Treatment of familial adenomatous polyposis with 13-membered macrolides.

Abstract

The present application also provides a method for treating FAP in a patient in need thereof, comprising administering to said patient a compound of Formula I: JPEG2025530776000087.jpg39151 [In the formula, R 9a is H, C 1-6 Alkyl, C 1-6 Alkylene-OH, C 1-6 Alkylene-OC 1-6 Alkyl, C(=O)C 1-6 Alkyl, C 1-6 is selected from the group consisting of alkylene-cycloalkyl, C(=O)cycloalkyl, and C(=O)NH-aryl; and R 10a is heteroaryl. or a pharmaceutically acceptable salt thereof, The method is disclosed.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to methods for treating familial adenomatous polyposis using macrolide compounds. [Background technology]

[0002] Background of the Invention Familial adenomatous polyposis coli (FAP) is characterized by the development of numerous nonmalignant adenomas (100–1,000) in the colon and rectum beginning during adolescence, which, if left untreated, eventually progress to colorectal cancer in middle age. Approximately 40% of patients typically experience clinical symptoms, such as rectal bleeding and associated anemia [1]. The disease is primarily caused by a deficiency in the activity of the familial adenomatous polyposis coli (APC) protein due to a germline mutation in the gene. Mutations are present in 80% of FAP patients and are inherited in an autosomal dominant pattern [2–4]. Mutations can be insertions, deletions, or nonsense mutations that result in loss of function of the APC protein and are primarily located in the 5′ region of the 8.5 kb coding region [5, 6]. Premature stop codons in the APC gene are commonly caused by nonsense mutations (30%) or frameshift mutations (68%), resulting in the synthesis of truncated, dysfunctional proteins and contributing to the majority of FAP patients [2-5]. In addition to colorectal cancer, the absence of functional APC protein has been associated with a predisposition to other diseases, such as desmoid tumors and Turcot syndrome associated with brain tumors [7].

[0003] The APC gene encodes a large 312 kDa protein composed of multiple functional domains that are involved in cell division, adhesion, and cell polarization during embryonic development [8, 9]. The protein's general structure consists of four domains, which allow it to interact with other proteins, such as b-catenin, axin inhibitor protein (AXIN), and microtubule plus-end binding protein (EB1), to form an active complex [8-10]. APC acts as a tumor suppressor gene, negatively regulating the b-catenin / WNT signaling pathway by mediating the degradation of b-catenin in the cytoplasm

[11] . The central region of APC between codons 1284 and 1580 is called the mutation cluster region (MCR), and approximately 60% of all APC mutations occur in this region, most of which are nonsense or frameshift mutations that generate truncated proteins [2, 12, 13]. The MCR region is involved in the ubiquitination of b-catenin, and its loss leads to increased cytoplasmic levels of b-catenin [8, 9]. Subsequently, b-catenin translocates to the nucleus, resulting in increased nuclear levels and increased activation of b-catenin / WNT pathway genes, such as c-myc and other proto-oncogenes [14-16]. Somatic mutations in the APC gene occur in 80% of colon cancers, 30% of which are nonsense [3, 12]. These somatic mutations are known to play an important role in cancer development [12, 17].

[0004] FAP APC min The (multiple intestinal neoplasia) mouse model was developed by exposing C57BL / 6 mice to the mutagen N-ethyl-N-nitrosourea and is a widely used disease model [18, 19]. The pathology is due to a nonsense mutation at codon 850 of the mouse homolog of the APC gene that results in a nonfunctional truncated protein [18, 20]. This mutation is similar to the mutation in the APC gene carried by human FAP patients

[21] . APC min The model is a heterozygous mutant mouse (homozygous APC minThe average lifespan is 119 days, and chronic anemia caused by intestinal polyps is the primary cause of death

[18] . min Mice develop numerous adenomas (approximately 30 / gastrointestinal tract), primarily located in the small intestine [18-20]. min Detectable in mice at 5 weeks of age, these early lesions appear as densely packed crypts with a morphology similar to that observed in low-grade colonic lesions seen in FAP patients [19, 21]. Accumulation of nuclear b-catenin was detected by immunohistochemistry in APC. min This can be observed in mouse adenomas

[19] . Furthermore, disruption of the nuclear translocation of b-catenin reduces the expression of genes that are transcriptionally co-regulated by b-catenin, such as c-myc, and leads to the development of APC. min It reduces polyp formation in mice.

[22]

[0005] Macrolides and other ribosome-targeting antibiotics have been shown to induce readthrough of premature stop codons in eukaryotic cells, leading to translation of full-length mRNA. A study by Zilberg et al.

