Piperidine inhibitors with SLC6A19 function

Abstract

This document discloses compounds and pharmaceutically acceptable salts thereof, pharmaceutical compositions, and related methods for treating or preventing diseases or disorders associated with abnormal amino acid concentrations through the modulation of the SLC6A19 transporter.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 472,979, filed on 14 June 2023. [Background technology]

[0002] Phenyletonuria (PKU) is a congenital metabolic disorder caused by mutations in phenylalanine hydroxylase (PAH), an enzyme responsible for the metabolism of phenylalanine. PKU is an autosomal recessive metabolic disorder in which phenylalanine is not properly metabolized, resulting in abnormally high plasma concentrations of phenylalanine. Individuals with PKU have abnormally high blood levels of phenylalanine, and if left untreated, this can lead to irreversible nerve damage and various complications, including intellectual disability, seizures, neurodevelopmental disorders, and behavioral problems. PKU is difficult to treat because blood phenylalanine levels are directly related to diet. Patients must adhere to a strict diet for life, which affects every aspect of their lives. Current standard treatments are enzyme cofactor therapy and enzyme replacement therapy, but these therapies are not effective for all patients and carry a potential risk of adverse events.

[0003] The enzyme responsible for metabolizing phenylalanine and maintaining its homeostasis is phenylalanine hydroxylase (PAH). Loss-of-function (LOF) mutations in the PAH gene on chromosome 12q23.2 are known to result in most PKU types. These LOF mutations causing PKU can be diagnosed as classical PKU (the most severe type) and less severe forms of "mild PKU" or "hyperphenylalaninemia." In addition to PAH, mutations in other enzymes that affect phenylalanine metabolism, such as dihydropteridine reductase (DHPR), an enzyme involved in the synthesis of cofactors necessary for PAH activity, can also increase phenylalanine levels. In addition to diet, blood amino acid concentrations, including phenylalanine levels, are regulated by SCL6A19. SCL6A19 is located in the proximal tubule of the kidney and is responsible for reabsorbing amino acids and returning them to the bloodstream. [Overview of the project]

[0004] This document discloses compounds and pharmaceutically acceptable salts thereof, pharmaceutical compositions, and related methods for treating or preventing diseases or disorders associated with abnormal amino acid concentrations through the modulation of the SLC6A19 transporter.

[0005] One aspect of the present invention relates to a compound of formula (I): [ka] or relating to a pharmaceutically acceptable salt thereof.

[0006] Another aspect of the present invention relates to a method for treating or preventing a disease or disorder associated with a genetic deficiency of phenylalanine hydroxylase in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula (I).

[0007] Another aspect of the present invention relates to a method for treating or preventing phenylketonuria, hyperphenylalaninemia, tyrosinemia, nonketotic hyperglycinemia, isovaleric acidemia, methylmalonic acidemia, propionic acidemia, maple syrup urine disease, DNAJC12 deficiency, urea cycle disorders, or hyperammonemia in a subject in need thereof, comprising administering to the subject an effective amount of a compound of formula (I).

[0008] Another aspect of the present invention relates to a method for regulating transport by SLC6A19 in an object requiring such regulation, comprising administering an effective amount of the compound of formula (I) to the object.

[0009] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art in which the present invention pertains. Methods and materials similar to or equivalent to those described herein may be used in the practice or testing of the present invention, but suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of any conflict, this specification shall prevail, including definitions. Furthermore, materials, methods, and examples are illustrative and not intended to limit the scope.

[0010] Other features, purposes, and advantages of the present invention will become apparent from the detailed description and the claims. [Modes for carrying out the invention]

[0011] definition For convenience, before further description of the present invention, the specific terms used in the specification, examples, and appended claims are summarized here. These definitions should be interpreted in the context of the remainder of this disclosure and understood as understood by those skilled in the art. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0012] For a more rapid understanding of the present invention, certain terms and phrases are defined below and throughout this specification.

[0013] The articles "a" and "an" are used herein for the purpose of indicating that the grammatical object of the article is one or more than one (i.e., at least one). By way of example, "an element" means one element or more than two elements.

[0014] The phrase "and / or" as used in this specification and the claims is to be understood to mean "either or both" of the elements so combined, i.e., elements that may be conjunctively or disjunctively present. Multiple elements listed using "and / or" are to be construed in the same fashion, i.e., as "one or more" of the elements so combined. Whether or not related to elements specifically identified by the "and / or" clause, other elements may optionally be present in addition to those specifically identified. Thus, by way of non-limiting example, when referring to "A and / or B", when used in combination with open-ended words such as "comprising", in one embodiment only A (optionally including elements other than B), in another embodiment only B (optionally including elements other than A), and in yet another embodiment both A and B (optionally including other elements), etc. can be referred to.

[0015] As used in this specification and the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" is inclusive, i.e., it should be construed to include not only at least one of a number or series of elements, but also two or more, and optionally additional items not in the list. Only terms clearly indicated otherwise, such as "only one of" or "exactly one of", or when used in the claims, "consisting of", will refer to including exactly one element out of a number or series of elements. Generally, as used in this specification, the term "or" should be construed as indicating an exclusive alternative (i.e., "one or the other but not both") only when preceded by terms indicating exclusivity, such as "either", "one of", "only one of" or "exactly one of". When used in the claims, "consisting essentially of" shall have the ordinary meaning as used in the field of patent law.

[0016] As used herein and in the claims, the phrase “at least one” should be understood to mean, with respect to a list of one or more elements, at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of all elements specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for the existence of any other elements besides those specifically identified in the list of elements, which the phrase “at least one” refers to, whether related to or unrelated to the elements specifically identified in the list of elements. Therefore, as a non-limiting example, "at least one of A and B" (or similarly, "at least one of A or B" or similarly, "at least one of A and / or B") may, in one embodiment, refer to the absence of B (and optionally including elements other than B) along with at least one A (including any two or more A's); in another embodiment, refer to the absence of A (and optionally including elements other than A) along with at least one B (including any two or more B's); and in yet another embodiment, refer to at least one A (including any two or more A's) and at least one B (including any two or more B's) (and optionally including other elements), and so on.

[0017] Unless otherwise clearly indicated, in any method claimed herein that includes two or more steps or operations, the order of the steps or operations of the method is not necessarily limited to the order in which the steps or operations of the method are shown.

[0018] In the claims and the above specification, all transitional clauses, such as “comprising,” “including,” “carrying,” “having,” “containing,” “accompanying,” “holding,” and “composed of,” should be understood to be open-ended, meaning “including, but not limited to, ….” As set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03, only the transitional clause “composed of” is a closed transitional clause, and only the transitional clause “essentially consisting of” is a semi-closed transitional clause.

[0019] Certain compounds included in the compositions of the present invention may exist in the form of specific geometric isomers or stereoisomers. In addition, the polymers of the present invention may also be optically active. In the present invention, all such compounds, including cis and trans isomers, R-enantiomers and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, racemic mixtures thereof, and other mixtures thereof, are intended to be within the scope of the invention. Additional chiral carbon atoms may be present in substituents, such as alkyl groups. All such isomers and mixtures thereof are intended to be included in the present invention.

[0020] "Geometric isomers" refer to isomers in which the orientation of substituent atoms differs in relation to a carbon-carbon double bond, cycloalkyl ring, or bridging bicyclic system. The atoms (other than H) on each side of the carbon-carbon double bond may be in the E-isomer configuration (substituents are on the opposite side of the carbon-carbon double bond) or the Z-isomer configuration (substituents are on the same side). "R", "S", "S*", "R*", "E", "Z", "cis", and "trans" indicate configuration relative to the core molecule. Certain of the disclosed compounds may exist in "atropisomer" form, i.e., as "atropisomers". Atropisomers are stereoisomers resulting from rotational impairment of single bonds that is high enough to isolate their conformational isomers due to steric strain impairment. The compounds of the present invention may be prepared as individual isomers by synthesis specific to each isomer, or by separation from an isomer mixture. Conventional resolution techniques include using optically active acids to form salts of the free bases of each isomer in an isomer pair (followed by fractional crystallization and regeneration of the free bases), using optically active amines to form salts of the acidic forms of each isomer in an isomer pair (followed by fractional crystallization and regeneration of the free acid), using optically pure acids, amines, or alcohols to form esters or amides of the isomers in an isomer pair (followed by chromatographic separation and removal of chiral auxiliaries), or using various well-known chromatographic methods to resolve a mixture of isomers of either the starting material or the final product.

