-95), 17 (APS-22-94), and 18 (APS-22-93) were more selective for hlAPP than flAPP. Compound 14 (APS-22-96) was the most potent anti-fibrillary agent in this series with a fluorescence intensity of 9.8 ± 0.3 and 15.4 ± 0.4% on hlAPP and flAPP, respectively. For the 4-aminoindole urea series, all compounds except compound 23 (APS-22-106) were found to be selective for both hlAPP and flAPP. Substitution of chlorine at the ortho position or two methoxy groups at ortho and para positions on the phenyl ring resulted in two highly potent compounds, 27 (APS-22-107) and 20 (APS-22-109), each proving selective for both hlAPP and flAPP. The overall range of fluorescence intensities for the 4-amino indole series was 7 - 45%, which was far superior to the ranges of the 2-aminofluorene (16 - 71%) and 4- morpholino urea (10 - 88%) series.
The ThT fluorescence assay is a biophysical method employed to study the kinetics of fibril formation in prone-to-aggregate proteins. In the assay, the diaryl derivatives were added followed by the addition of ThT. After this, hlAPP or flAPP was mixed in the buffer. The kinetics of fibril formation begin when the protein is dissolved in the buffer and introduced onto the plate. In the ThT assay graph, the plateau phase represents the formation of mature fibrils, where an equilibrium is achieved between the aggregation and disaggregation process. The anti-fibrilization property of novel compounds is determined at the plateau phase, at which fluorescence intensity (%) of the tested compounds is compared for different incubations. The kinetic curves obtained for compounds 1-27 are depicted in Fig. 2. As mentioned previously, the 4-indolyl series of compounds seem to be the most potent, followed by the 4-morpholino series and the 2-aminofluorene series. Compounds which scored below 10% in the ThT assay for both conditions (hlAPP and fLAPP), were selected for further testing. These compounds were 14 (APS-22-96), 27 (APS-22-107), and 20 (APS-22-
109). Compounds 14 (APS-22-96), 27 (APS-22-107), 20 (APS-22-109) and 26 (APS-22-
110) were selected to be tested for their dose responsiveness as they had the lowest fluorescence.
Example 3
Dose responsiveness
The dose dependency of the anti-fibrillar effect of the best compounds was evaluated with different concentrations. Four best compounds were added at various concentrations for the anti-aggregation effect on hlAPP and flAPP: 14 (APS-22-96), 20 (APS-22-109), 26 (APS-22-110), and 27 (APS-22-107) were selected to be tested as they had the lowest fluorescence. The anti-aggregation effect is more prominent when tested with hlAPP. However, for both peptides compound 14 (APS-22-96) was the most effective at reducing fibril formation even at low doses. Compound 27 (APS-22-107) also reduced fibril formation at low doses. Interestingly, low doses of compound 26 (APS-22-110) increased the fibril formation. This effect was observed to a lesser extent in compound 20 (APS-22-109).
Example 4
Effect on size and distribution of amylin proteins
DLS was used to determine the size and distribution of amylin proteins after one hour of incubation with the three most promising compounds. Before testing, 10 pM of hlAPP and flAPP mix were incubated with 100 pM of one of four treatments: DMSO (control), Compound 14 (APS-22-96), Compound 20 (APS-22-109), or Compound 27 (APS-22-107). The particle diameters were analyzed via DLS after 60 minutes. When measured at 0 minutes, the stock solution predominantly contained monomers, with some pre-fibrillar proteins present. The amylin misfolds quickly, with pre-fibrillar proteins forming within 15 minutes. After 60 minutes of incubation at 25°C, the control contained fibrils, as indicated by the steep peak around 1,000 nm. All compounds reduced the percentage of intensity of the peak at around 1,000 nm with compounds 14 and 27 resulting in a slight left shift confirming a slight reduction of particle size. Compounds 14 (APS-22-96) and 20 (APS-22-109) exhibited peaks near 1,000 nm as well, indicating that neither of these compounds decreased the presence of aggregates in solution. Compound 27 (APS-22-107) proved more effective at reducing fibrils, but a peak around 660 nm indicates that this compound was not entirely effective at reducing large aggregates. Interestingly, small peaks around 1 nm and 35 nm suggest the presence of monomeric species in the sample treated with Compound 27 (APS- 22-107), indicating its partial success in breaking apart amylin aggregates.
