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WO2024233922A2 - Échafaudages à base de pyridine-pipérazine en tant qu'activateurs de neurolysine hautement puissants et sélectifs - Google Patents

Échafaudages à base de pyridine-pipérazine en tant qu'activateurs de neurolysine hautement puissants et sélectifs Download PDF

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Publication number
WO2024233922A2
WO2024233922A2 PCT/US2024/028847 US2024028847W WO2024233922A2 WO 2024233922 A2 WO2024233922 A2 WO 2024233922A2 US 2024028847 W US2024028847 W US 2024028847W WO 2024233922 A2 WO2024233922 A2 WO 2024233922A2
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arch
nmr
mhz
neurolysin
amine
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WO2024233922A3 (fr
Inventor
Thomas J. ABBRUSCATO
Saideh NOZOHOURI
Yon Zhang
Paul TRIPPIER
Shikha KUMARI
Vardan T. Karamyan
Shiva Hadi ESFAHANI
Srinidhi JAYARAMAN
Krishnaiah Maddeboina
Shirisha JONNALAGADDA
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University of Nebraska Lincoln
Texas Tech University TTU
Texas Tech University System
University of Nebraska System
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University of Nebraska Lincoln
Texas Tech University TTU
Texas Tech University System
University of Nebraska System
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Publication of WO2024233922A2 publication Critical patent/WO2024233922A2/fr
Publication of WO2024233922A3 publication Critical patent/WO2024233922A3/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—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
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol

Definitions

  • the present invention relates in general to the field of allosteric potentiators of Neurolysin (Nln), and more particularly, to a novel pyridine-piperazine (Py-Pip) scaffold as highly potent and selective neurolysin activators.
  • Stroke is the second leading cause of death in the world, causing an estimated 5.5 million deaths every year. 1,2 Stroke can be classified into two major categories, ischemic and hemorrhagic. Ischemic stroke results from interruption of the blood supply to a particular area of the brain, while hemorrhagic stroke is caused by the rupture of a blood vessel or abnormal blood vessel structure. 3 Ischemic stroke, accounting for about 80% of all strokes, causes severe disability and/or death, and its management is burdensome for modern societies because of increasing aging populations.
  • IV rtPA tissue plasminogen activator
  • Peptidase neurolysin (Nln; EC 3.4.24.16), a zinc metalloendopeptidase that contains a His- Glu-X-X-His domain, belongs to the M3 family of peptidases responsible for hydrolytic processing of bioactive peptides that are expressed by neuronal and glial cells in the extracellular environment. 11,12 The most established role of neurolysin is in the metabolism of neurotensin, a 13-residue neuropeptide. 4 ’ 5 It hydrolyzes this peptide between residues of Pro and Tyr, forming shorter fragments that are considered to be inactive.
  • Neurotensin is found in the peripheral nervous system (PNS) and central nervous system (CNS) where it mediates several effects, including modulation of central dopaminergic, serotonergic, glutamatergic thermoregulation, intestinal motility, and cholinergic systems. 6 Additionally, neurolysin is well described with known endogenous substrates that are part of peptidergic systems, including: bradykinin, angiotensin I/II, hemopressin, dynorphin A (1-8), substrate P, metorphamide, and somatostatin. These peptidergic systems are involved in the pathogenesis of stroke, resistance to ischemic injury and/or post-stroke brain recovery.
  • Nln is an adaptive cerebroprotective mechanism regulating excitotoxicity, oxidative stress, edema formation, blood-brain barrier (BBB) hyperpermeability, and inflammation in the post-stroke brain.
  • BBB blood-brain barrier
  • dipeptides that enhance the catalytic efficacy of Nln. These dipeptides were identified using in silico screening of -140,000 molecules from the National Cancer Institute Developmental Therapeutics Program database. 1 In an Nln enzymatic assay, both dipeptides enhanced the rate of synthetic substrate hydrolysis by recombinant (human and rat) and mouse brain-purified Nln in a concentration- dependent manner with negligible effect on the activity of other closely related peptides. Moreover, drug affinity responsive target stability (DARTS) and differential scanning fluorimetry (DSF) assays have confirmed the concentration-dependent interaction of Nln with these dipeptides.
  • DARTS drug affinity responsive target stability
  • DSF differential scanning fluorimetry
  • an aspect of the present disclosure relates to a composition for enhancing neurolysin activity comprising: a molecule of Formula I, II, III, or IV a salt, or enantiomer thereof:
  • R2 is selected from:
  • the molecule is selected from at least one of:
  • the composition is adapted for oral, intravenous, subcutaneous, parenteral, enteral, transcutaneous, transdermal, pulmonary, or rectal administration.
  • the activator is adapted for at least one of immediate release, delayed release, or prolonged release.
  • the molecule comprises a homologation of a linker that can include 0,
  • the molecule further comprises bioisosteres of the piperazine ring, wherein the bioisosteres are selected from spirocyclics, pyridazine, pyrimidine, or triazinane.
  • an aspect of the present disclosure relates to an allosteric activator of neurolysin selected from: a molecule of Formula I, II, III, or IV, a salt, or enantiomer thereof: Formula I,
  • R2 is selected from:
  • R1 and R2 are selected from the following combinations:
  • the molecule is selected from at least one of:
  • the composition is adapted for oral, intravenous, subcutaneous, parenteral, enteral, transcutaneous, transdermal, pulmonary, or rectal administration.
  • the composition is adapted for at least one of immediate release, delayed release, or prolonged release.
  • the molecule further comprises homologation of a linker that can include 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more carbons on either side of the amine, e.g., between amine and piperazine and bioisosteric replacement of the linker amine with C, O, S.
  • the molecule further comprises bioisosteres of the piperazine ring, wherein the bioisosteres are selected from spirocyclics, pyridazine, pyrimidine, or triazinane.