[0021] For example, if a specific enantiomer of the compound of the present invention is desired, it may be prepared by asymmetric synthesis or by induction using a chiral auxiliary agent, in which case the resulting diastereomer mixture is separated and the auxiliary groups are cleaved to obtain the pure desired enantiomer. Alternatively, if the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, a diastereomer salt is formed using an optically active suitable acid or base, and then the diastereomer thus formed is separated by fractional crystallization or chromatography, which is well known in the art, and then the pure enantiomer is recovered.

[0022] Purity by mole fraction (%) is the ratio of moles of an enantiomer (or diastereomer), or the ratio of moles of an enantiomer (or diastereomer) to moles of its optical isomer. When the stereochemistry of a disclosed compound is named or described by structure, the named or described stereoisomer has a purity of at least about 60%, about 70%, about 80%, about 90%, about 99%, or about 99.9% in mole fraction relative to the other stereoisomer. When a single enantiomer is named or described by structure, the described or named enantiomer has a purity of at least about 60%, about 70%, about 80%, about 90%, about 99%, or about 99.9% in mole fraction. When a single diastereomer is named or described by its structure, the described or named diastereomer has a purity of at least about 60%, about 70%, about 80%, about 90%, about 99%, or about 99.9% in mole fraction.

[0023] When a disclosed compound is named or described by structure without showing its stereochemistry, and the compound has at least one chiral center, the name or structure should be understood to include any of the enantiomers of the compound that do not contain the corresponding optical isomer, a racemic mixture of the compound, or a mixture in which one enantiomer is concentrated relative to the corresponding optical isomer. When a disclosed compound is named or described by structure without showing its stereochemistry, and has two or more chiral centers, the name or structure should be understood to include a diastereomer that does not contain other diastereomers, a considerable number of diastereomers that do not contain other diastereomer pairs, a mixture of diastereomers, a mixture of diastereomer pairs, a mixture of diastereomers in which one diastereomer is concentrated relative to the other diastereomer(s), or a mixture of diastereomers in which one or more diastereomers are concentrated relative to the other diastereomer. The present invention includes all of these forms.

[0024] The structures shown herein are also intended to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, hydrogen may be replaced with deuterium or tritium, or carbon may be replaced with 13 C concentrated carbon or 14 The compounds produced by replacing carbon-enriched carbon fall within the scope of the present invention.

[0025] The term "prodrug," as used herein, includes compounds that are converted into therapeutic agents under physiological conditions. A common method for preparing prodrugs involves including a selective moiety that is hydrolyzed under physiological conditions to expose the desired molecule. In other embodiments, prodrugs are converted by the enzymatic activity of a host animal.

[0026] The terms “pharmaceutically acceptable excipient” or “pharmaceutically acceptable carrier” as used herein mean a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, which is involved in transporting or delivering the chemical substance from one organ or body part to another. Each carrier must be “acceptable” in the sense that it is compatible with the other components of the formulation, is not harmful to the patient, and is substantially nonpyrogenic. Some examples of materials that can function as pharmaceutically acceptable carriers include: (1) sugars, e.g., lactose, glucose, and sucrose; (2) starches, e.g., corn starch and potato starch; (3) cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, e.g., cocoa butter and suppository wax; (9) oils, e.g., peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and sesame oil. Examples include (10) soybean oil, 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) buffers 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, and (21) other non-toxic suitable substances used in pharmaceutical formulations. In certain embodiments, the pharmaceutical compositions of the present invention are non-pyrogenic, i.e., they do not cause a significant rise in temperature when administered to a patient.

[0027] The term "pharmaceutically acceptable salt" refers to relatively non-toxic inorganic and organic acid addition salts of a compound(s). These salts can be prepared in situ during the final isolation and purification of the compound(s), or by reacting the purified compound(s) in free base form separately with a suitable organic or inorganic acid and isolating the resulting salts. Representative salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitic acid, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, and lauryl sulfate. (For example, see Berge et al. (1977) “Pharmaceutical Salts”, J.Pharm.Sci. 66:1-19.)

[0028] In other cases, compounds useful in the methods of the present invention may contain one or more acidic functional groups, and thus pharmaceutically acceptable salts can be formed with pharmaceutically acceptable bases. In these cases, the term “pharmaceutically acceptable salt” refers to relatively non-toxic inorganic base addition salts and organic base addition salts of the compound(s). These salts can similarly be prepared in situ during the final isolation and purification of the compound(s), or by separately reacting the purified compound(s) in free acid form with a suitable base, such as a pharmaceutically acceptable metal cation hydroxide, carbonate, or bicarbonate, with ammonia, or with a pharmaceutically acceptable organic primary, organic secondary, or organic tertiary amine. Typical alkali salts or alkaline earth salts include lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, and aluminum salts. Typical organic amines useful for forming base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, and piperazine (see, for example, Berge et al.).

[0029] The term "pharmaceutically acceptable cocrystal" refers to a solid coformer that does not form formal ionic interactions with small molecules.

[0030] The “therapeutic effective dose” (or “effective dose”) of a compound relating to therapeutic use refers to the amount of the compound in preparation form that, when administered (to a mammal, preferably a human) as part of a desired administration regimen, reduces symptoms, improves a condition, or delays the onset of a condition, according to a clinically acceptable standard for the disorder or condition being treated, or for cosmetic purposes, for example, in a reasonable benefit / risk ratio applicable to any medical treatment.

[0031] The terms “preventive or therapeutic” treatment are recognized in the art and include administration to one or more hosts of the composition. Treatment is preventive (i.e., the treatment protects the host from developing the undesirable condition) if it is performed before the appearance of clinical symptoms of the undesirable condition (e.g., disease or other undesirable condition in the host animal), and therapeutic (i.e., the treatment is intended to reduce, improve or stabilize the existing undesirable condition or its side effects) if it is performed after the appearance of the undesirable condition.

[0032] The terms “patient” or “subject” refer to a mammal requiring a specific treatment. In certain embodiments, the patient may be a primate, dog, cat, or horse. In certain embodiments, the patient may be a human.

[0033] An "effective dose" is an amount sufficient to achieve a beneficial or desired outcome. For example, a therapeutic dose is an amount that achieves a desired therapeutic effect. This amount may be the same as or different from a prophylactic effective dose, which is the amount required to prevent the onset of a disease or symptoms of a disease. An effective dose can be administered in one or more doses, applications, or medications. The therapeutic effective dose of a composition varies depending on the composition selected. The compositions of the present invention can be administered at least once a day to at least once a week (including once every other day). It will be apparent to those skilled in the art that certain factors (including, but not limited to, the severity of the disease or disorder, past treatment history, the subject's overall health and / or age, and other pre-existing diseases) may influence the dose and timing required to effectively treat the subject. Furthermore, treating a subject with a therapeutic effective dose of a composition described herein may include a single treatment or a series of treatments.

[0034] The terms “decrease,” “reduce,” “reduced,” “reduce,” “decrease,” and “inhibit” are all used herein to generally mean a statistically significant reduction compared to a reference. However, to avoid any doubt, “decrease,” “reduce,” or “decrease” or “inhibit” typically mean a reduction of at least 10% compared to a reference level, and may include reductions of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, and at least about 99% (including these values) compared to a reference level, for example, the complete absence of a given element or parameter, or a reduction of 10 to 99% compared to no treatment being performed.

[0035] The terms “increased,” “increased,” “improved,” or “activated” are all used herein to generally mean an increase of a statistically significant amount, and to avoid any ambiguity, the terms “increased,” “increased,” “improved,” or “activated” mean an increase of at least 10% compared to a reference level, for example, an increase of at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or 100% (including this value), or any increase of 10 to 100% compared to a reference level, or an increase of at least about 2 times, at least about 3 times, at least about 4 times, at least about 5 times or at least about 10 times compared to a reference level, or any increase of 2 to 10 times or more.