TEM is one of the leading microscopy methods for imaging small particles, including proteinaceous fibrils, as the image is generated through the bombardment of electrons on the sample deposited onto a copper grid. Both the hlAPP and flAPP samples treated with DMSO exhibited large clusters of dense fibrils. When treated with Compound 27, hlAPP fibrils were not significantly different from treatment with DMSO, but flAPP fibrils were narrow and isolated, refusing to accumulate in large clusters. Compound 20 was the most effective compound on both hlAPP and flAPP samples. hlAPP samples exposed to Compound 20 depicted a fracturing of the fibrils, with small clusters of proteins being spread out across the field of view. flAPP treated with the same compound yielded small accumulations of thin, thread-like fibrils; a clear reduction from the dense accumulations visualized in the control. Such data support the anti-fibrillization properties of Compound 20 for both hlAPP and flAPP. Finally, flAPP samples treated with Compound 14 also exhibited a great reduction in the clustered accumulation of fibrils, as much of the field was empty except for occasional small, isolated packets.
With the prevalence of diabetes mellitus rising rapidly in both felines and humans, it is of great necessity to develop therapeutic(s) to reduce the severity of disease. Analysis of 27 di-aryl urea derivatives yielded three potential candidates, Compounds 14, 20, and 27, which have promise for future development. These three compounds prompted significant reduction of IAPP fibrils when analyzed via previously described biophysical methods. Ideally, one or more of these compounds could serve as candidate(s) for clinical trials in the future. Ideally, the selected compound(s) would be paired with current disease management techniques (i.e., insulin injection and dietary restrictions).
Example 5 The anti-fibrillar activity of compounds from series 3 at low pH condition. ThT experiment has been performed with the best compounds, which are part of series 3 (4- aminoindole-linked urea derivatives) using a Tris buffer at 20 mM with pH adjusted at 5.5. The experimental conditions were similar as described in the materials and methods. The only difference is that the Tris buffer was utilized instead of PBS (at pH 7.4). This was achieved to represent physiological milieu with low pH. The anti-fibrillar effects of series 3 are represented in the histograms in Figure 6. The ThT maximum fluorescence activity was lower with all compound treatments at 100 pM in comparison with the control DMSO (Figure 6-A hlAPP and Figure 6-B flAPP conditions). This is indicative of the reduction of both hlAPP and flAPP fibril formation by compounds 17-24. The best compound 24 was tested at 25, 50 and 100 pM with both peptides at 10 pM (Figure 6-C hlAPP and -D flAPP). Substantial anti-fibrillar activity remained at the lowest concentration used with the Tris buffer at pH 5.5.
Example 6
Compound 24 is nontoxic to Min-6 cell. The cytoprotective effect of compound 24 was evaluated using mouse insulinoma cell line (Min-6). FlAPP fibrils were preformed 12 hours prior the experiment by incubating the peptide at a concentration of 1 mM in PBS (pH 7.4) with 4.8% DMSO for 48 hours. FlAPP treated cells (at 20 pM) demonstrated a reduction of survival (Figure 7) as assessed with the resazurin assay, which is metabolized by mitochondria resulting in a fluorescent byproduct and allows for detection of alive cells. The fluorescence intensity of Min-6 cells alone was set arbitrarily at 100%. Compound 24 resulted in an increase of fluorescent signal compared to the flAPP treated cells. Resveratrol (res, a positive control) resulted in comparative effect compared to compound 24 at equivalent concentration. As expected, compound 12 did not result in an increase in the cell survival. However, the one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test did not show significant differences with the flAPP data abpve. Compounds 24 and 12 were not toxic to Min-6 cells at a concentration of 12.5 pM.
Example 7
Synthesis of urea/thiourea-based small molecules
The synthesis of comparable bio-isosteric analogs of urea (denoted with the letter U) and thiourea (denoted with the letter T) are achieved through a reaction of corresponding isocyanate or isothiocyanate with respective amine via nucleophilic addition shown in Fig. 8. The reaction workup has been done with a simple crystallization with ethyl acetate-hexane to obtain a pure product with acceptable yield ranging from 11-100 %.
A variety of substituents was applied on one side of the urea/thiourea linker (Px in Fig. 8) which consisted of a phenyl, p-chlorophenyl, 3,5-dichlorophenyl, p-bromophenyl, p- iodophenyl, p-acetylphenyl, p-methylphenyl, 3 -methylphenyl, 3,5-methylphenyl, p- methoxyphenyl, 3 -thiomethylphenyl, 7V,7V-dimethylphenyl, biphenyls, ethyl, cyclohexyl. The aromatic moiety was sometime distanced from the linker by a methyl, ethyl, or carbonyl. In addition, different aromatic moieties were attached to the urea/thiourea linker to the other side (Py in Fig. 8) of the molecules; the moieties being incorporated mainly consisted of an indole, benzothiazole, aminofluorene, 4-morpholino, 7V,7V-dimethylphenyl, p-trifluoromethyphenyl, cyclohexyl. The nitrogen present in the urea/thiourea linker has been coupled with different substituents including methyl, ethyl, and isopropyl in some instances (Table 2).