  • an aspect of the present disclosure relates to a method of treating the symptoms of inflammatory disorder comprising: identifying a subject in need of treatment for ischemia; and providing the subject with an amount of an allosteric potentiator of neurolysin sufficient to increase the activity of neurolysin, wherein the allosteric potentiator is not a peptide or peptidomimetic.
  • the allosteric potentiator of neurolysin is a molecule of Formula I, II, III, or IV, a salt, or enantiomer thereof:
  • R is selected from: pyridazine, pyrimidine, pyrazine, heterocycles that are substituted or unsubstituted (four, five and six-membered), naphthyl, mono, di, tri substituted,
  • R2 is selected from:
  • R1 and R2 are selected from the following combinations:
  • the molecule is selected from at least one of:
  • the inflammatory disorder is selected from at least one of ischemic stroke, traumatic brain injury, autism, Alzheimer's Disease, dementias or Parkinson's Disease, brain edema, asthma, chronic obstructive pulmonary disease, or neuroinflammation.
  • the neurolysin is murine or human.
  • the molecule comprises a homologation of a linker that can include 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more carbons on either side of the amine, e.g., between amine and piperazine and bioisosteric replacement of the linker amine with C, O, or S.
  • the molecule further comprises bioisosteres of the piperazine ring, wherein the bioisosteres are selected from spirocyclics, pyridazine, pyrimidine, or triazinane.
  • an aspect of the present disclosure relates to a method of making a pyridine-piperazine scaffold comprising at least one of: or R is selected from: pyridazine, pyrimidine, pyrazine, heterocycles that are substituted or unsubstituted (four, five and six-membered), naphthyl, mono, di, tri substituted,
  • the molecule is selected from at least one of:
  • the molecule comprises a homologation of a linker that can include 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more carbons on either side of the amine, e.g., between amine and piperazine and bioisosteric replacement of the linker amine with C, O, or S.
  • the molecule further comprises bioisosteres of the piperazine ring, wherein the bioisosteres are selected from spirocyclics, pyridazine, pyrimidine, or triazinane.
  • FIG. 1 A The effect of pyridine-piperazine (Py-Pip) obtained from NCI DTP on catalytic activity of recombinant neurolysin (Nln).
  • Py-Pip pyridine-piperazine obtained from NCI DTP
  • FIG. 1 A representative reaction progress curves of QFS (15 ⁇ M) hydrolysis by rat recombinant Nln (0.3 nM) in the presence of different concentrations of Py-Pip.
  • concentration-dependent effect of Py-Pip on QFS hydrolysis by Nln (n 3, mean ⁇ SD are presented). Note that the initial velocity of the hydrolysis in the absence of Py-Pip corresponds to 100% on the vertical axis and to -13 on the horizontal axis.
  • FIG. IB The effect of Py-Pip obtained from NCI DTP on fluorescence signal of Mca-Pro- Leu-OH.
  • a representative concentration-dependent effect of Py-Pip on fluorescence signal of Mca-Pro-Leu-OH, the product of QFS hydrolysis by Nln, under the same assay conditions as presented in FIG. 1 is shown. The only difference was that Mca-Pro-Leu-OH, instead of QFS, was present in the assay at 2 ⁇ M final concentration.
  • Each data point represents the average fluorescence signal measured every minute for duration of 10 min. Note that -13 on the horizontal axis corresponds to the condition where neither compound was present.
  • FIG. 2 The modulatory site on Nln is different from the substrate-binding site.
  • FIG. 4 The effect of Py-Pip obtained from NCI-DTP on catalytic activity of human recombinant Nln.
  • the initial velocity of the hydrolysis in the absence of either compound corresponds to 100% on the vertical axis and to -13 on the horizontal
  • FIG. 6 The effect of Py-Pip obtained from NCI DTP on hydrolysis of endogenous substrates by Nln.
  • Column 1 in all panels represents the amount of respective peptide fragment, i.e.
  • FIG. 7 is a summary of the SAR of the pyridine-piperazine scaffold for Nln activation.
  • FIGS. 8A to 8D show the permeability of compound 7c (KS52) and compound 7g (KS73) through MDR1-MDCA monolayer.
  • FIG. 8 A Apical to basolateral transport of non- peptidomimetic Nln activators through a BBB-mimicking model.
  • FIG. 8 A In vitro permeability coefficient (Pe) of compounds KS52 (compound 7c) and KS73 (compound 7g) compared to the dipeptide activator His-Tyr.
  • FIG. 8B, FIG. 8C Permeability coefficient (Pe) of KS52 (compound 7c) and KS73 (compound 7g) in the presence and absence of P-gp inhibitor CsA, in either apical to basolateral (A > B) (FIG. 8C) or basolateral to apical (B > A) (FIG. 8C) direction.
  • FIG. 8D Comparison of influx (A > B direction) and efflux (B > A direction) mediated by P-gp.
  • FIG. 9A and 9B show the pharmacokinetic profile of KS52 (compound 7c) and KS73 (compound 7g) after intravenous bolus injection in healthy mice.
  • FIG. 9 A Concentration-time profiles of KS52 and KS73 (compounds 7c and 7g, respectively) showed biexponential decline in plasma. Left panel showed the absoluteive values, right panel showed the relative portion to injection dose.
  • FIG. 9B Concentration-time profiles of KS52 an KS73 (compounds 7c and 7g, respectively) showed biexponential decline in brains. Left panel showed the absoluteive values, right panel showed the relative portion to injection dose.
  • FIGS. 11A to 11C shows plasma pharmacokinetic profile of KS73 (compound 7g) in stroke mice.
  • FIG. 11 A Representative TTC staining of brain slices after 1-h tMCAO-induced occlusion and 3-h reperfusion (Left panel) and cerebral blood flow measured by a laser Doppler probe presented as percentages of the baseline.