[0036] As used herein, the term “regulate” includes upregulation and downregulation, for example, improving or inhibiting a response.

[0037] As defined herein, “radioactive pharmaceutical agent” means a pharmaceutical agent containing at least one radioactive isotope that emits radiation. Radioactive pharmaceutical agents are conventionally used in nuclear medicine for the diagnosis and / or treatment of various diseases. Radiolabeled pharmaceutical agents, such as radiolabeled antibodies, contain radioisotopes (RI) that function as radiation sources. As intended herein, the term “radioisotope” includes metallic and nonmetallic radioisotopes. Radioisotopes are selected based on the medical use of the radiolabeled pharmaceutical agent. When the radioisotope is a metallic radioisotope, chelating agents are typically used to bind the metallic radioisotope to the remainder of the molecule. When the radioisotope is a nonmetallic radioisotope, the nonmetallic radioisotope is typically bound to the remainder of the molecule directly or via a linker.

[0038] For the purposes of this invention, chemical elements are identified according to the inside cover of the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed., 1986-87.

[0039] The compound of the present invention One aspect of the present invention relates to a compound of formula (I): [ka] or relating to a pharmaceutically acceptable salt thereof.

[0040] In certain embodiments, the compound has the relative stereochemistry shown above.

[0041] In certain embodiments, the compound has the absolute stereochemistry shown above.

[0042] Pharmaceutical composition, route of administration, and dosage In certain embodiments, the present invention relates to a pharmaceutical composition comprising the compound of the present invention and a pharmaceutically acceptable carrier.

[0043] In certain embodiments, the pharmaceutical composition of the present invention further comprises at least one additional pharmaceutically active agent other than the compound of the present invention. The at least one additional pharmaceutically active agent may be an agent useful for treating ischemia-reperfusion injury.

[0044] The pharmaceutical compositions of the present invention can be prepared by combining one or more compounds of the present invention with a pharmaceutically acceptable carrier and, optionally, one or more additional pharmaceutically active agents.

[0045] As stated above, “effective dose” refers to any amount sufficient to achieve the desired biological effect. By combining the teachings provided in this invention, selecting from a variety of active compounds, and carefully considering factors such as efficacy, relative bioavailability, patient weight, severity of adverse side effects, and method of administration, an effective prophylactic or therapeutic regimen can be planned that does not cause substantially undesirable toxicity and is effective in treating a particular target. The effective dose for any particular use may vary depending on factors such as the disease or condition being treated, the specific compound of this invention being administered, the size of the target, or the severity of the disease or condition. Those skilled in the art can empirically determine the effective dose of the specific compound and / or other therapeutic agents of this invention without requiring excessive experimentation. The maximum dose, i.e., the safest dose based on several medical judgments, may be used. Multiple doses per day may be intended to achieve an appropriate systemic level of the compound. An appropriate systemic level can be determined, for example, by measuring the patient's peak or sustained plasma level of the drug. “Dose” and “administered dose” are used synonymously herein.

[0046] In certain embodiments, intravenous administration of the compound may typically be 0.1 mg / kg / day to 20 mg / kg / day. In one embodiment, intravenous administration of the compound may typically be 0.1 mg / kg / day to 2 mg / kg / day. In one embodiment, intravenous administration of the compound may typically be 0.5 mg / kg / day to 5 mg / kg / day. In one embodiment, intravenous administration of the compound may typically be 1 mg / kg / day to 20 mg / kg / day. In one embodiment, intravenous administration of the compound may typically be 1 mg / kg / day to 10 mg / kg / day.

[0047] Generally, the daily oral dose of the compound in human subjects ranges from approximately 0.01 mg / kg to 1000 mg / kg per day. Therapeutic effects are expected with oral doses ranging from 0.5 to 50 mg / kg, administered once or more times daily. Depending on the method of administration, the dose may be adjusted as appropriate to achieve the desired drug level locally or systemically. For example, intravenous administration is expected to result in daily doses that are one to several orders of magnitude smaller. If the response in the subject is insufficient at such doses, higher doses (or more effective higher doses via a different, more localized delivery route) may be used, to the extent that patient tolerance allows. Multiple daily administrations are intended to achieve appropriate systemic levels of the compound of the present invention.

[0048] For any of the compounds described herein, the therapeutic dose can initially be determined from animal models. The therapeutic dose can also be determined from human data of compounds tested in humans and compounds known to exhibit similar pharmacological activity, such as other relevant activators. Higher doses may be required for parenteral administration. The dose applied can be adjusted based on the relative bioavailability and efficacy of the compound being administered. Adjusting the dose to achieve maximum efficacy based on the above methods and other methods well known in the art is well within the capabilities of those skilled in the art.

[0049] The formulations of the present invention can be administered in a pharmaceutically acceptable solution, which may conventionally contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, suitable carriers, adjuvants, and optionally other therapeutic components.

[0050] When used in therapeutic applications, the compound can be administered to a target in an effective amount by any means of delivering the compound to the desired surface. Administration of the pharmaceutical composition may be carried out by any means known to those skilled in the art. Methods of administration include, but are not limited to, intravenous, intramuscular, intraperitoneal, intravesical (bladder), oral, subcutaneous, direct injection (e.g., injection into a tumor or abscess), mucosal administration (e.g., local administration into the eye), inhalation, and topical administration.

[0051] For intravenous and other parenteral administration methods, the compounds of the present invention can be formulated as lyophilized preparations, as lyophilized preparations of active compounds intercalated or encapsulated in liposomes, as lipid complexes in aqueous suspensions, or as salt complexes. The lyophilized preparations are generally reconstituted immediately before administration with a suitable aqueous solution, such as sterile water or physiological saline.

[0052] For oral administration, the compound can be readily formulated by combining the active compound(s) with a pharmaceutically acceptable carrier known in the art. Such carriers allow the compound of the present invention to be formulated as tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral intake by the target of treatment. Pharmaceutical preparations for oral use can be obtained as solid excipients, and optionally, if desired, appropriate adjuvants may be added, after which the resulting mixture may be pulverized and the granular mixture processed to obtain a tablet or sugar-coated tablet core. Suitable excipients include fillers, such as sugars containing lactose, sucrose, mannitol, or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, a disintegrant, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid, or a salt thereof, such as sodium alginate, may be added. Optionally, the oral formulation may be formulated in saline or a buffer, such as EDTA, to neutralize the acidic state in the body, or it may be administered without any carrier.

[0053] Furthermore, the specific target is an oral dosage form of the above-mentioned component(s). The component(s) may be chemically modified to facilitate oral delivery of its derivatives. Generally, the intended chemical modification involves attaching at least one moiety to the component molecule itself, which enables (a) inhibition of acid hydrolysis and (b) uptake into the bloodstream from the stomach or intestines. Also desired is improved overall stability of the component(s) and extended circulation time in the body. Examples of such moieties include polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, and polyproline. Abuchowski and Davis, “Soluble Polymer-Enzyme Adducts”, In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, NY, pp.367-383 (1981); Newmark et al., J Appl Biochem 4:185-9 (1982). Other polymers that can be used are poly-1,3-dioxolane and poly-1,3,6-trioxocan. For pharmaceutical applications, the polyethylene glycol portion is suitable, as shown above.

[0054] The release site for the component (or derivative) may be the stomach, small intestine (duodenum, jejunum, or ileum), or large intestine. Those skilled in the art will know that formulations are available that do not dissolve in the stomach but release the substance in the duodenum or other part of the intestine. Preferably, the release avoids adverse effects in the gastric environment by either protecting the compound (or derivative) of the present invention or by passing through the gastric environment and releasing the bioactive substance, for example, in the intestines.

[0055] To ensure complete gastric tolerance, a coating that is impermeable to at least pH 5.0 is essential. Examples of more common inert components used as enteric coatings include cellulose acetate trimellilate (CAT), hydroxypropyl methylcellulose phthalate (HPMCP), HPMCP50, HPMCP55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and shellac. These coatings may be used as mixed coatings.