The kinetics of a-syn fibril formation were assessed in the presence of high micromolar urea/thiourea derivatives (Table 2). For the selection of the best compounds, the cut-off was set using a maximum ThT fluorescence intensity below 15% after the subtraction of the standard error of the mean (SEM). Compounds bearing an indole, benzothiazole, or N,N- dimethylphenyl on one side with halo-substituted aromatic moieties had shown less than 15% cut-off fluorescence obtained with the ThT assay and were then moved to the advanced biophysical and biological testing. The aminoindole small molecule derivatives were better than the morpholino and aminofluorene analogs (Table 2).
Table 2: structure-activity relationships for various compounds.
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
ThT anti-fibrillization kinetics. The ThT assay serves as the first line of testing to evaluate the potential of synthesized compounds for their ant-fibril effect. This assay employs thioflavin T, which is a fluorescent dye that recognizes and binds to the P-pleated sheet structures of misfolded protein aggregates. Proteins that are susceptible to misfolding, such as tau and a-syn, typically undergo a structural transformation from their native conformation to adopt a P-sheet structure when aggregated. We screened our compounds for their anti-fibril effect on a-syn, and three isoforms of tau (0N3R, 2N3R, and 2N4R). Compounds 6T and 14T showed the best anti-fibril action on a-syn (Fig. 9A), with compounds 14T and 19U having the lowest percentage (Table 2). 19U has been extensively studied prior to this work and only used for the mice study. Prism analysis yielded a LogEC50 of 29.8 ± 3.7 for compound 14T in the correlation between Log(agonist) and normalized response (variable slope) (Fig. 9B). To explore the dual target ability of the compounds, we tested them for their tau anti-fibril effects. The first of the tau series was the 0N3R isoform, where only compounds 6T and 14T were tested, and compound 14T was deemed the best, having a 66% reduction in fibril formation as against 7% for compound 6T after 18 h of incubation (Fig. 10A). In addition to evaluating compounds 6T and 14T, we further examined additional sets of compounds (38T and 18T) on tau 2N3R. At the conclusion of the experiment, it was observed that virtually all the compounds effectively halted tau 2N3R fibrillization (Fig. 10B) The compounds demonstrated a significant reduction in tau 2N3R, with the following order of efficacy: 18T (97%), 6T (96%), 14T (79%), and 38T (70%). As for tau 2N4R, we observed a decrease of 34.2% and 67.1% for 6T and 14T respectively (Figure 2B).
PICUP assay assessment of the anti-oligomer effect of compounds.
The PICUP assay utilizes the action of Tris(2,2'-bipyridyl)ruthenium(II) chloride (Ru(bpy)3) and ammonium persulfate (APS), which are electron and radical -generating compounds respectively, to induce oligomer formation. This photochemical assay has been utilized to examine the impact of compounds on the formation of oligomers, which are the initial events preceding fibril formation. Compounds that showed an anti-fibrillization effect from the ThT experiment with a cut off below 15% (including the subtraction of the SEM) were selected for the PICUP assay, i.e., compounds 6T, 14U, 14T, 15U, and 16T. Compared to the control, compounds 6T, and 14T prevented the formation of oligomers. Compounds 14U and 15U did not inhibit oligomerization. Among the promising compounds, 14T, and 6T were the most effective. Compounds 14T and 16T were further tested to determine if they have a dose-dependent effect. The compounds proved to be effective from 12.5 to 50 pM.
The effect of compounds 14T and 6T was assessed in a dose-dependent manner at different concentrations (50, 100, and 200 pM) on oligomerization of tau 0N4R and 2N4R isoforms. Across the tested concentrations, compound 14T showed no effect on stopping the formation of both tau 0N4R and 2N4R oligomers, and a similar result was observed for the effect of compound 6T on tau 2N4R. At 100 and 200 pM, compound 6T reduced very slightly oligomerization of tau 0N4R. The prospect of the anti-oligomer effect of compound 6T was further explored on tau 0N3R. When compared to the control with no compound treatment, compound 6T showed no effect on stopping tau 0N3R oligomerization.
Direct visualization of fibril formation.
For the validation of the anti-fibrillar effect compounds 6T and 14T (the best inhibitors), the transmission electron microscope (TEM) was used as a direct method to identify both a-syn and tau 2N4R fibrils. After the completion of the a-syn ThT assay measuring the kinetics of aggregation, samples were obtained to examine fibrils and assess the effect of the best compounds (6T and 14T) in comparison to the control (DMSO). Both compounds significantly reduced a-syn fibrilization after visualization at 40k magnification. Compounds 6T and 14T were further evaluated at 6 or 12.5 pM, 25 pM, and 100 pM to determine its dose-response effect, and it was observed that its greatest effect occurred at a concentration of 100 pM.