  • FIG. 1 IB Plasma profile in stroke and healthy animals. Left panel showed the absoluteive values, right panel showed the relative portion to total injection dose.
  • FIG. 11C The area under the plasma concentration-time curves (AUC 0-30 min) in stroke (tMCAO) and healthy (Control) mice.
  • TTC 2, 3, 5 -triphenyl tetrazolium chloride.
  • %ID/ml percentage of injection dose/ml.
  • FIGS. 12A to 12 C shows brain uptake of KS73 (compound 7g) at a single time point analysis in stroke mice.
  • FIG. 12A Brain uptake among stroke ipsilateral hemisphere, stroke contralateral hemisphere, and healthy brain. Left panel showed the absoluteive values, right panel showed the relative portion to total injection dose.
  • FIG. 12B Brain uptake clearance kinetics (Kin).
  • FIG. 12C Achieved brain concentration relative to A 50 .
  • Cbr concentration in brain
  • %ID/ml percentage of injection dose/ml
  • tMCAO-Ips ipsilateral hemisphere of stroke animal, contralateral hemisphere of stroke animal, and healthy brains.
  • FIGS. 14A and 14B show the half-life of KS compounds in plasma and brains.
  • FIGS. 15A and 15B show brain concentrations of KS compounds.
  • KS52 compound 7c
  • KS73 compound 7g
  • FIGS. 15A and 15B show significantly higher total brain concentrations relative to their A 50 .
  • the dashed line indicates the A 50 for each compound, respectively.
  • FIGS. 17A and 17B show that there was no in vivo heart toxicity from KS73 (compound 7g) in heart weight (FIG. 17A) and body weight (FIG. 17B).
  • This disclosure focuses on the discovery of small molecule neurolysin (Nln) activators, which are used to treat ischemic stroke.
  • the molecules taught herein are organic small molecules (not peptides), have much better drug-like properties, and have been evaluated in in vivo models of ischemic stroke.
  • the present invention includes the identification of a small organic molecule that is pyridine-piperazine (Py-Pip) compound that enhances activity of Nln.
  • Pharmacological studies characterize and confirm the activity of Py-Pip.
  • a detailed structure-activity relationship is described using Structure-Activity Relationship (SAR) studies that explored the structure of Py-Pip and led to the development of higher potency analogs.
  • SAR Structure-Activity Relationship
  • a dosage unit for use of the allosteric activator of neurolysin of the present invention may be a single compound or mixtures thereof with other compounds.
  • the compound may be mixed together, form ionic or even covalent bonds.
  • the allosteric activator of neurolysin of the present invention may be administered in oral, intravenous (bolus or infusion), intraperitoneal, subcutaneous, or intramuscular form, all using dosage forms well known to those of ordinary skill in the pharmaceutical arts.
  • different dosage forms e.g., tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups, and emulsions may be used to provide the allosteric activator of neurolysin of the present invention to a patient in need of therapy that includes ischemic stroke.
  • the allosteric activator of neurolysin may also be administered as any one of known salt forms.
  • the allosteric activator of neurolysin is typically administered in admixture with suitable pharmaceutical salts, buffers, diluents, extenders, excipients and/or carriers (collectively referred to herein as a pharmaceutically acceptable carrier or carrier materials) selected based on the intended form of administration and as consistent with conventional pharmaceutical practices.
  • a pharmaceutically acceptable carrier or carrier materials selected based on the intended form of administration and as consistent with conventional pharmaceutical practices.
  • the allosteric activator of neurolysin may be formulated to provide, e.g., maximum and/or consistent dosing for the particular form for oral, rectal, topical, intravenous injection or parenteral administration. While the allosteric activator of neurolysin may be administered alone, it will generally be provided in a stable salt form mixed with a pharmaceutically acceptable carrier.
  • the carrier may be solid or liquid, depending on the type and/or location of administration selected.
  • Techniques and compositions for making useful dosage forms using the present invention are described in one or more of the following references: Anderson, Philip O.; Knoben, James E.; Troutman, William G, eds., Handbook of Clinical Drug Data, Tenth Edition, McGraw-Hill, 2002; Pratt and Taylor, eds., Principles of Drug Action, Third Edition, Churchill Livingston, New York, 1990; Katzung, ed., Basic and Clinical Pharmacology, Ninth Edition, McGraw Hill, 2007; Goodman and Gilman, eds., The Pharmacological Basis of Therapeutics, Tenth Edition, McGraw Hill, 2001; Remington’s Pharmaceutical Sciences, 20th Ed., Lippincott Williams & Wilkins., 2000, and updates thereto; Martindale, The Extra Pharmacopoeia, Thirty-Second Edition (The Pharmaceutical Press, London, 1999); all of which are incorporated by reference, and the like, relevant portions
  • the allosteric activator of neurolysin may be included in a tablet.
  • Tablets may contain, e.g., suitable binders, lubricants, disintegrating agents, coloring agents, flavoring agents, flow-inducing agents and/or melting agents.
  • oral administration may be in a dosage unit form of a tablet, gelcap, caplet or capsule, the active drug component being combined with a non-toxic, pharmaceutically acceptable, inert carrier such as lactose, gelatin, agar, starch, sucrose, glucose, methylcellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, mixtures thereof, and the like.
  • Suitable binders for use with the present invention include: starch, gelatin, natural sugars (e.g., glucose or beta-lactose), com sweeteners, natural and synthetic gums (e.g., acacia, tragacanth or sodium alginate), carboxymethylcellulose, polyethylene glycol, waxes, and the like.
  • Lubricants for use with the invention may include: sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, mixtures thereof, and the like.
  • Disintegrators may include: starch, methyl cellulose, agar, bentonite, xanthan gum, mixtures thereof, and the like.