[0056] Coatings or coating mixtures can also be used for tablets that are not intended to protect from the stomach. Examples of such coatings include sugar coatings or coatings that facilitate swallowing tablets. Capsules may consist of a hard shell (such as gelatin) for the delivery of dry therapeutic agents (e.g., powders), while liquid forms may use a soft gelatin shell. The shell material for cachets may be thick starch or other food paper. Wet mashing techniques can be used for pills, lozenges, molded tablets, or powder tablets.

[0057] The therapeutic agent can be incorporated into the formulation as fine multiparticles in the form of granules or pellets with a particle size of approximately 1 mm. Formulations for capsule administration can also be in the form of powders, lightly compressed plugs, or tablets. The therapeutic agent can be prepared by compression.

[0058] The compound may include both a coloring agent and a flavoring agent. For example, the compound (or derivative) of the present invention may be formulated (such as by encapsulation in liposomes or microspheres) and then further included in an edible product, such as a chilled beverage containing a coloring agent and a flavoring agent.

[0059] The therapeutic agent may be diluted or its volume increased using an inert material. Examples of such diluents include carbohydrates, particularly mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextran, and starch. Certain inorganic salts, including calcium triphosphate, magnesium carbonate, and sodium chloride, may also be used as fillers. Some commercially available diluents include Fast-Flo, Emdex, STA-Rx1500, Emcompress, and Avicell.

[0060] The disintegrant may be included in the formulation of the therapeutic agent of the present invention to form a solid dosage form. Materials used as disintegrants include, but are not limited to, starch, including commercially available starch-based disintegrants such as Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, carboxymethylcellulose acid, natural sponges, and bentonite may all be used. Another form of disintegrant is an insoluble cation exchange resin. Powdered gum may be used as both a disintegrant and binder; examples of such gums include powdered gums such as agar, karaya, or tragacanth. Alginic acid and its sodium salts are also useful as disintegrants.

[0061] The therapeutic agents may be bound together with a binder to form a hard tablet, and examples of such binders include natural products such as acacia, tragacanth, starch, and gelatin. Other examples include methylcellulose (MC), ethylcellulose (EC), and carboxymethylcellulose (CMC). Both polyvinylpyrrolidone (PVP) and hydroxypropyl methylcellulose (HPMC) can be used in an alcoholic solution to granulate the therapeutic agents of the present invention.

[0062] To prevent sticking during the formulation process, antifriction agents may be included in the formulation of the therapeutic agent. Lubricants may be used as a layer between the therapeutic agent and the acetabular wall, and these lubricants include, but are not limited to, stearic acid (including its magnesium and calcium salts), polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils, and waxes. Soluble lubricants, such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000, and Carbowax 6000, may also be used.

[0063] A flow enhancer may be added to improve the fluidity of the drug during formulation and to assist in rearrangement during compression. Examples of such flow enhancers include starch, talc, calcined silica, and hydrated aluminosilicate.

[0064] To aid in the dissolution of the therapeutic agent in an aqueous environment, surfactants may be added as wetting agents. Examples of surfactants include anionic detergents, such as sodium lauryl sulfate, sodium dioctyl sulfosuccinate, and sodium dioctyl sulfonate. Cationic detergents may be used, such as benzalkonium chloride and benzethonium chloride. Potential nonionic detergents that can be included in the formulations of the present invention as surfactants include lauromacrogol 400, polyoxyl stearate 40, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. These surfactants can be present in the formulations of the compounds or derivatives of the present invention either alone or in mixtures of various proportions.

[0065] Pharmaceutical preparations for oral administration include push-fit capsules made of gelatin, and soft, sealable capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. Push-fit capsules may contain the active ingredient in an additive mixture with a filler, such as lactose, a binder, such as starch, and / or a lubricant, such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the active compound may be dissolved or suspended in a suitable liquid, such as fatty oil, liquid paraffin, or liquid polyethylene glycol. Stabilizers may also be added. Microspheres formulated for oral administration may also be used. Such microspheres are well defined in the art. All oral formulations must be in a dose suitable for oral administration.

[0066] For oral administration, the composition may take the form of tablets or lozenges formulated using conventional methods.

[0067] For topical administration, the compound may be formulated as a solution, gel, ointment, cream, suspension, etc., as is well known in the art. Systemic formulations include formulations designed for administration by injection, such as subcutaneous injection, intravenous injection, intramuscular injection, intrathecal injection, or intraperitoneal injection, and formulations designed for transdermal administration, transmucosal administration, oral administration, or pulmonary administration.

[0068] The compounds for use according to the present invention may also be conveniently delivered for inhalation administration in the form of an aerosol spray from a pressurized pack or nebulizer using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the unit of the dose may be determined by incorporating a valve that delivers a quantified amount. For use in an inhaler or injector, for example, gelatin capsules and gelatin cartridges may be formulated to contain a powder mix of the compounds of the present invention and a suitable powder base, such as lactose or starch.

[0069] Furthermore, the present invention aims at the pulmonary delivery of the compounds (or salts thereof) disclosed herein. The compounds are delivered to the lungs of mammals during inhalation, pass through the lining of the pulmonary epithelium, and reach the bloodstream. Other reports on inhalation 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) (α1-antitrypsin), Smith et al., 1989, J Clin Invest 84:1145-1146 (α-1-proteinase), and Oswein et al., 1990, “Aerosolization of Proteins”, Proceedings of Symposium on Respiratory Drug Delivery. Examples include II, Keystone, Colorado, March, (recombinant human growth hormone), Debs et al., 1988, J Immunol 140:3482-3488 (interferon-gamma and tumor necrosis factor alpha), and Platz et al., U.S. Patent No. 5,284,656 (granulocyte colony-stimulating factor, as incorporated by reference). Methods and compositions for delivering systemic drugs to the lungs are described in U.S. Patent No. 5,451,569, issued to Wong et al. on September 19, 1995 (as incorporated by reference).

[0070] The invention is intended for use in its implementation, but is not limited to, a variety of mechanical devices designed for the pulmonary delivery of therapeutic products, including nebulizers, metered-dose inhalers, and powder inhalers, all of which are well known to those skilled in the art.

[0071] Some specific examples of commercially available devices suitable for carrying out the present invention include the Ultravent nebulizer from Mallinckrodt, Inc., St. Louis, Mo., the Acorn II nebulizer from Marquest Medical Products, Englewood, Col., the Ventolin metered-dose inhaler from Glaxo Inc., Research Triangle Park, North Carolina, and the Spinhaler powder inhaler from Fisons Corp., Bedford, Mass.

[0072] Any such apparatus requires the use of a formulation suitable for dispensing the compounds of the present invention. Typically, each formulation is specific to the type of apparatus used and may involve the use of an appropriate propellant in addition to conventional diluents, adjuvants, and / or carriers useful for the treatment. The use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is also intended. The chemically modified compounds of the present invention may be prepared in different formulations depending on the type of chemical modification or the type of apparatus used.

[0073] Formulations suitable for use in either jet or ultrasonic nebulizers typically contain the compound (or derivative) of the present invention dissolved in water at a concentration of about 0.1 to 25 mg per 1 mL of solution. The formulation may also contain buffers and monosaccharides (for example, for stabilizing the inhibitor and regulating osmotic pressure). The nebulizer formulation may also contain surfactants to reduce or prevent surface-induced aggregation of the compound of the present invention as the solution is atomized when forming an aerosol.

[0074] Formulations used in metered-dose inhalers generally consist of a finely powdered substance containing the compound (or derivative) of the present invention suspended in a propellant with the help of a surfactant. The propellant may be any of the conventional materials used for this purpose, such as chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons, or hydrocarbons (including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof). Suitable surfactants include sorbitan trioleate and soy lecithin. Oleic acid is also useful as a surfactant.

[0075] The formulation administered from the powder inhalation device contains a finely dried powder containing the compound (or derivative) of the present invention, but may also contain an extender such as lactose, sorbitol, sucrose, or mannitol in an amount that facilitates the dispersion of the powder from the device, for example, 50 to 90% by weight of the formulation. The compound (or derivative) of the present invention is often and beneficially prepared in the form of fine particles with an average particle size of less than 10 micrometers (μm), most preferably 0.5 to 5 μm, so that it can be delivered most effectively to the deep parts of the lungs.