[23] demonstrated that tylosin can suppress translation termination caused by nonsense mutations in the APC gene using colorectal cancer cell lines and an in vivo colon cancer model. Other studies have shown that gentamicin and erythromycin treatment also suppress nonsense mutations in the APC gene in human colon cancer cells, inhibit the in vivo growth of human colorectal xenografts carrying APC nonsense mutations in a mouse model of FAP, and reduce the number of intestinal polyps [23, 24]. Subsequently, in a clinical study of FAP patients carrying APC nonsense mutations, Kariv et al.

[25] demonstrated that erythromycin resulted in a reduction in adenoma burden. Recent studies using aminoglycosides have shown that these antibiotics promote readthrough of premature stop codons in the APC and cystic fibrosis transmembrane conductance regulator genes, resulting in partial alleviation of the disease state

[23] . In general, the extent of full-length functional protein induced by macrolide- and aminoglycoside-mediated readthrough ranges from 2–10% of normal levels, which has been shown to be sufficient to restore normal cellular function [26, 27]. However, aminoglycoside and macrolide antibiotics that have demonstrated readthrough effects have low efficacy, insufficient exposure to target tissues, and side effects that limit chronic administration, thus hindering their clinical use in FAP treatment [28-30].

[0006] As a result, there remains a need for new macrolide compounds for treating FAP. Summary of the Invention

[0007] Summary of the Invention This need and others are met by a method for treating FAP in a patient in need thereof, comprising administering to said patient a compound of Formula I: [ka] [During the ceremony, R 9a is H or optionally substituted C 1-6 alkyl; and R 10a is an optionally substituted heteroaryl. This is achieved by the present invention which is directed to a method comprising administering to a patient a compound of formula (I) or a pharmaceutically acceptable salt thereof. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 summarizes the antiproliferative activity of compounds of the invention in human colon cancer cells. [Figure 2] FIG. 2 summarizes the pharmacokinetic parameters of compounds of the invention after oral administration at 100 mg / kg bw in CD1 mice. [Figure 3] FIG. 3 shows that treatment with compounds of the invention reduces the nuclear translocation of beta-catenin in colon cancer cells harboring a nonsense mutation in APC. [Figure 4] FIG. 4 shows the reduction of c-myc protein levels in colon cancer cells treated with compounds of the invention. [Figure 5] FIG. 4 shows the long-term survival and reduced anemic phenotype of APCmin mice treated with compounds of the invention. [Figure 6] FIG. 5 shows the reduction in the number of intestinal polyps and dysplastic lesions observed in APCmin mice treated with compounds of the invention. [Figure 7] FIG. 7 shows that compounds of the invention reduce nuclear levels of beta-catenin in epithelial cells of the small intestine. DETAILED DESCRIPTION OF THE INVENTION

[0009] Detailed Description of the Invention definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in practicing or testing the present invention, suitable methods and materials are described below. Furthermore, the materials, methods, and examples are merely illustrative and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety, including U.S. Patent No. 2013 / 0090326. In case of conflict, the present specification, including these definitions, will control.

[0010] As used herein, the terms "singular," "a," "an," and similar expressions include not only embodiments having one member but also embodiments having multiple members. Thus, singular, "a," "an," and similar expressions can mean one or more, unless indicated to the contrary or otherwise clear from the context. A claim or description including "or" between one or more members of a group is deemed satisfied when one, more than one, or all of the members of the group are present in, employed in, or otherwise relevant to a given product or process, unless indicated to the contrary or otherwise clear from the context. The invention includes embodiments in which only one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one or all of the members of the group are present in, employed in, or otherwise relevant to a given product or process.

[0011] As used herein, the term "about" means "approximately" and is used to modify a numerical value and indicate a defined range around that value. When "X" is a value, "about X" typically refers to a value from 0.95X to 1.05X. Reference to "about X" specifically refers to values ​​of at least X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, "about X" is intended to provide instruction and descriptive support for a claim limitation, such as "0.98X." When the quantity "X" includes only integer values ​​(e.g., "X carbons"), "about X" refers to (X-1) to (X+1). In this case, "about X" as used herein specifically refers to at least the values ​​X, X-1, and X+1.

[0012] When "about" is applied to the beginning of a numerical range, it applies to both ends of the range. Thus, "about 5 to 20%" is equivalent to "about 5% to about 20%." When "about" is applied to the first value in a set of numerical values, it applies to every value in that set. Thus, "about 7, 9, or 11%" is equivalent to "about 7%, about 9%, or about 11%."