  • the allosteric activator of neurolysin may be administered in the form of liposome delivery systems, e.g., small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles, whether charged or uncharged.
  • Liposomes may include one or more: phospholipids (e.g., cholesterol), stearylamine and/or phosphatidylcholines, mixtures thereof, and the like.
  • the allosteric activator of neurolysin may also be coupled to one or more soluble, biodegradable, bioacceptable polymers as drug carriers or as a prodrug.
  • Such polymers may include: polyvinylpyrrolidone, pyran copolymer, polyhydroxylpropylmethacrylamide-phenol, polyhydroxyethylasparta-midephenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues, mixtures thereof, and the like.
  • biodegradable polymers for use with the present invention include: polylactic acid, polyglycolic acid, copolymers of polylactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, and crosslinked or amphipathic block copolymers of hydrogels, mixtures thereof, and the like.
  • gelatin capsules may include the allosteric activator of neurolysin and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like.
  • diluents may be used to make compressed tablets. Both tablets and capsules may be manufactured as immediate-release, mixed-release or sustained- release formulations to provide for a range of release of medication over a period of minutes to hours.
  • Compressed tablets may be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere.
  • An enteric coating may be used to provide selective disintegration in, e.g., the gastrointestinal tract.
  • the oral drug components may be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like.
  • suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules.
  • Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents, mixtures thereof, and the like.
  • Liquid dosage forms for oral administration may also include coloring and flavoring agents that increase patient acceptance and therefore compliance with a dosing regimen.
  • water a suitable oil, saline, aqueous dextrose (e.g., glucose, lactose and related sugar solutions) and glycols (e.g., propylene glycol or polyethylene glycols) may be used as suitable carriers for parenteral solutions.
  • Solutions for parenteral administration include generally, a water-soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffering salts.
  • Antioxidizing agents such as sodium bisulfite, sodium sulfite and/or ascorbic acid, either alone or in combination, are suitable stabilizing agents.
  • Citric acid and its salts and sodium EDTA may also be included to increase stability.
  • parenteral solutions may include pharmaceutically acceptable preservatives, e.g., benzalkonium chloride, methyl- or propyl- paraben, and/or chlorobutanol.
  • Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field, relevant portions incorporated herein by reference.
  • the allosteric activator of neurolysin may also be delivered as an intranasal form via use of a suitable intranasal vehicle.
  • the allosteric activator of neurolysin may be delivered using lotions, creams, oils, elixirs, serums, transdermal skin patches and the like, as are well known to those of ordinary skill in that art.
  • Parenteral and intravenous forms may also include pharmaceutically acceptable salts and/or minerals and other materials to make them compatible with the type of injection or delivery system chosen, e.g., a buffered, isotonic solution.
  • useful pharmaceutical dosage forms for administration of allosteric activator of neurolysin may include the following forms.
  • Capsules may be prepared by filling standard two-piece hard gelatin capsules each with 10 to 500 milligrams of powdered active ingredient, 5 to 150 milligrams of lactose, 5 to 50 milligrams of cellulose and 6 milligrams magnesium stearate.
  • Soft Gelatin Capsules A mixture of active ingredient is dissolved in a digestible oil such as soybean oil, cottonseed oil or olive oil. The active ingredient is prepared and injected by using a positive displacement pump into gelatin to form soft gelatin capsules containing, e.g., 100-500 milligrams of the active ingredient. The capsules are washed and dried.
  • a digestible oil such as soybean oil, cottonseed oil or olive oil.
  • the active ingredient is prepared and injected by using a positive displacement pump into gelatin to form soft gelatin capsules containing, e.g., 100-500 milligrams of the active ingredient. The capsules are washed and dried.
  • Tablets A large number of tablets are prepared by conventional procedures so that the dosage unit was 100-500 milligrams of active ingredient, 0.2 milligrams of colloidal silicon dioxide, 5 milligrams of magnesium stearate, 50-275 milligrams of microcrystalline cellulose, 11 milligrams of starch and 98.8 milligrams of lactose. Appropriate coatings may be applied to increase palatability or delay absorption.
  • effervescent tablet To provide an effervescent tablet appropriate amounts of, e.g., monosodium citrate and sodium bicarbonate, are blended together and then roller compacted, in the absence of water, to form flakes that are then crushed to give granulates. The granulates are then combined with the active ingredient, drug and/or salt thereof, conventional beading or filling agents and, optionally, sweeteners, flavors and lubricants.
  • active ingredient, drug and/or salt thereof conventional beading or filling agents and, optionally, sweeteners, flavors and lubricants.
  • a parenteral composition suitable for administration by injection is prepared by stirring 1.5% by weight of active ingredient in deionized water and mixed with, e.g., up to 10% by volume propylene glycol and water.
  • the solution is made isotonic with sodium chloride and sterilized using, e.g., ultrafiltration.
  • aqueous suspension is prepared for oral administration so that each 5 ml contain 100 mg of finely divided active ingredient, 200 mg of sodium carboxymethyl cellulose, 5 mg of sodium benzoate, 1.0 g of sorbitol solution, U.S.P., and 0.025 ml of vanillin.
  • the active ingredient is compressed into a hardness in the range 6 to 12 Kp.
  • the hardness of the final tablets is influenced by the linear roller compaction strength used in preparing the granulates, which are influenced by the particle size of, e.g., the monosodium hydrogen carbonate and sodium hydrogen carbonate. For smaller particle sizes, a linear roller compaction strength of about 15 to 20 KN/cm may be used.
  • kits useful, for example, for the treatment of cancer, which comprise one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of allosteric activator of neurolysin.
  • kits may further include, if desired, one or more of various conventional pharmaceutical kit components, such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as will be readily apparent to those skilled in the art.