[0076] Nasal delivery of the pharmaceutical composition of the present invention is also intended. Nasal delivery allows the pharmaceutical composition of the present invention to enter the bloodstream directly after the therapeutic product has been administered through the nose, without the need for the product to be deposited in the lungs. Nasal delivery formulations include formulations containing dextran or cyclodextran.

[0077] For nasal administration, a useful device is a small, rigid bottle fitted with a metered-dose sprayer. In one embodiment, the metered dose is delivered by drawing a solution of the pharmaceutical composition of the present invention into a chamber of a predetermined volume, which has holes sized to aerosolize the aerosol formulation by forming a spray when the liquid inside the chamber is compressed. The pharmaceutical composition of the present invention is administered by compressing the chamber. In one specific embodiment, the chamber is an array of pistons. Such devices are commercially available.

[0078] Alternatively, it is a plastic squeeze bottle with a hole or opening sized to aerosolize an aerosol formulation by forming a spray when squeezed. The opening is usually located at the top of the bottle, and this top is usually tapered so that it partially fits into the nasal cavity for efficient administration of the aerosol formulation. Preferably, the nasal inhaler will supply a quantified amount of the aerosol formulation to administer a measured dose of the drug.

[0079] The compound may be formulated for parenteral administration by injection, such as bolus injection or continuous infusion, when systemic delivery is desired. The injectable formulation may be provided in unit dosage forms, for example in ampoules or multi-dose containers, with preservatives added. The composition may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and may contain formulation agents, such as suspending agents, stabilizers and / or dispersants.

[0080] The parenteral pharmaceutical formulation comprises an aqueous solution of the active compound in a water-soluble form. In addition, the suspension of the active compound may be prepared as a suitable oily injectable suspension. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. The aqueous injectable suspension may contain a substance that improves the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension may also contain a suitable stabilizer or an agent that increases the solubility of the compound of the present invention to enable the preparation of a highly concentrated solution.

[0081] Alternatively, the active compound may be in powder form for preparation with a suitable vehicle, such as sterile pyrogen-free water, before use.

[0082] The compound may be formulated into a composition for rectal or vaginal use, such as a conventional suppository base, such as a suppository or retaining enema containing cocoa butter or other glycerides.

[0083] In addition to the above formulations, the compound may also be formulated as a depot preparation. Such long-acting formulations may be formulated using a suitable polymer or hydrophobic material or ion exchange resin (for example, as an emulsion in an acceptable oil), or as a sparingly soluble derivative, for example, as a sparingly soluble salt.

[0084] The pharmaceutical composition may also include a suitable solid or gel phase carrier or excipient. 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 glycol.

[0085] Suitable liquid or solid pharmaceutical preparations include, for example, aqueous or saline solutions for inhalation, microencapsulated, spiral-shaped, coated on microscopic gold particles, contained in liposomes, atomized, aerosol, pellets for skin implantation, or dried on a sharp object for rubbing onto the skin. Examples of pharmaceutical compositions include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops, or preparations for sustained release of active compounds, in which excipients, additives and / or auxiliaries, such as disintegrants, binders, coatings, swelling agents, lubricants, flavoring agents, sweeteners, or solubilizers, are conventionally used as described above. These pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief discussion of drug delivery methods, see Langer R, Science 249:1527-33 (1990).

[0086] The compounds of the present invention and optionally other therapeutic agents may be administered neat or in the form of pharmaceutically acceptable salts or cocrystals. When used in medicine, the salts or cocrystals must be pharmaceutically acceptable, but conventionally, pharmaceutically acceptable salts or cocrystals can be prepared using salts or cocrystals that are not pharmaceutically acceptable. Examples of such salts include, but are not limited to, those prepared from hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, p-toluenesulfonic acid, tartaric acid, citric acid, methanesulfonic acid, formic acid, malonic acid, succinic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid. Furthermore, such salts can be prepared as alkali metal salts or alkaline earth metal salts, for example, sodium, potassium, or calcium salts of the carboxylic acid group.

[0087] Suitable buffering agents include acetic acid and salts (1-2% w / v), citric acid and salts (1-3% w / v), boric acid and salts (0.5-2.5% w / v), and phosphoric acid and salts (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).

[0088] The pharmaceutical compositions of the present invention include compounds as described herein and optionally a therapeutic agent contained in an effective amount in a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" means one or more suitable solid or liquid fillers, diluents, or encapsulating materials that are appropriate for administration to humans or other vertebrates. The term "carrier" means a natural or synthetic organic or inorganic component that, in combination with its active ingredient, facilitates application. The components of the pharmaceutical composition may also be mixed with and with the compounds of the present invention in such a way that there are no interactions that substantially impair the desired pharmaceutical effect.

[0089] Therapeutic agents (multiple) (specifically, the compounds of the present invention, but not limited to them) may be provided as particles. As used herein, particles mean nanoparticles or microparticles (or, in some cases, larger particles) that may consist, in whole or in part, of the compounds of the present invention or other therapeutic agents (multiple) as described herein. The particles may contain the therapeutic agent (multiple) in a core surrounded by a coating (including, but not limited to, enteric coatings). The therapeutic agent (multiple) may be dispersed throughout the particles. The therapeutic agent (multiple) may be adsorbed onto the particles. The particles may have a release kinetic sequence in any order, including zero-order release, primary release, secondary release, delayed release, sustained release, immediate release, and any combination thereof. In addition to the therapeutic agent (multiple), the particles may contain any substance commonly used in the fields of pharmacy and medicine, such as disintegrating, non-disintegrating, biodegradable, or non-biodegradable substances, or combinations thereof. The particles may be microcapsules containing the compounds of the present invention in solution or semi-solid state. The particles can take virtually any form.

[0090] Both non-biodegradable and biodegradable polymer materials can be used to manufacture particles for delivering the therapeutic agent(s). Such polymers may be natural or synthetic. The polymer is selected based on the desired release period. Of particular interest are the biodegradable hydrogels described in Sawhney HS et al. (1993) Macromolecules 26:581-7, the teachings of which are incorporated herein by reference. Examples of these polymers include polyhyaluronic acid, casein, gelatin, glutin, polyanhydride, polyacrylic acid, alginate, chitosan, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).

[0091] The therapeutic agent(s) may be included in a controlled-release system. The term “controlled-release” is intended to refer to any drug-containing formulation that controls the mode and profile of drug release from the formulation. This formulation includes, but is not limited to, immediate-release and non-immediate-release formulations, including sustained-release and delayed-release formulations. The term “sustained-release” (also called “sustained-release”) is used in its conventional sense to refer to a drug formulation that releases the drug gradually over a long period of time, preferably, but not necessarily, delivers the drug at substantially constant blood levels over a long period of time. The term “delayed-release” is used in its conventional sense to refer to a drug formulation in which there is a time lag between the administration of the formulation and the release of the drug from that formulation. “Delayed-release” may or may not involve sustained-release of the drug over a long period of time, and therefore may not be “sustained-release”.

[0092] For the treatment of chronic conditions, the use of long-term sustained-release implants may be particularly appropriate. “Long-term” release, as used herein, means that the implant is configured and positioned to deliver therapeutic levels of the active ingredient for at least 7 days, preferably 30 to 60 days. Long-term sustained-release implants are well known to those skilled in the art, and examples of such implants include some of the release systems described above.

[0093] Other suitable modifications and alterations to the compositions and methods described herein are readily apparent from the description of the invention contained herein, in light of information known to those skilled in the art, and may be made without departing from the scope of the invention or any of its embodiments, as will be understood by those skilled in the art in the relevant field. While the invention has been described in detail above, the invention will be more clearly understood by referring to the following examples, which are included herein for illustrative purposes only and are not intended to limit the invention. [Examples]

[0094] The present invention is further described in the following embodiments, but this does not limit the scope of the invention as described in the claims.