[0013] The following abbreviations and terms have the indicated meanings throughout: [Table 1-1] [Table 1-2]

[0014] The symbol "-" means a single bond, "=" means a double bond, and "≡" means a triple bond. [ka] means a single or double bond. [ka] refers to a group on a double bond occupying either position on the end of the double bond to which the symbol is attached; i.e., the geometry of the double bond, E- or Z-, is ambiguous. When a group is shown removed from its parent structural formula, the symbol "~" is used at the end of the theoretically broken bond to separate the group from its parent structural formula.

[0015] When chemical structures are shown or described, unless otherwise explicitly stated, all carbon atoms are assumed to be hydrogen-substituted to match tetravalent.For example, in the structure on the left side of the schematic diagram below, nine hydrogen atoms are implied.These nine hydrogen atoms are shown in the structure on the right side.A specific atom in a structural formula may be described in the structural formula as a letter, with hydrogen atoms as substitutions (explicitly defined hydrogen atoms), for example, -CH2CH2-.Those skilled in the art will understand that the above-mentioned description technique is common in the chemical field to simplify and simplify the description of complex structures. [ka]

[0016] For example, the following expression: [ka] As used herein, when a group "R" is shown as "floating" on a ring system, unless otherwise defined, the substituent "R" may be present on any atom of the ring system and may be assumed to replace a shown, implied, or explicitly defined hydrogen from one of the ring atoms so long as a stable structure is created.

[0017] For example, the following expression: [ka] As in, when the group "R" is shown as floating on a fused or bridged ring system, unless otherwise defined, the substituent "R" can be present on any atom of the fused or bridged ring system and can be assumed to replace a shown hydrogen (e.g., -NH- in the formula above), an implied hydrogen (e.g., in the formula above when a hydrogen is not shown but is understood to be present), or an explicitly defined hydrogen (e.g., in the formula above when "Z" equals =CH-) from one of the ring atoms so long as a stable structure is formed. In the example shown, the "R" group can be present on either the 5-membered or 6-membered ring of the fused or bridged ring system.

[0018] When the group "R" is shown as being on a ring system containing saturated carbons, for example, as in the formula: [ka] (wherein, in this example, "y" can be a number greater than 1), each intended to replace a hydrogen on the ring indicated, implied, or explicitly defined therein; unless otherwise defined, two "R"s can be present on the same carbon when the resulting structure is stable. In another example, two Rs on the same carbon can form a ring with that carbon, thereby forming, for example, a ring of the following formula: [ka] Spiro ring structures can be formed with the depicted rings such as in:

[0019] As used herein, the term "alkyl" includes an aliphatic hydrocarbon chain that may be straight or branched. The chain may contain the indicated number of carbon atoms: for example, C1 to C6. 10indicates that the group can have 1 to 10 (inclusive) carbon atoms in it. Unless otherwise specified, alkyl groups contain 1 to about 20 carbon atoms. In some embodiments, alkyl groups have 1 to about 10 carbon atoms. In some embodiments, alkyl groups ("lower alkyl") have 1 to 8, 1 to 6, or 1 to 3 carbon atoms in the chain. Examples can include, but are not limited to, methyl, ethyl, propyl, isopropyl (iPr), 1-butyl, 2-butyl, isobutyl (iBu), tert-butyl, pentyl, 2-methylbutyl, 1,1-dimethylpropyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, cyclopentyl, or cyclohexyl.

[0020] An alkyl group can be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms (e.g., 1 to 4, 1 to 2, or 1) of the alkyl group can be independently substituted with a moiety selected from the group consisting of chloro, fluoro, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio. In some embodiments, the alkyl group is unsubstituted or not optionally substituted.

[0021] The term "aryl" as used herein includes cyclic aromatic carbocyclic ring systems containing 6 to 18 carbons. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl, tetracenyl, biphenyl, and phenanthrenyl.

[0022] An aryl group can be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms (e.g., 1 to 5, 1 to 2, or 1) of the aryl group can be independently replaced with a moiety selected from the group consisting of alkyl, cyano, acyl, halo, haloalkyl, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio. In some embodiments, an alkoxy group is unsubstituted or not optionally substituted.

[0023] As used herein, the term "arylalkyl" or "aralkyl" includes an alkyl group, as defined herein, in which at least one hydrogen substituent is replaced with an aryl group, as defined herein. Examples include, but are not limited to, benzyl, 1-phenylethyl, 4-methylbenzyl, and 1,1,-dimethyl-1-phenylmethyl.