  • Printed instructions either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, may also be included in the kit. It should be understood that although the specified materials and conditions are important in practicing the invention, unspecified materials and conditions are not excluded so long as they do not prevent the benefits of the invention from being realized.
  • suitable liquid dosage forms include solutions or suspensions in water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions reconstituted from non- effervescent granules and effervescent preparations reconstituted from effervescent granules.
  • Such liquid dosage forms may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.
  • Oral dosage forms optionally contain flavorants and coloring agents.
  • Parenteral and intravenous forms may also include minerals and other materials to make them compatible with the type of injection or delivery system chosen.
  • the term “chewable” refers to semi-soft, palatable and stable chewable treat without addition of water. It should be appreciated to the skilled artisan that a chewable composition will be stable and palatable, fast disintegrating, semi-soft medicated chewable tablets (treats) by extrusion without the addition of extraneous water. Soft chewable tablets do not harden on storage and are resistant to microbial contamination.
  • a semi-soft chewable contains a blend of any one or more binders, flavors, palatability enhancers, humectants, disintegrating agents, non- aqueous solvents, and diluents that are plasticized with liquid plasticizers, such as glycols and polyols to make them ductile and extrudable.
  • the chewable can be made by extrusion, e.g., including fats or lipids as plasticizers and binding agents, is manufactured in the absence of added water, uses plasticizers to replace water in extrudable matrices, contains humectants to maintain the extrudable chew in a pliant and soft state during its shelflife, or any combination thereof.
  • the chewable form may be provided in conjunction with one or more flavorings and/or taste-masking agents that improve the taste of the formulation greater than 10, 20, 30, 40, 50, 60, 70, 80, or 90%.
  • the chewable can include the active agent and the ion exchange resin to enhance taste masking.
  • Neurolysin is a recently recognized peptidase functioning to preserve the brain from ischemic injury.
  • the inventors identified novel small molecule activators of Nln. Using a computational approach and in silico screening data from ⁇ 140,000 molecules from the National Cancer Institute Developmental Therapeutics Program database, the inventors evaluated the top-ranking compounds in an Nln enzymatic assay, and identified a Pyridine-Piperazine molecule (Py-Pip) that enhances activity of Nln.
  • Py-Pip enhanced the rate of synthetic substrate hydrolysis by recombinant human and rat Nln in a concentration-dependent manner (micromolar A 50 and Amax ⁇ 300%) but had negligible effect on activity of closely related peptidases. Py-Pip also enhanced hydrolysis of Nln endogenous substrates neurotensin, angiotensin I, and bradykinin and increased efficiency of the synthetic substrate hydrolysis (Vmax/Km ratio) in a concentration-dependent manner. Py-Pip and competitive inhibitor dynorphin A (1-13) did not affect each other's affinity for Nln, showing the differing nature of their respective binding sites. This is the first small organic molecule that can enhance Nln activity. As shown in this and other examples herein, Py-Pip provides a chemical scaffold to develop high-potency, drug-like molecules as research tools and potential drug leads.
  • This example describes the discovery of a small organic molecule that selectively enhances activity of peptidase Nln - a newly recognized cerebroprotective mechanism in the post-stroke brain.
  • the identified molecule also serves as a chemical scaffold for development of drug-like molecules to further study Nln and may become lead structures for a new class of drugs.
  • Py-Pip enhanced the Nln activity (rat and human) in a concentration dependent manner. The enhanced activity could be observed with both synthetic substrate as well as endogenous substrates.
  • Py-Pip demonstrated high specificity and selectivity towards Nln, when screened against thimet oligopeptidase (TOP), angiotensin-converting enzyme (ACE), ACE2 and neutral endopeptidase (NEP).
  • TOP thimet oligopeptidase
  • ACE angiotensin-converting enzyme
  • NEP neutral endopeptidase
  • Concentration-response experiments using Py- Pip in the presence and absence of a competitive inhibitor of Nln Dynorphin A (1-13) showed that the activators and the inhibitor can function independent of each other.
  • Saturation experiments using a Nln synthetic substrate revealed a concentration-dependent increase in catalytic efficiency of Nln (i.e, increased Vmax/Km ratio) in the presence of Py-Pip.
  • Recombinant peptidases Recombinant rat Nln and recombinant human Nln and thimet oligopeptidase (TOP) containing an N-terminal poly-histidine tag were produced and purified in house as detailed by the present inventors (Naomi J Wangler, Srinidhi Jayaraman, Rui Zhu, Yehia Mechref, Thomas J Abbruscato, Ulrich Bickel, Vardan T Karamyan. Preparation and preliminary characterization of recombinant neurolysin for in vivo studies. J Biotechnol. 2016;234: 105-115.
  • ACE angiotensin-converting enzyme
  • ACE2 angiotensin-converting enzyme 2
  • NEP neprilysin
  • Enzymatic assays Activity of neurolysin was measured in a continuous assay by documenting the increase in fluorescence occurring upon cleavage of a quenched fluorescent substrate (referred to as QFS in the text) as described in detail in the inventors’ previous publications (See PMID: 27496565, above; Mamoon Rashid, Harold J Wangler, Li Yang, Kaushik Shah, Thiruma V Arumugam, Thomas J Abbruscato, Vardan T Karamyan. Functional up-regulation of endopeptidase neurolysin during post-acute and early recovery phases of experimental stroke in mouse brain. J Neurochem. 2014; 129(1): 179-89. PMID: 24164478; and see PMID: 34389655, above).
  • a fixed concentration of recombinant or mouse brain purified Nln was incubated with 25 ⁇ M (for primary screening and determination of A 50 values) or 2.5 to 75 ⁇ M (for determination of Km and Vmax values) QFS in artificial cerebrospinal fluid (NaCl 126 mM, NaHCO 3 26 mM, KC1 3 mM, KH 2 PO 4 1.4 mM, HEPES 25 mM, glucose 4 mM, MgCI 2 1.3 mM, CaCI 2 1.4mM, ZnSO 4 0.0002 mM, pH 7.2) containing 0.01% final assay concentration of Triton X-100 at 37 °C.