[0095] Example 1: SLC6A19 Isoleucine Transport Assay Cell line generation and maintenance The Flp-In®T-REx®293 cell line was purchased from Thermo Fisher Scientific. Using this cell line, a stable cell line was constructed that inductively expresses human SLC6A19 with a V5 tag at the C-terminus and stably expresses human TMEM27 (also known as Collectrin) with a myc-DDK tag at the C-terminus. The stable cell line was constructed by transfecting plasmids encoding SLC6A19 and TMEM27 using a standard protocol, followed by antibiotic selection. Stable cells were maintained in DMEM / F12 supplemented with Glutamax, 10% fetal bovine serum, 100 U / mL penicillin, 100 ug / mL streptomycin, 200 ug / mL hygromycin, 10 ug / mL blastosidine, and 300 ug / mL neomycin (Thermo Fisher).

[0096] Assay: Isoleucine transport assay in 96-well format On day 0, stable cell lines were seeded at a density of 35,000 cells / well into 96-well cell culture-treated plates coated with poly-D-lysine. On day 1, SLC6A19 expression was induced by dispensing tetracycline at a final concentration of 1 ug / mL using a Tecan D300e digital dispenser. On day 2, a transport assay was performed. The medium was removed from the plates using the GentleSpin setting of a Centrifugal Blue Washer (Blue Cat Bio), and the cells were washed with 175 uL of live cell imaging solution (Thermo Fisher) using the Blue Washer. After washing, the cells were treated at room temperature with either 70 uL of DMSO diluted in Krebs buffer (140 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 11 mM HEPES, 10 mM glucose, pH 7.4), a positive control, or a compound. 20-60 minutes later, 13 C6, 1530 μL of 3.3 mM NL-isoleucine solution (Cambridge Isotope Laboratories) was added. After incubation with the isoleucine substrate at room temperature for 20 minutes, the cells were washed with 175 μL of live cell imaging solution using a Blue Washer. The cells were then lysed with 150 μL of 15 μM D-Leucine-d10 in ultrapure water (CDN Isotopes). To promote lysis, the plate was shaken in a shaker at 700 rpm for at least 40 minutes. After lysis, the standard dilution curve was obtained. 13 C6, 15 NL-isoleucine was added to the wells containing lysates of untreated cells. The plate was returned to the shaker for at least 2 minutes to ensure proper mixing of the standard curve. The plate was then centrifuged at 4,000 rpm for 5 minutes to pelletize the cell debris and precipitate. The supernatant was diluted 1:10 with acetonitrile + 0.1% formic acid in a polypropylene plate.

[0097] Assay: Isoleucine transport assay in 384-well format On day 0, stable cell lines were seeded at a density of 20,000 cells / well using a Viaflo 384-well pipette into poly-D-lysine coated 384-well cell culture-treated plates containing medium with 1 ug / mL of tetracycline. The transport assay was performed the following day (day 1). The medium was removed from the plates using the GentleSpin setting of a Centrifugal Blue Washer (Blue Cat Bio), and the cells were washed with 80 μL of live cell imaging solution (Thermo Fisher) using the Blue Washer. After washing, the cells were treated with either 20 μL of DMSO diluted in Krebs buffer (140 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgCl2, 11 mM HEPES, 10 mM glucose, pH 7.4), a positive control, or a compound using a TECAN liquid handler. After incubation at room temperature for 20–60 minutes, 13 C6, 158.6 μL of a 3.3 mM solution of N-L-isoleucine (Cambridge Isotope Laboratories) was added. After incubating with the isoleucine substrate for 20 minutes at room temperature, the cells were washed with 80 μL of live cell imaging solution using a Blue Washer. Next, 15 μM of D-Leucine-d 10 (80 μL) (CDN Isotopes) was used to lyse the cells. To facilitate lysis, the plates were shaken on a shaker at 700 rpm for a minimum of 2 hours. After lysis, 13 C6, 15 N-L-isoleucine was added to the wells containing lysates of untreated cells. To ensure proper mixing of the standard curve, the plates were returned to the shaker for a minimum of 5 minutes. The plates were then centrifuged at 4,000 rpm for 10 minutes to pellet cell debris and precipitates. The supernatant was diluted 1:10 with acetonitrile + 0.1% formic acid in a polypropylene plate.

[0098] Analysis of 13 C6, 15 N-L-isoleucine was performed using RapidFire365-QTOF6545 (Agilent). For quantitative sample analysis, automated solid-phase extraction (HILIC H6 cartridge) was utilized followed by mass spectrometry injection. Samples were loaded using 95% acetonitrile, 0.1% formic acid and eluted directly from the cartridge using 5% acetonitrile, 0.1% formic acid for ESI-MS (electrospray ionization) analysis. Quantification of the analyte was performed using Agilent Masshunter Quant software from high-resolution full scan data.

[0099] Example 2: Clearance in human LM The clearance of compound (I) in human liver microsomes (LM) was evaluated by the substrate depletion method. Compound (I) (1 μM) was incubated with human liver microsomes (0.5 mg / mL protein) at 37°C in the presence of 1 mM NADPH and 100 mM phosphate buffer. At various time points from 0 to 60 minutes, a fixed amount was taken from the reaction mixture, mixed with 5 times the volume of cold acetonitrile (ACN) containing an internal standard (IS), and then centrifuged. The resulting supernatant was diluted with 1 times the volume of ultrapure water, and the parent compound was analyzed by LC / MS. The elimination constant rate, denoted as "ke", was determined by applying linear regression to the natural logarithm of the compound (I) retention rate versus incubation time curve. The clearance of compound (I) (μL / min / mg protein) was calculated using the formula: CLint = ke × incubation volume (μL) / protein amount (mg).

[0100] Example 3: CYP3A inhibition Midazolam was used as a substrate to investigate CYP3A inhibition in human liver microsomes. Midazolam (1 μM) was incubated with human liver microsomes (0.2 mg / mL protein) at 37°C for 5 minutes in the presence of 30 μM compound (I), 1 mM NADPH, and 100 mM phosphate buffer. The reaction was terminated by adding 1.5 times the volume of cold ACN containing an internal standard, and then centrifuged. The resulting supernatant was diluted with 1 times the volume of ultrapure water, and LC / MS analysis was performed for 1-hydroxylumidazolam, the major metabolite of CYP3A. The inhibition rate by compound (I) was determined by comparing the decrease in 1-hydroxylumidazolam formation with that of a solvent control.

[0101] Example 4: Bidirectional permeability assay in MDCK-MDR1 cells and Caco2 cells Bidirectional experiments were performed using Caco2 cells and MDCKII cells (MDCKII-MDR1) stably transfected with human MDR1 encoding Pgp. These cells were cultured in 96-well transwell plates at 37°C in 5% CO2. The test compounds were prepared in Hanks equilibrium salt solution containing 10 mM HEPES (pH 7.4) and 4% BSA to a concentration of 1 μM for the MDCKII-MDR1 assay and 10 μM for the Caco2 assay. To measure the transport rate from the apical membrane side (A) to the basement membrane side (B), 75 μL of the compound solution was added to the apical membrane side (donor), and 235 μL of a blank solution without the test compound was added to the basement membrane side compartment (acceptor). To evaluate the transport rate from the basement membrane side (B) to the apical membrane side (A), 235 μL of compound solution was added to the basement membrane side (donor), and 75 μL of a blank solution without the compound was added to the apical membrane side (acceptor). After incubation at 37°C for 2 hours, 50 μL samples were taken from both compartments A and B, mixed with cold ACN containing IS, and then centrifuged. The supernatant was analyzed by LC / MS to detect the parent compound. Furthermore, to investigate whether the compound is a substrate of Pgp, a bidirectional assay in MDCKII-MDR1 was performed in the presence of a 100 μM Pgp inhibitor, verapamil.

[0102] The apparent transmission coefficient (P) expressed in centimeters per second. app ) is formula: P app =(V A ×[drugs] アクセプター ) / (Area × Time × [Drug] 初期濃度、ドナー It can be calculated using ).

[0103] In this formula, V A The volume (in milliliters) in the acceptor-side well is represented by the area, and the area is the surface area of ​​the membrane (0.143 cm² for Transwell-96 6-well permeable support). 2 ) corresponds to the time, and the time indicates the total transport time (seconds).