For tau 2N4R, TEM analyses were performed on tau 2N4R at 6 pM from commercial source (Rpeptide) and home-made in the lab. Compounds 6T and 14T were evaluated at 100 pM. After incubation for 24h at 37°C, the tau 2N4R from the commercial source resulted in multifocal packets of intertwined fibrils, sometime in row and chain conformation after incubation. Compound 14T resulted in less fibrils, with shorter appearance. Compound 6T had minimal impact on the reduction of tau 2N4R fibril formation. TEM analyses of the reaction solution extracted at the end of the ThT assays shown in Fig. 10C, were performed with the homemade tau 2N4R. The fibrils obtained from the kinetics are individualized and elongated. Similar trend was observed from the two compound treatments. The compound 14T showed an effect that resulted in the formation of short and less dense 2N4R fibrils when compared to the control. At the opposite, compound 6T failed to reduce the density of fibrils.
Disaggregation of amyloid-beta plaques isolated from AD patients.
The multi-target effect of compounds 6T and 14T were further explored to determine if the compounds had a disaggregation potential on amyloid-beta (AP) plaques isolated from AD patients. Samples were obtained from the brain of an Alzheimer’s disease patient and treated with the compounds 6T and 14T at 50 pM. TEM micrographs for Ap plaques were taken at both 2500, 20k, and 40k magnifications. The 2500 magnification demonstrates the plaque-like materials present after the incubation with different treatments. The 20k magnification vividly shows the compact arrangement (density) of the formed fibrils, while the 40k magnification helps to distinguish the changes in the fibrillar structures postcompound treatment. In comparison to the control (0.25% DMSO), the micrographs demonstrated the disaggregation effects resulting from compounds 6T and 14T (predominantly 6T) on the Ap plaques extracted from AD brains. a-Syn Inclusion-Forming Neuroblastoma Cell Assay.
M17D neuroblastoma cells expressing the fusion protein S3K::YFP were utilized to assess the impact of compounds 6T and 14T on both cell viability and inclusion formation. The presence of the triple K mutations (aS E35K + E46K + E61K ( = aS3K)) ‘amplifies’ the aS missense mutation E46K found in the familial PD. aS3K mutations make the protein, a- syn, readily prone to form round cytoplasmic inclusions in the cell system utilized, herein the M17D. The neuroblastoma cells expressing the aS3K have delayed growth and present more cell stress and toxicity. Several compounds were capable to reduce the formation of the aS inclusion formation and aS-induced cytotoxicity. The neuroblastoma cells utilized in this assay expressed an S3K::YFP fusion protein in a doxycycline inducible-dependent manner. The induction caused a pronounced round YFP-positive inclusions for the cell treated with the vehicle (DMSO) in the absence of compound. Compounds 14U, 15U, and 16T exhibited anti-fibrillary activity below 15% but were unable to reduce the oligomer formation. These compounds were used as negative controls and did not exhibit a significant anti-inclusion activity below a concentration of 40 pM.
Compounds 6T and 14T have been identified for their anti-fibril and anti-oligomer abilities using ThT and PICUP assays. Using the u-syn inclusion-forming neuroblastoma assay, both compounds exhibited a significant reduction in the formation of inclusions (Fig. 11). Compound 6T had a greater effect at 2.5, 5, 10, and 40 pM while compound 14T showed the most significant effect at 40 pM. The two compounds did not show any effect on the confluency of the cell.
Antiseeding Effect of Compound 14T and 6T in TauRD P301S FRET Biosensor Cells.
Using the TauRD P301S FRET biosensor model, we next looked at whether compounds 6T and 14T reduce tau seeding. The cell-based experiment entails the production of tau seeds and treatment of biosensor cells with the tau seeds. The production of tau seeds is achieved by expression of htau P301S in vitro. Human embryonic kidney (HEK) 293T cells were transfected with the htauP301S plasmid to overexpress human P301S tau. The compounds (14T and 6T) or vehicle (control, 0.01% DMSO) were applied to HEK 293T cells post 24 h of transfection. Following 48 h of exposure to the compounds at 5, and 20 pM doses, cell viability and tau seeding activity were evaluated (Fig. 12A). HEK 293T cell viability was not impacted by treatment with vehicle (0.01% DMSO) or compounds 14T and 6T (Fig. 12B). Cell lysates from P301S tau overexpressing cells treated with compounds 14T and 6T exhibited reduced seeding activity when compared to lysates from cells treated with the vehicle (0.01% DMSO) alone (Figs. 12C-D).