  • artificial cerebrospinal fluid NaCl 126 mM, NaHCO 3 26 mM, KC1 3 mM, KH 2 PO 4 1.4 mM, HEPES 25 mM, glucose 4 mM, MgCI 2 1.3 mM, CaCI 2 1.4mM, Z
  • Activity of recombinant human TOP was assessed in the same way as for neurolysin, except that the assay was carried out in the presence of 0.1 mM dithiothreitol (PMID: 34389655).
  • activities of recombinant human ACE, NEP and ACE2 were measured similar to Nln, except that quenched fluorescent substrate Mca-Arg-Pro-Pro-Gly-Phe-Ser-Ala-Phe-Lys(Dnp)- OH was used for ACE and NEP, and Mca-Ala-Pro-Lys-(Dnp)-OH was used for ACE2 (both substrates at 10 ⁇ M final assay concentration; PMID: 34389655 and 34436882).
  • test compounds Primary screening of top-ranked compounds (received from NCI DTP) was carried out at 10 and 100 ⁇ M final assay concentrations. Determination of A 50 values was conducted in the presence of 0.1 to 300 ⁇ M final assay concentrations. In all experiments, test compounds were incubated with Nln for 10 min at 37 °C before addition of the substrate to start the reaction. All test compounds were dissolved in DMSO at 10 to 50 mM stock concentrations. Presence of DMSO (up to 3.5%) did not affect activity of the enzyme.
  • Endogenous substrates of Nln and mass spectrometry analysis Endogenous substrates of Nln and mass spectrometry analysis. Hydrolysis of neurotensin, bradykinin and angiotensin I (20 ⁇ M; obtained from American Peptides) by recombinant rat Nln (2 nM in artificial CSF containing 0.01% Triton X-100) was carried out in the presence and absence of the Py-Pip (30 ⁇ M). The reaction with bradykinin lasted 10 minutes, whereas it was 20 minutes for Ang I and NT (optimized to yield -10% substrate hydrolysis). The reaction was stopped with -32 mM HC1 followed by freezing at -80 °C.
  • LC-MS/MS analysis was carried out at Texas Tech University Health Sciences Center, School of Pharmacy, Amarillo, where each sample was subjected to LC-MS/MS analysis using a Shimadzu Nexera Ultra High-performance LC system and a triple quadrupole Ion-Trap AB SCIEX QTRAP 5500 mass spectrometer. Samples were separated using 1.7 ⁇ m kinetex EVO C18 100A LC column (50mm x 2.1mm) (Phenomenex). An injection volume of 5 ⁇ L and a flow rate of 300 ⁇ L/min. Solvent A contained HPLC grade water with 0.1% formic acid and Solvent B contained 100% acetonitrile with 0.1% formic acid.
  • the elution gradient of solvent B applied was 5% over 0.1 minutes, 5% - 50% over 2.9 minutes, 80% over 4.8 minutes, 95% over 0.2 minutes.
  • the peptides were ionized by electrospray ionization (Turbo Spray) and analyzed by using an AB Sciex QTRAP 5500 mass spectrometer.
  • the precursor ion m/z of charged state (m+l)/l, (m+2)/2 or (m+3)/3 were identified as and subjected to fragmentation by a collision induced dissociation (CID) gas.
  • CID collision induced dissociation
  • the modulatory site on Nln is different from the substrate-binding site.
  • a set of experiments were carried out using an active-site inhibitor of Nln, dynorphin A (1-13) (Srinidhi Jayaraman, Joanna Kocot, Shiva Hadi Esfahani, Harold J Wangler, Arzu Uyar, Yehia Mechref, Paul C Trippier, Thomas J Abbruscato, Alex Dickson, Hideki Aihara, David A Ostrov, Vardan T Karamyan. Identification and Characterization of Two Structurally Related Dipeptides that Enhance Catalytic Efficiency of Neurolysin.
  • Ki value was ⁇ 1.5 ⁇ M (95% CI: 0.9 - 2.5 ⁇ M) in the absence of Py-Pip and it was 1.14 ⁇ M (95% CI: 0.50 - 2.69 ⁇ M) in the presence of 30 ⁇ M Py-Pip.
  • concentration-response effect of Py-Pip on activity of Nln was studied in the absence and presence of a fixed concentration of dynorphin A (1-13) (FIG. 2B). In this experiment, dynorphin A (1-13) inhibited activity of Nln and decreased the Amax value of Py-Pip.
  • a 50 value for Py-Pip was 9.55 ⁇ M (95% CI: 5.76 - 15.9 ⁇ M) and 9.08 ⁇ M (95% CI: 4.2 -20.04 ⁇ M) in the absence and presence of dynorphin A (1-13).
  • the observations made in this set of experiments show that dynorphin A (1-13) and Py-Pip interact with different binding sites on Nln, because they did not affect each other’s affinity for the peptidase.
  • the A 50 value is not a direct indicator of the affinity of a ligand
  • all variables were maintained the same (concentrations of Nln, substrate, Py- Pip, etc.)
  • the observed similar A 50 values for the modulators in the absence and presence of dynorphin A (1-13) show that affinity of the modulators remained unchanged.
  • dynorphin A (1-13) is a competitive inhibitor of Nln, these data also show that the binding site of the modulators is different from the substrate binding site.
  • Example 2 Discovery of the Pyridine-Piperazine Scaffold as Highly Potent and Selective Neurolysin Activators
  • the peptidase neurolysin (Nln) is upregulated following ischemic stroke to activate endogenous cerebroprotective mechanisms and has subsequently emerged as a therapeutic target for drug discovery efforts to treat ischemic stroke.