[0104] The emission ratio is given by the formula: Emission ratio = P app(B→A) / P app It was calculated using (A→B). In the formula, P app (B→A) represents the apparent transmittance coefficient in the direction from the base to the apex. P app (A→B) represents the apparent transmittance coefficient in the direction from the apex to the base.

[0105] Example 5: Measurement of hERG inhibition hERG inhibition was assayed using a slightly modified protocol described in Haraguchi, Y. et al. Electrophysiological analysis of mammalian cells expressing hERG using automated 384-well-patch-clamp. BMC Pharmacol Toxicol. 2015;16:39.

[0106] Cell culture and cell preparation for patch clamp analysis:In this study, we used HEK293-hERG cells (Merck Millipore, Billerica, MA, USA), which are HEK293 cells that stably express hERG, and CHO-hERG cells (Merck Millipore), which are CHO cells that stably express hERG. HEK293 cells were cultured in an equal mixture of Dulbecco's Modified Eagle Medium (DMEM) and Nutrient Mixture F-12 (Invitrogen Life Technologies), supplemented with 10% fetal bovine serum (FBS) (Invitrogen Life Technologies) and 1% penicillin / streptomycin (Invitrogen Life Technologies). CHO cells were cultured at 37°C in Ham's F12 medium (Invitrogen Life Technologies), supplemented with 10% FBS and 1% penicillin / streptomycin, under a 5% CO2 humidified atmosphere. For the patch-clamp experiment, cultured cells on a polystyrene culture dish (Sumitomo Bakelite, Tokyo, Japan) were isolated at room temperature for several minutes using Accutase (Innovative Cell Technologies, Inc., CA, USA). To avoid cell damage, the cells were gently pipetted and resuspended in the patch-clamp solution to obtain a viable single-cell suspension.

[0107] Patch clamp analysis Patch clamp measurements were performed using the SyncroPatch 384 Patch Engine (PE) (Nanion Technologies, Munich, Germany), an automated multi-well planar patch clamp system (Figure 1a). The SyncroPatch 384 PE is a patch clamp module integrated into a liquid handling robot equipped with a Biomek FX (Beckman Coulter, Brea, CA, USA) with 384 pipetting heads. The system was controlled by dedicated software PatchControl 384 (Nanion Technologies). 384 cells were measured simultaneously using this device. Patch clamp recordings were performed at room temperature using intracellular solution [50 mM KCl, 10 mM NaCl, 60 mM KF, 20 mM EGTA, 10 mM HEPES (pH: 7.2): containing 25 μM escin in perforated patches (HEK293-hERG cells) and escin-free in whole-cell recordings (CHO-hERG cells)] and standard bath solution [140 mM NaCl, 4 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM D-glucose, 10 mM HEPES (pH: 7.4)]. Freshly prepared suspended single cells and drugs were added to a Teflon reservoir (Nanion Technologies). The following processes were automated using an auto patch clamp system: Suspended single cells were aspirated from the reservoir using a pipette and transferred to a flat 384-well patch clamp tip, where they were automatically vacuumed and captured in the wells. Seal formation, perforation, or establishment of a standard whole-cell mode, and electrophysiological recording were controlled by PatchControl 384. Drug addition and washing of each well were also performed automatically by a liquid handling robot. Concentration-response curves and IC50 values ​​were automatically calculated by separate dedicated software, DataControl 384 (Nanion Technologies). The kv current was induced using a voltage step of 1 second from the holding potential (-80mV) to +20mV, followed by a voltage step of 1 second to -50mV.

[0108] Example 6: In vivo pharmacokinetics PK studies were conducted in SD rats (n=3 per group). The inhibitor was administered intravenously (IV) at 1 mg / kg over 15 minutes and orally (PO) at 10 mg / kg. Blood samples were collected at 0.17, 0.25, 0.42, 0.75, 1, 2, 4, 8, and 24 hours after administration for IV administration, and at 0.25, 0.5, 1, 2, 4, 8, and 24 hours after administration for PO administration. Blood samples were centrifuged immediately after collection, and the separated plasma fractions were processed for LC / MS analysis. Logarithmically transformed plasma concentrations were plotted against the corresponding sample collection times. PK parameters were determined using the non-compartmental method. IV plasma clearance was calculated by dividing the dose by AUCinf. Elimination half-lives were determined by linear regression of logarithmically transformed data. Cmax was determined by graphical analysis of plasma concentration-time data, and oral bioavailability was determined from the AUCinf ratio of dose-adjusted IV and PO.

[0109] Example 7: Synthesis of the compound of formula (I) [ka]

[0110] Step 1:2 Synthesis A mixture containing 1 (1 g, 9.61 mmol) and PPTS (121 mg, 0.48 mmol) in DCM (30 mL) was to which DHP (1.13 g, 13.45 mmol) was added dropwise at 0°C. The resulting mixture was stirred at room temperature for 2 hours. Next, the mixture was concentrated to dryness under reduced pressure. The residue was diluted with RINKAN (80 mL), washed with water (80 mL) and brine (80 mL), the organic layer was separated and dried over anhydrous Na2SO4, and concentrated to obtain crude 2 (1.78 g, yield 98.5%), a colorless oil, which was used directly in the next step without further purification.

[0111] Step 2:3 Synthesis A mixture containing 20 mL of MeOH and 8 mL of H2O, along with 2 (1.78 g, 9.47 mmol), was gradually to which 453 mg of LiOH (18.91 mmol) was added. The resulting mixture was stirred at room temperature for 3 hours. Next, the mixture was diluted with 30 mL of water and extracted with 60 mL of MTBE. The aqueous layer was separated and adjusted to pH 5 with 1 N HCl aqueous solution. The mixture was then washed twice with 60 mL of HCl. The organic layers were combined and washed with 60 mL of water and 60 mL of brine. The mixture was dried over anhydrous Na2SO4 and concentrated to obtain crude 3 (1.59 g, yield 96.5%), a colorless oil, which was used directly in the next step without further purification.

[0112] Step 3: Synthesis of 5 A mixture containing 4 (4.0 g, 18.35 mmol) and TEA (3.7 g, 36.7 mmol) in anhydrous DCM (50 mL) was to be mixed with NsCl (4.48 g, 20.19 mmol) dropwise at 0°C, and the resulting mixture was stirred at room temperature for 2 hours. Next, the mixture was diluted with saturated NaHCO3 solution (100 mL) and extracted twice with DCM (100 mL). The organic layers were combined and washed with brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated to dryness. The residue was purified by flash column chromatography (eluting at PE / siRNA = 100:0~1:1) to obtain the yellow solid 5 (6.5 g, yield 87.8%). LC / MS (ESI) m / z: 404 (M+H) + .

[0113] Step 4: Synthesis of 6 A solution containing 5 (6.5 g, 16.13 mmol) in anhydrous DCM (80 mL) was added dropwise with HCl (4 M in dioxane, 8 mL, 32.2 mmol) at 0°C, and the resulting mixture was stirred at room temperature for 2 hours. Next, the mixture was concentrated to dryness to obtain crude 6 (3.9 g, 79.9% yield), a white solid, which was used directly in the next step without further purification. LC / MS (ESI) m / z: 304 (M+H) + .

[0114] Step 5: Synthesis of 7 A mixture containing 6 (3.9 g, 12.85 mmol) and AcOH (77 mg, 1.29 mmol) in MeOH (60 mL) was gradually mixed with 2,4-dimethoxybenzaldehyde (2.35 g, 14.14 mmol) and NaBH(OAc)3 (5.45 g, 25.7 mmol) at 0°C. The resulting mixture was stirred at room temperature under an N2 atmosphere for 4 hours. Next, the mixture was diluted with ELISA (100 mL) and washed with saturated NaHCO3 solution (100 mL) and brine (100 mL). The organic layer was separated, dried over anhydrous Na2SO4, filtered, and concentrated to dryness. The residue was purified by flash column chromatography (eluting at DCM / MeOH = 100:0~12:1) to obtain 7 (5.1 g, yield 87.9%), which was a yellow solid. LC / MS(ESI) m / z:454(M+H) + .