In vitro ADME testing: solubility and microsomal stability.
The MSU Medicinal Chemistry Core facility conducted kinetic solubility assessments for the most promising compounds. Compounds 6T and 14T (best compounds) were solubilized in ThT buffer comprised of lx PBS (pH = 7.4) with an additional 300 mM NaCl and 0.5 mM SDS. Compared to the 100 pM concentration used in the ThT assay (all within the same buffer solution), compound 6T has a solubility greater than 300 pM, and compound 14T has a slighter lesser solubility of 90 pM (control compound mebendazole had a measured solubility of 42 pM). This indicates a notable disparity in solubility between the two compounds, with compound 6T showcasing considerably higher solubility compared to compound 14T. The anti-fibrillization effect observed with compounds 6T and 14T is independent of the solubility.
In the microsomal stability assay, the control (imipramine) has a retention capacity of 44% of its initial amount after 30 minutes. When compared to the control, compound 6T demonstrates a higher value of 74% indicating potentially higher stability while compound 14T exhibits a lower percentage remaining at 25%, suggesting it might be more prone to degradation.
Single dose pharmacokinetics. The pharmacokinetics (PK) study for the best drug candidate was carried out using CD1 male mouse and single intravenous administration. The PK studies were performed to determine the potential of the best compounds to cross the blood-brain barrier. In addition to the best compounds found herein, compound 19U, a previously characterized in vitro, was included for the PK study due to its excellent a-syn anti-oligomer, anti-fibril, anti-inclusion activities. Compounds 6T, 14T, and 19U resulted in a plasma: brain ratio of about 1. When compared to 14T, compounds 19U and 6T exhibited an extended half-life, increased exposure, reduced clearance rate, and a larger volume of distribution in both the plasma and the brain, suggesting a superior pharmacokinetic characteristic. A small amount of compound was administered which may have resulted in short half-life. However, microsomal stability is below 70% which may indicate involvement of phase 1 metabolism. Optimization of compounds will address microsomal stability.
Examination of the anti-aggregation effect of 140 small molecules containing a urea/thiourea linkage, with either an indole or phenyl benzothiazole analog, against the aggregation of a-syn. Analysis of a-syn ThT fluorescence reveals that the compounds exhibiting indole or N,N-dimethylphenyl on one end, along with halo- substituted aromatic groups on the other, emerge as the most potent inhibitors of fibril formation. The most promising compounds were examined for their ability to inhibit both anti-oligomer and anti- fibrillar activities on different tau isoforms. Compound 6T emerged as the best inhibitor or tau 0N4R oligomerization. Compounds 6T and 14T both showed a great effect against 2N3R (ThT assay) and 2N4R (TEM) fibrillization. Also, 6T was the best in disaggregating the formation of Ap plaques, 14T showed notable effectiveness in reducing the plaques. Compound 14T showed notable effectiveness in reducing the number of M17D a-syn inclusions. Both compounds 6T and 14T reduced the seeding of htauP301S in biosensor cells and crossed the blood brain barrier in CD1 mice. Optimization will be performed to improve pharmacokinetic properties in rodents, i.e., metabolic stability and half-life. All patents, patent application publications, journal articles, textbooks, and other publications mentioned in the specification are indicative of the level of skill of those in the art to which the disclosure pertains. All such publications are incorporated herein by reference to the same extent as if each individual publication were specifically and individually indicated to be incorporated by reference.
The invention illustratively described herein may be suitably practiced in the absence of any element(s) or limitation(s), which is/are not specifically disclosed herein. Thus, for example, each instance herein of any of the terms "comprising," "consisting essentially of," and "consisting of may be replaced with either of the other two terms. Likewise, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "the method" includes one or more methods and/or steps of the type, which are described herein and/or which will become apparent to those ordinarily skilled in the art upon reading the disclosure.
The term "about," when referring to a number or a numerical value or range (including, for example, whole numbers, fractions, and percentages), means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error) and thus the numerical value or range can vary between 1% and 15% of the stated number or numerical range (e.g., +/- 5 % to 15% of the recited value) provided that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). The term “substantially” can allow for a degree of variability in a value or range, for example, within 90%, within 95%, or within 99% of a stated value or of a stated limit of a range.
The terms and expressions, which have been employed, are used as terms of description and not of limitation. In this regard, where certain terms are defined under "Definitions" and are otherwise defined, described, or discussed elsewhere in the "Detailed Description," all such definitions, descriptions, and discussions are intended to be attributed to such terms. There also is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof. Furthermore, while subheadings, e.g., "Definitions," are used in the "Detailed Description," such use is solely for ease of reference and is not intended to limit any disclosure made in one section to that section only; rather, any disclosure made under one subheading is intended to constitute a disclosure under each and every other subheading. It is recognized that various modifications are possible within the scope of the claimed invention. Thus, although the present invention has been specifically disclosed in the context of preferred embodiments and optional features, those skilled in the art may resort to modifications and variations of the concepts disclosed herein. Such modifications and variations are considered within the scope of the invention as claimed herein.