  • Overexpression of Nln in a mouse model of ischemic stroke showed proof-of-concept with significant improvement of stroke outcomes.
  • a rational drug-design approach was used on a hit scaffold identified in a high- throughput screen to identify Nln activators having a central pyridine-piperazine (Py-Pip) scaffold, tethered with an amine linker.
  • This example includes the initial SAR around the HTS hit NCI66183 (24c, Table 3) leading to Nln activators consisting of a central pyridine-piperazine (Py-Pip) scaffold and the identification of potent, stable, selective, brain penetrant and ‘drug-like’ small-molecule Nln activators with superior profile to current peptidomimetic activators for the potential treatment of ischemic stroke and other related neurodegenerative diseases. 18 ' 20
  • Scheme 1.1 a Synthesis of pyridine amine 4-substituted piperazine analogs (7a-7g, 7i, 7k- 71, 7n-7p and 8a-8d).
  • Reagents and conditions (i) Nal, Na 2 CO 3 , THF, 60 °C, 12 h; (ii) N H 2 NH 2 H 2 O, ethanol, reflux, 3 h; (iii) (iii-a) AcOH, THF/MeOH (4: 1), 65 °C, 3 h; (iii-b) NaBH 4 , 0 °C to rt, 2 h.
  • Scheme 1.2 a Synthesis of compounds 8c and 8d (alternative synthetic route) using 2- chloromethyl pyridine hydrochloride salt.
  • Scheme 2 a Synthesis of 3 -substituted (14) phenyl functionalized piperazine analog. “Reagents and conditions: (i) Et 3 N, THF, 0 °C to RT, 4 h; (ii) K 2 CO 3 , DMF 60 °C, 4 h; (iii) LAH 5eq, THF 50 °C, overnight.
  • Reagents and conditions (i) DMF, K + (CH 3 ) 3 CO , RT, 12 h.
  • Scheme 4 “Synthetic route to amide containing analog 18. “Reagents and conditions: (i) EDC.HC1, HOBt, Et 3 N, DCM, RT, 4 h
  • Reagents and conditions (ia) AcOH, THF/MeOH (4: 1), 65 °C, 3 h; (ib) NaBH 4 , 0 °C to rt, 2h.
  • the m-Br analogue (14) lead to an approximate two-fold reduction of activity.
  • FIG. 7 is a summary of the SAR of the pyridine-piperazine scaffold for Nln activation.
  • KS52 possessed a half-life in mouse plasma of >1000 minutes with enhanced stability in mouse brain of approximately nine hours.
  • the p-nitrophenyl compound 7g showed similar mouse plasma stability with further improved mouse brain half-life of 11.5 hours.
  • Table 4 Stability of selected compounds in mouse plasma and mouse brain.
  • FIGS. 8A to 8D show the permeability of compound 7c (KS52) and compound 7g (KS73) through MDR1-MDCA monolayer.
  • FIG. 8 A Apical to basolateral transport of non- peptidomimetic Nln activators through a BBB-mimicking model. Chemical structure of non- peptidomimetic Nln activators, KS52 and KS73 (compounds 7c and 7g, respectively).
  • FIG. 8A In vitro permeability coefficient (Pe) of compounds KS52 and KS73 (compounds 7c and 7g, respectively) compared to the endogenous activator His-Tyr.
  • FIG. 8B, FIG. 8C Permeability coefficient (Pe) of KS52 and KS73 (compounds 7c and 7g, respectively) in the presence and absence of P-gp inhibitor CsA, in either apical to basolateral (A
  • FIG. 8C Basolateral to apical (B > A) (B) direction.
  • FIG. 8D Comparison of influx
  • Table 5 In vitro blood-brain barrier penetration and fraction unbound in mouse plasma and brain of selected compounds.
  • Solvent B is gradually increased to 95% at 5 min, held at 95% until 6 min, then gradually ramped back down to 5% at 8.0 min.
  • HPLC purity data for all final compounds were performed on a Waters ACQUITY ultra-performance liquid chromatography (UPLC) H-Class System with TUV (254 nm) detector and Empower 2 software (Milford, MA, USA) using an Agilent Eclipse plus C185 ⁇ column (4.6 X 150 mm) using solvent A (water with 0.1 % Trifluoroacetic acid), solvent B
  • Compound 12 was synthesized and characterized by the literature-reported procedure of Jun Yin, Xuefeng Guan, Di Wang, and Shiyong Liu, “Metal-Chelating and Dansyl-Labeled Poly(N-isopropylacrylamide) Microgels as Fluorescent Cu2+ Sensors with Thermo-Enhanced Detection Sensitivity” Langmuir 2009 25 (19), 11367-11374; DOI: 10.1021/la901377h.
  • Compound 3m was purchased from commercial source and used without further purification.
  • reaction was extracted using DCM-water and the organic layer was collected and dried using anhydrous sodium sulfate, and evaporated under vacuum to obtain crude product.
  • the pure product was isolated via column chromatography (5% MeOH, 95% DCM). Yellow solid, yield (133 mg, 47%).
  • CD 3 OD ⁇ 151.25, 131.46, 129.63, 129.26, 128.93, 128.68, 116.11, 53.23, 52.65, 50.76, 48.98,
  • Example 3 In-Vivo Brain Uptake Studies of Pyridine-Piperazine-based Neurolysin Activators in Healthy and Stroke Animals
  • peptidase neurolysin functions to preserve the brain following ischemic stroke by hydrolyzing various neuropeptides. Nln activation has emerged as an attractive drug discovery target for treatment of ischemic stroke.