[0115] Step 6: Synthesis of 8 To a mixture containing 7 (5.1 g, 11.26 mmol) and AcOH (2.02 g, 33.8 mmol) in EtOH (120 mL), (1-ethoxycyclopropoxy)trimethylsilane (3.7 g, 22.52 mmol) and NaCNBH3 (1.4 g, 22.52 mmol) were gradually added. The resulting mixture was stirred at 80°C for 4 hours under an N2 atmosphere. Next, the mixture was concentrated to dryness. The residue was diluted with HCl (100 mL) and washed with saturated NaHCO3 solution (100 mL x 2) and brine (100 mL). The organic layer was separated, dried over anhydrous Na2SO4, filtered, and concentrated to dryness. The residue was purified by flash column chromatography (eluting at PE / HCl = 100:0~3:1) to obtain 8 (4.9 g, yield 88.3%), which was a yellow solid. LC / MS(ESI)m / z:494(M+H) + .

[0116] Step 7:9 Synthesis Adding 8 (4.9 g, 9.94 mmol) to TFA (50 mL) and stirring the resulting mixture at 80°C for 3 hours, the mixture was then concentrated to dryness. The residue was diluted with RINKAN (80 mL) and washed with saturated NaHCO3 solution (100 mL) and brine (100 mL). The organic layer was separated, dried over anhydrous Na2SO4, filtered, and concentrated to obtain crude 9 (2.7 g, yield 79.4%), a yellow solid, which was used directly in the next step without further purification. LC / MS(ESI) m / z: 344(M+H) + .

[0117] Step 8: Synthesis of 10 To a solution containing CDI (1.54 g, 9.53 mmol) and diisopropylethylamine (3.0 mL, 17.3 mmol) in DMF (17 mL), (2-fluoro-4-(trifluoromethoxy)phenyl)methaneamine (1.81 g, 8.66 mmol) was gradually added. The mixture was stirred at room temperature for 2 hours, and LC-MS indicated that the starting materials were completely consumed. The crude solution (0.5 M) was used directly in the next step. LC / MS (ESI) m / z: 304 (M+H) + .

[0118] Step 9:11 Synthesis A mixture containing 9 (2.7 g, 7.87 mmol) and DIPEA (2.0 g, 15.74 mmol) in DMF (50 mL) was mixed with 10 (0.5 M, 8.66 mmol). The resulting mixture was stirred at 70°C for 6 hours. Next, the mixture was diluted with saturated NH4Cl solution (100 mL) and extracted twice with HCl (100 mL). The organic layers were combined and washed with brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated to dryness. The residue was purified by flash column chromatography (eluting at PE / HCl = 100:0 to 0:100) to obtain 11 (3.1 g, yield 68.9%), a yellow solid. LC / MS (ESI) m / z: 579 (M+H) + .

[0119] Step 10: Synthesis of 12 A mixture containing 11 (3.1 g, 5.36 mmol) and K2CO3 (2.22 g, 16.08 mmol) in DMF (50 mL) was mixed with PhSH (1.2 g, 10.72 mmol). The resulting mixture was stirred at 60°C for 2 hours. After cooling, the mixture was diluted with water (150 mL) and extracted twice with RINKAN (100 mL). The organic layers were combined and washed with brine (100 mL), dried over anhydrous Na2SO4, filtered, and concentrated to dryness. The residue was purified by flash column chromatography (eluting at DCM / MeOH = 100:0 to 10:1) to obtain 12 (1.6 g, yield 76.2%), a colorless oil. LC / MS (ESI) m / z: 394 (M+H) + .

[0120] Step 11:13 Synthesis A mixture containing 3 (266 mg, 1.53 mmol) and 12 (600 mg, 1.53 mmol) in DMF (6 mL) was to which DIPEA (394 mg, 3.05 mmol) and HATU (696 mg, 1.83 mmol) were added. The resulting mixture was stirred at room temperature for 2 hours. This reaction mixture was concentrated under vacuum and used directly in the next step. LC / MS (ESI) m / z: 572 (M + Na) + .

[0121] Step 12: Synthesis of (I) A crude mixture containing 13 in MeOH (10 mL) was to which TFA (40 mg, 0.30 mmol) was added. The resulting mixture was stirred at room temperature for 2 hours. The solution was then concentrated under vacuum to dryness, and the residue was purified by preparative HPLC to obtain a white solid (I) (390 mg, 54% yield in 2 steps). LC / MS (ESI) m / z: 466 (M+H) + . 1H δ of NMR (400MHz, MeOD) is 7.49~7.40 (m, 1H), 7.10 (t, J=8.5Hz, 2H), 4.83~4. 75(m, 1H), 4.69~4.57(m, 1H), 4.55~4.48(m, 1H), 4.46~4.21(m, 3H), 3.92~3 .75(m, 3H), 3.52(d, J=32.0Hz, 1H), 3.18~2.94(m, 1H), 2.73~2.63(m, 1H), 2 .62~2.52(m, 3H), 2.45~2.25(m, 2H), 1.04~0.91(m, 2H), 0.88~0.70(m, 2H).

[0122] Example 8: Pharmacokinetic (PK) and absorption, distribution, metabolism, excretion, and toxicity (ADMET) data for the compound of formula (I). Various tests were performed on the compound of formula (I) in in vitro and in vivo PK assays. The results are summarized in Tables 1 and 2. [Table 1] [Table 2]

[0123] Example 9: Pharmacokinetic (PK) and absorption, distribution, metabolism, excretion, and toxicity (ADMET) data of the control compound. Various tests were performed on comparative compounds 1 and 2 using ADMET assays in vitro and in vivo. The results are summarized in Tables 3 and 4. [Table 3] [Table 4]

[0124] The structures of comparative compounds 1 and 2 are as follows. [ka]

[0125] Example 10: Isoleucine transport assay data for SLC6A19 The compound of formula (I) and a control compound were tested using an isoleucine transport assay. The results are summarized in Table 5 below. [Table 5]

[0126] Reference All U.S. patents, U.S. patent application publications, and international patent application publications cited herein are incorporated herein by reference.

[0127] Equal parts Those skilled in the art will recognize, or can confirm by mere ordinary experimentation, many equivalents to the specific embodiments of the present invention described herein. Such equivalents are intended to be included in the following claims.

Claims

1. Compound of formula (I): 【Chemistry 1】 or a pharmaceutically acceptable salt thereof, which may have the absolute stereochemistry shown above, the compound or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition comprising the compound described in claim 1 and a pharmaceutically acceptable excipient.

3. A method for treating or preventing a disease or disorder associated with a genetic deficiency of phenylalanine hydroxylase, comprising administering an effective amount of the compound described in claim 1 to a subject in need thereof.

4. A method for treating or preventing phenylketonuria, comprising administering an effective amount of the compound described in claim 1 to a subject in need thereof.

5. A method for treating or preventing hyperphenylalaninemia, comprising administering an effective amount of the compound described in claim 1 to a subject in need thereof.

6. The method according to any one of claims 3 to 5, wherein the compound reduces the whole-body phenylalanine concentration of the subject.

7. A method for treating or preventing tyrosinemia (type I, type II, or type III), comprising administering an effective amount of the compound described in claim 1 to a subject in need thereof.

8. The method according to claim 7, wherein the compound reduces the whole-body tyrosine concentration of the subject.

9. A method for treating or preventing nonketotic hyperglycinemia, comprising administering an effective amount of the compound described in claim 1 to a subject in need thereof.

10. The method according to claim 9, wherein the compound reduces the whole-body glycine concentration of the subject.

11. A method for treating or preventing isovaleric acidemia, methylmalonic acidemia, propionic acidemia, maple syrup urine disease, DNA JC12 deficiency, urea cycle disorder, or hyperammonemia, comprising administering an effective amount of the compound described in claim 1 to a subject in need thereof.

12. A method for treating or preventing diabetes mellitus, chronic kidney disease, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, metabolic syndrome, obesity-related disorders, or neurodevelopmental disorders and autism spectrum disorders, comprising administering an effective amount of the compound described in claim 1 to a subject in need thereof.

13. The method according to any one of claims 1 to 12, wherein the compound inhibits SLC6A19 in the subject.

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