Claims

WHAT IS CLAIMED IS:
1. A compound of formula IV:
Figure imgf000093_0001
Formula IV wherein:
R3 and R4 are each independently H, alkyl or acyl; R3 and R4, together with the atoms to which they are each attached, form an aryl or heteroaryl group; or the group NR5 is connected at R3 or R4;
R5 and R6 are each independently H or alkyl or, R5 and R6, together with the atoms to which they are attached, form a heterocyclyl group;
R7 and R8 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy; or
R7 and R8, together with the atoms to which they are each attached, form an aryl or heteroaryl group;
X1 is NR5, O, S or CR92, wherein each R9 is independently, H, alkyl or acyl; and
X2 is O or S.
2. A compound of the formula:
Figure imgf000093_0002
Formula IV wherein:
R3 and R4 are each independently H, alkyl or acyl or, R3 and R4, together with the atoms to which they are each attached, form an aryl or heteroaryl group;
R5 and R6 are each independently H or alkyl or, R5 and R6, together with the atoms to which they are attached, form a heterocyclyl group;
R7 and R8 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy; or R7 and R8, together with the atoms to which they are each attached, form an aryl or heteroaryl group;
X1 is NR5, O, S or CR92, wherein each R9 is independently, H, alkyl or acyl; and
X2 is O or S.
3. The compound of claim 1, wherein the compound of formula IV is a compound of the formula:
Figure imgf000094_0001
4 . The compound of claim 1, wherein the compound of the formula IV is a compound of the formula:
Figure imgf000094_0002
5. The compound of claim 1, wherein the compound is of the formula:
Figure imgf000095_0001
6. The compound of claim 1, wherein the compound is of the formula:
Figure imgf000095_0002
7. The compound of claim 1, wherein the compound of formula IV is a compound of the formula:
Figure imgf000096_0001
8. The compound of claim 1, wherein the compound is of the formula:
Figure imgf000096_0002
9. The compound of claim 1, wherein the compound is of the formula:
Figure imgf000097_0001
10. The compound of claim 1 or 2, wherein X2 is S.
11. The compound of any of claim 7, 8, or 9, wherein one R9 is H.
12. The compound of any of claims 7, 8 or 9, wherein both R9 groups are H.
13. A compound of formula I:
Figure imgf000097_0002
Formula I wherein R1 and R2 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy.
14. The compound of claim 13, wherein halo is F, I, Cl, or Br.
15. The compound of claim 14, wherein mono-halo methyl is -C(H2)F, -C(H2)I, - C(H2)C1 or -C(H2)Br.
16. The compound of claim 14, wherein di-halo methyl is -C(H)F2, -C(H)l2, -C(H)C12 or -C(H)Br2.
17. The compound of claim 14, wherein tri-halo methyl is -CF3, -CI3, -CCI3 or -CBrs.
18. The compound of claim 13, wherein the Ci-Ce alkyl is -CH3.
19. The compound of claim 13, wherein the Ci-Ce alkoxy is -OCH3.
20. The compound of claim 13, which has the structure:
Figure imgf000098_0001
21. The compound of claim 13, which has the structure:
Figure imgf000098_0002
22. A compound of formula Ila:
Figure imgf000098_0003
Formula Ila wherein R1 and R2 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy; and R10 is heteroaryl or heterocycloalkyl.
23. The compound of claim 22, wherein the compound is of the formula:
Figure imgf000098_0004
24. The compound of claim 22, wherein the compound is of the formula:
Figure imgf000099_0001
25. The compound of any one of claims 22-24, wherein halo is F, I, Cl, or Br.
26. The compound of any one of claims 22-24, wherein mono-halo methyl is -C(H2)F, -C(H2)I, -C(H2)C1 or -C(H2)Br.
27. The compound any one of claims 22-24, wherein di-halo methyl is -C(H)F2, - C(H)I2, -C(H)C12 or -C(H)Br2.
28. The compound any one of claims 22-24, wherein tri-halo methyl is -CF3, -CI3, - CCI3 or -CBr3.
29. The compound of any one of claims 22-24, wherein the Ci-Ce alkyl is -CH3.
30. The compound of any one of claims 22-24, wherein the Ci-Ce alkoxy is -OCH3.
31. The compound of any one of claims 22-24, which has the structure:
Figure imgf000099_0002
32. A compound of formula III:
Figure imgf000099_0003
Formula III wherein R1 and R2 are each independently selected from the group consisting of hydrogen, halo, mono-halo methyl, di-halo methyl, tri-halo methyl, -NO2, a Ci-Ce alkyl, and a Ci-Ce alkoxy.