  • two Pyridine- Piperazine-based lead compounds (7c also known as KS52 and 7g also known as KS73) were used for quantitative pharmacokinetic analysis to provide valuable information for subsequent precl i ni cal devel opment .
  • FIG. 9A and 9B show the pharmacokinetic profile of KS52 and KS73 (compounds 7c and 7g, respectively) after intravenous bolus injection in healthy mice.
  • FIG. 9 A Concentration-time profiles of KS52 an KS73 (compounds 7c and 7g, respectively) showed biexponential decline in plasma. Left panel showed the absoluteive values, right panel showed the relative portion to injection dose.
  • FIG. 9B Concentration-time profiles of KS52 an KS73 (compounds 7c and 7g, respectively) showed biexponential decline in brains. Left panel showed the absoluteive values, right panel showed the relative portion to injection dose.
  • FIGS. 11A to 11C shows plasma pharmacokinetic profile of KS73 (compound 7g) in stroke mice.
  • FIG. 11 A Representative TTC staining of brain slices after 1-h tMCAO-induced occlusion and 3-h reperfusion (Left panel) and cerebral blood flow measured by a laser Doppler probe presented as percentages of the baseline.
  • FIG. 1 IB Plasma profile in stroke and healthy animals. Left panel showed the absoluteive values, right panel showed the relative portion to total injection dose.
  • FIG. 11C The area under the plasma concentration-time curves (AUC 0-30 min) in stroke (tMCAO) and healthy (Control) mice.
  • TTC 2, 3, 5 -triphenyl tetrazolium chloride.
  • %ID/ml percentage of injection dose/ml.
  • KS73 Compound 7g
  • Stroke Mice Single time point brain uptake for KS73 (compound 7g) in stroke mice was quantitated as aforementioned in healthy mice. After 30 minutes of dosing, the brain uptake was not significantly different among healthy brains, ipsilateral hemispheres and contralateral hemispheres of stroke mice (10.32 +/- 0.46 vs.10.48 +/- 0.63 and 11.22 +/- 0.76% ID/ml, p > 0.05) (FIG. 5 A).
  • the apparent Kin values of ipsilateral and contralateral hemispheres were comparable (396.07 +/- 14.21 and 426.88 +/- 33.65 pl.min-l.g-1, p > 0.05) at 30 minutes, but were lower than that of healthy brains (863.71 +/- 31.28 pl. min-l.g-1) (p ⁇ 0.001) (FIG. 5B).
  • the lower apparent Kin values in stroke brains might be due to the aforementioned relatively higher plasma AUC 0-30 minutes in stroke animals instead of absoluteive brain concentrations, i.e., increased plasma exposure lowers the brain uptake clearance when the brain concentration did not significantly change.
  • FIGS. 12A to 12 C shows brain uptake of KS73 (compound 7g) at a single time point analysis in stroke mice.
  • FIG. 12A Brain uptake among stroke ipsilateral hemisphere, stroke contralateral hemisphere, and healthy brain. Left panel showed the absoluteive values, right panel showed the relative portion to total injection dose.
  • FIG. 12B Brain uptake clearance kinetics (Kin).
  • FIG. 12C Achieved brain concentration relative to A 50 .
  • Cbr total concentration in brain
  • %ID/ml percentage of injection dose/ml
  • tMCAO-Ips, tMCAO-Con and Control ipsilateral hemisphere of stroke animal, contralateral hemisphere of stroke animal, and healthy brains.
  • In vitro BBB permeability assays A co-culture system composed of mouse brain endothelial bEnd.3 cells and primary astrocytes was used to evaluate the in vitro BBB penetration of new Nln activators. KS compounds or endogenous Nln activator histidine-tyrosine (HY) dipeptide was added to the top chambers of the transwells and a fraction of the solution in the bottom chambers was collected at the indicated time points for LCMS/MS assays.
  • KS compounds or endogenous Nln activator histidine-tyrosine (HY) dipeptide was added to the top chambers of the transwells and a fraction of the solution in the bottom chambers was collected at the indicated time points for LCMS/MS assays.
  • FIGS. 14A and 14B show the half-life of KS compounds in plasma and brains.
  • FIGS. 15A and 15B shows brain concentrations of KS compounds.
  • KS52 compound 7c
  • KS73 compound 7g
  • FIGS. 15A and 15B show significantly higher brain total concentrations relative to their A 50 .
  • the dashed line indicate the A 50 for each compound, respectively.
  • FIGS. 17A and 17B show that there was no in vivo heart toxicity from KS73 (compound 7g) in heart weight (FIG. 17A) and body weight (FIG. 17B).
  • Table 7 shows the results from a hERG channel inhibition assay, a highly sensitive measurement which will identify compounds exhibiting cardiotoxicity related to hERG inhibition in vivo.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open- ended and do not exclude additional, unrecited elements or method steps.
  • “comprising” may be replaced with “consisting essentially of’ or “consisting of’.
  • the phrase “consisting essentially of’ requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention.
  • the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), propertie(s), method/process steps or limitation(s)) only.
  • A, B, C, or combinations thereof refers to all permutations and combinations of the listed items preceding the term.
  • “A, B, C, or combinations thereof’ is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB.
  • expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth.
  • the skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
  • words of approximation such as, without limitation, “about”, “substantial” or “substantially” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present.
  • the extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature.
  • a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ⁇ 1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.
  • each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.
  • compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit, and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

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Abstract

L'invention propose de nouvelles compositions et de nouvelles méthodes d'amélioration de l'activité de la neurolysine comprenant : une molécule de formule I, II, III ou IV, un sel, ou un énantiomère de celui-ci.
PCT/US2024/028847 2023-05-10 2024-05-10 Échafaudages à base de pyridine-pipérazine en tant qu'activateurs de neurolysine hautement puissants et sélectifs Pending WO2024233922A2 (fr)

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