[0024] Arylalkyl group or aralkyl group can be unsubstituted or optionally substituted for each of its constituent groups.For example, but not limited to, the aryl group of arylalkyl group can be substituted, for example, as in 4-methylbenzyl.In some embodiments, the group is unsubstituted or not optionally substituted, especially when it contains defined substituents such as hydroxyalkyl or alkylaminoalkoxy group.

[0025] As used herein, "fluoroalkyl" includes alkyl groups in which the alkyl group contains one or more fluoro substituents. Examples include, but are not limited to, trifluoromethyl.

[0026] As used herein, a "geminal" substitution includes two or more substituents directly attached to the same atom. An example is 3,3-dimethyl substitution on a cyclohexyl or spirocyclohexyl ring.

[0027] As used herein, "halo" or "halogen" includes fluoro, chloro, bromo and iodo.

[0028] The term "heteroaryl" or "heterocycloaryl" includes fully unsaturated or partially unsaturated monocyclic and bicyclic groups of about 4 to about 14 ring atoms (e.g., 4 to 10 or 5 to 10 atoms) containing at least one heteroatom. Heteroatoms as used in the term heteroaryl refer to oxygen, sulfur, and nitrogen. The nitrogen atom of a heteroaryl is optionally oxidized to the corresponding N-oxide. Examples include, but are not limited to, pyrazinyl, furanyl, thienyl, pyridyl, pyrimidinyl, isoxazolyl, isothiazolyl, oxazolyl, thiazolyl, pyrazolyl, furazanyl, pyrrolyl, pyrazolyl, triazolyl, 1,2,4-thiadiazolyl, pyrazinyl, pyridazinyl, quinoxalinyl, phthalazinyl, imidazo[1,2-a]pyridine, imidazo[2,1-b]thiazolyl, benzofurazanyl, indolyl, azaindolyl, benzimidazolyl, benzothienyl, quinolinyl, imidazolyl, thienopyridyl, quinazolinyl, thienopyrimidyl, pyrrolopyridyl, imidazopyridyl, isoquinolinyl, benzoazaindolyl, 1,2,4-triazinyl, and benzothiazolyl. Other examples include [ka] and the like.

[0029] As used herein, the term "heteroarylene" or "heterocycloarylene" includes di-substituted heteroaryl groups.

[0030] Heteroaryl groups can be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms (e.g., 1 to 5, 1 to 2, or 1) of the heteroaryl group can be independently substituted with a moiety selected from the group consisting of alkyl, cyano, acyl, halo, haloalkyl, hydroxy, oxo, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio. In some embodiments, heteroaryl groups are unsubstituted or not optionally substituted.

[0031] As used herein, the term "heteroaroyl" includes heteroaryl-C(O)- groups, where heteroaryl is as defined herein. Heteroaroyl groups include, but are not limited to, thiophenoyl, nicotinoyl, pyrrol-2-ylcarbonyl, and pyridinoyl.

[0032] The term "heterocycloalkyl" may be used interchangeably herein and as used herein includes heterocyclyl-C(O)- groups, where heterocyclyl is as defined herein. Examples include, but are not limited to, N-methylprolinoyl and tetrahydrofuranoyl.

[0033] As used herein, "heterocyclyl" (heterocyclo; heterocyclic; heterocycloalkyl) includes a non-aromatic saturated ring of about 3 to about 8 ring atoms (e.g., 5 to about 10 ring atoms, or 3 to about 6 ring atoms), in which one or more of the atoms in the ring system is an element other than carbon, e.g., nitrogen, oxygen, or sulfur. A heterocyclyl group optionally contains at least one sp 2It contains a hybrid atom (e.g., a ring incorporating a carbonyl, an endocyclic olefin, or an exocyclic olefin). In some embodiments, the nitrogen or sulfur atom of the heterocyclyl is optionally oxidized to the corresponding N-oxide, S-oxide, or S,S-dioxide. Monocyclic heterocycle refers to a 3-, 4-, 5-, 6-, 7-, or 8-membered ring containing at least one heteroatom independently selected from the group consisting of O, N, and S. A 3- or 4-membered ring contains zero or one double bond and one heteroatom selected from the group consisting of O, N, and S. A 5-membered ring contains zero or one double bond and one, two, or three heteroatoms selected from the group consisting of O, N, and S. A 6-membered ring contains zero, one, or two double bonds and one, two, or three heteroatoms selected from the group consisting of O, N, and S. The 7- and 8-membered rings contain zero, one, two, or three double bonds, and one, two, or three heteroatoms selected from the group consisting of O, N, and S. Representative examples of monocyclic heterocycles include azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolin ... These include, but are not limited to, thiazolinyl, pyridazin-3(2H)-onyl, pyridin-2(1H)-onyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidethiomorpholinyl (thiomorpholinesulfone), thiopyranyl, and trithianyl.