33. The compound of claim 32, wherein halo is F, I, Cl, or Br.
34. The compound of claim 33, wherein mono-halo methyl is -C(H2)F, -C(H2)I, -C(H2)C1 or -C(H2)Br.
35. The compound of claim 33, wherein di-halo methyl is -C(H)F2, -C(H)l2, -C(H)C12 or
-C(H)Br2.
36. The compound of claim 34, wherein tri-halo methyl is -CF3, -Ch, -CCI3 or -CBrs.
37. The compound of claim 33, wherein the Ci-Ce alkyl is -CH3.
38. The compound of claim 32, wherein the Ci-Ce alkoxy is -OCH3.
39. A compound of formula V:
X2
R11 R12 N^'N' 1 l R R Formula V wherein:
R11 is alkyl, cycloalkyl, aryl, arylakyl, or arylacyl;
R12 is cycloakyl, aryl, or heterocyclyl;
R5 and R6 are each independently H or alkyl or, R5 and R6, together with the atoms to which they are attached, form a heterocyclyl group; and
X2 is O or S.
40. A pharmaceutical composition comprising at least one compound of claims 1-9, 13, 22, 32, or 39 and a pharmaceutically acceptable carrier.
41. The pharmaceutical composition of claim 40, which further comprises at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus, wherein the at least one other compound is selected from the group consisting of a sulfonylurea, a meglitinide, a biguanide, a thiazolidinedione (TZD), an a- glucosidase inhibitor, a dipeptidyl peptidase IV (DPP-4) inhibitor, a bile acid sequestrant, a dopamine agonist, a sodium-glucose transport protein 2 (SGLT2) inhibitor, and a glucagon like peptide 1 (GLP-1) receptor agonist.
42. The pharmaceutical composition of claim 41, wherein:
(a) the sulfonylurea is at least one compound selected from the group consisting of chlorpropamide, tolazamide, tolbutamide, acetohexamide, glyburide, glipizide, glimepiride, and gliclazide,
(b) the meglitinide is repaglinide, netaglinide, or a combination thereof,
(c) the biguanide is metformin,
(d) the TZD is rosiglitazone, pioglitazone, or a combination thereof,
(e) the a-glucosidase inhibitor is at least one compound selected from the group consisting of acarbose, miglitol and voglibose,
(f) the DPP-4 inhibitor is at least one compound selected from the group consisting of alogliptin, linagliptin, sitagliptin, saxagliptin and vldagliptin,
(g) the bile acid sequestrant is colesevelam,
(h) the dopamine agonist is bromocriptine,
(i) the SGLT2 inhibitor is at least one compound selected from the group consisting of cangliflozin, dapagliflozin, empagliflozin and ertugliflozin, and
(j) the GLP-1 receptor agonist is semaglutide.
43. The pharmaceutical composition of claim 41, wherein the at least one other compound is a combination of compounds selected from:
(a) metformin and at least one of glyburide, glipizide, sitagliptin, saxagliptin, pioglitazone, repaglinide, rosiglitazone, linagliptin, alogliptin, canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin,
(b) glimepiride and either or both of pioglitazone and rosiglitazone,
(c) alogliptin and pioglitazone,
(d) dapagliflozin and saxagliptin,
(e) empagliflozin and linagliptin, (f) ertugliflozin and sitagliptin,
(g) lixisenatide and glargine insulin, and
(h) liraglutide and degludec insulin.
44. The pharmaceutical composition of any one of claims 40-44, which is formulated for oral administration.
45. The pharmaceutical composition of claim 40, which further comprises at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus, wherein the at least one other compound is selected from the group consisting of a GLP-1 receptor agonist and a dual GLP-1 receptor and gastric inhibitory polypeptide (GIP) receptor agonist.
46. The pharmaceutical composition of claim 45, wherein:
(a) the GLP-1 receptor agonist is exenatide, liraglutide, dulaglutide, lixisenatide or semaglutide, and
(b) the dual GLP-1 receptor and GIP receptor agonist is tirzepatide.
47. The pharmaceutical composition of any one of claims 45 or 46, which is formulated for administration by injection.
48. The pharmaceutical composition of claim 40, which further comprises bexagliflozin.
49. The pharmaceutical composition of claim 48, which is formulated for oral administration. a pharmaceutically acceptable carrier.
50. A method of inhibiting aggregation of misfolded islet amyloid protein (IAPP) in a human with, or at risk for, diabetes mellitus, which method comprises administering to the human the compound of any one of claims 1-9, 13, 22, 32, or 39, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit aggregation of misfolded IAPP, whereupon aggregation of misfolded IAPP is inhibited in the human with, or at risk for, diabetes mellitus.
51. The method of claim 50, which further comprises administering, simultaneously or sequentially by the same or different routes, at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus and which is selected from the group consisting of a sulfonylurea, a meglitinide, a biguanide, a thiazolidinedione (TZD), an a-glucosidase inhibitor, a dipeptidyl peptidase IV (DPP-4) inhibitor, a bile acid sequestrant, a dopamine agonist, a sodium-glucose transport protein 2 (SGLT2) inhibitor, and a glucagon like peptide 1 (GLP-1) receptor agonist.
52. The method of claim 51, wherein:
(a) the sulfonylurea is at least one compound selected from the group consisting of chlorpropamide, tolazamide, tolbutamide, acetohexamide, glyburide, glipizide, glimepiride, and gliclazide,
(b) the meglitinide is repaglinide, netaglinide, or a combination thereof,
(c) the biguanide is metformin,
(d) the TZD is rosiglitazone, pioglitazone, or a combination thereof,
(e) the a-glucosidase inhibitor is at least one compound selected from the group consisting of acarbose, miglitol and voglibose,
(f) the DPP-4 inhibitor is at least one compound selected from the group consisting of alogliptin, linagliptin, sitagliptin, saxagliptin and vldagliptin,
(g) the bile acid sequestrant is colesevelam,
(h) the dopamine agonist is bromocriptine,
(i) the SGLT2 inhibitor is at least one compound selected from the group consisting of cangliflozin, dapagliflozin, empagliflozin and ertugliflozin, and
(j) the GLP-1 receptor agonist is semaglutide.
53. The method of claim 51, wherein the at least one other compound is a combination of compounds selected from:
(a) metformin and at least one of glyburide, glipizide, sitagliptin, saxagliptin, pioglitazone, repaglinide, rosiglitazone, linagliptin, alogliptin, canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin,
(b) glimepiride and either or both of pioglitazone and rosiglitazone,
(c) alogliptin and pioglitazone,
(d) dapagliflozin and saxagliptin, (e) empagliflozin and linagliptin,
(f) ertugliflozin and sitagliptin,
(g) lixisenatide and glargine insulin, and
(h) liraglutide and degludec insulin, wherein the compounds in the combination can be administered simultaneously or sequentially by the same or different routes.
54. The method of any one of claims 50-53, wherein administering is orally administering.
55. The method of claim 50, which further comprises administering at least one other compound, which is prophylactically or therapeutically effective for the treatment of diabetes mellitus, wherein the at least one other compound is selected from the group consisting of a GLP-1 receptor agonist and a dual GLP-1 receptor and gastric inhibitory polypeptide (GIP) receptor agonist.
56. The method of claim 55, wherein:
(a) the GLP-1 receptor agonist is exenatide, liraglutide, dulaglutide, lixisenatide or semaglutide, and
(b) the dual GLP-1 receptor and GIP receptor agonist is tirzepatide.
57. The method of 55 or 56, wherein administering is by injection.
58. A method of inhibiting aggregation of misfolded islet amyloid protein (IAPP) in a cat with, or at risk for, diabetes mellitus, which method comprises administering to the cat the compound of any one of claims 1-9, 13, 22, 32, or 39, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit aggregation of misfolded IAPP, whereupon aggregation of misfolded IAPP is inhibited in the cat with, or at risk for, diabetes mellitus.
59. The method of claim 58, which further comprises administering bexafliflozin.
60. The method of claim 58, wherein administering is orally administering.
61. The method of claim 58, which further comprises administering insulin.
62. The method of claim 61, wherein the compound of any one of claims 1-9, 13, 22, 32, or 39, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, is administered orally and insulin is administered by injection.
63. A method of inhibiting tubulin-associated unit (tau) protein aggregation in a subject having, or at risk for, tau protein aggregation, which method comprises administering to subject any one of claims 39, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit tubulin-associated unit (tau) protein aggregation
64. The method of claim 63, wherein the subject has, or is at risk for, Alzheimer’s disease.
65. A method of inhibiting alpha-synuclein (a-syn) protein aggregation in a subject having, or at risk for a-syn, which method comprises administering to subject any one of claims 39, or a pharmaceutical composition comprising same and a pharmaceutically acceptable carrier, in an amount effective to inhibit alpha-synuclein (a-syn) protein aggregation.
66. The method of claim 65, wherein the subject has, or is at risk for, Parkinson’s disease.
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