[0034] As used herein, the term "heterocycloalkylene" includes di-substituted heterocyclyl (heterocyclo; heterocyclic) groups.

[0035] The term "heterocyclyl" also includes polycyclic rings, such as bicyclic or tricyclic heterocycles, which may be fused, bridged, or spiro-oriented. A bicyclic heterocycle is a monocyclic heterocycle fused to a phenyl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle, or a bridged monocyclic heterocyclic ring system in which two non-adjacent atoms of the ring are joined by an alkylene bridge of 1, 2, 3, or 4 carbon atoms or an alkenylene bridge of 2, 3, or 4 carbon atoms. Representative examples of bicyclic heterocycles include, but are not limited to, 3-azabicyclo[3.1.0]hexane, 3-azabicyclo[4.1.0]heptane, 3-azabicyclo[3.2.0]heptane, (3aR,6aS)-hexahydro-1H-2λ2-cyclopenta[c]pyrrole, and (3aR,7aS)-octahydro-2λ2-isoindole.

[0036] A tricyclic heterocycle is exemplified by a bicyclic heterocycle fused to a phenyl group, or a bicyclic heterocycle fused to a monocyclic cycloalkyl, or a bicyclic heterocycle fused to a monocyclic cycloalkenyl, or a bicyclic heterocycle fused to a monocyclic heterocycle, or a bicyclic heterocycle in which two non-adjacent atoms of the bicyclic ring are joined by an alkylene bridge of 1, 2, 3, or 4 carbon atoms or an alkenylene bridge of 2, 3, or 4 carbon atoms.

[0037] Heterocyclyl groups can be unsubstituted or optionally substituted. When optionally substituted, one or more hydrogen atoms (e.g., 1 to 4, 1 to 2, or 1) of the group can be independently replaced with a moiety selected from the group consisting of alkyl, halo, haloalkyl, oxo, acetyl, hydroxy, alkoxy, amino, alkylamino, acylamino, thio, and alkylthio. In some embodiments, substituted heterocyclyl groups can incorporate exocyclic or endocyclic alkenes (e.g., cyclohex-2-en-1-yl). In some embodiments, heterocyclyl groups are unsubstituted or not optionally substituted.

[0038] Monocyclic, bicyclic, and tricyclic heterocycles are attached to the parent molecule through any carbon atom or any nitrogen atom contained within the ring and can be unsubstituted or substituted.

[0039] As used herein, the term "hydrophilic moiety" or "hydrophilic group" includes moieties or functional groups that have a strong affinity for water. Examples can include, but are not limited to, charged moieties, such as cationic or anionic moieties, or polar uncharged moieties, such as alkoxy or amine groups.

[0040] As used herein, the term "hydroxyalkyl" includes an alkyl group in which at least one hydrogen substituent is replaced with an alcohol (-OH) group. In certain embodiments, a hydroxyalkyl group has one alcohol group. In certain embodiments, a hydroxyalkyl group has one or two alcohol groups, each on a different carbon atom. In certain embodiments, a hydroxyalkyl group has one, two, three, four, five, or six alcohol groups. Examples can include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, and 1-hydroxyethyl.

[0041] When any two substituents or any two instances of the same substituent are "independently selected" from a list of alternatives, the groups can be the same or different. For example, R a and R b are independently selected from the group consisting of alkyl, fluoro, amino, and hydroxyalkyl, two R a group and two R b A molecule having groups can have all groups be alkyl groups (e.g., four different alkyl groups). Alternatively, the first R a can be alkyl and the second R a can be fluoro and the first R b can be hydroxyalkyl, and the second R b can be amino (or any other substituent selected from the group). Alternatively, both R a and the first R b can be fluoro, while the second R b can be alkyl (i.e., some pairs of substituents can be the same and other pairs can be different).

[0042] An "amino-protecting group" is a protecting group suitable for preventing undesired reactions at an amino nitrogen. Representative amino-protecting groups include, but are not limited to, formyl; acyl groups, e.g., alkanoyl groups such as acetyl; alkoxycarbonyl groups such as tert-butoxycarbonyl (Boc); arylmethoxycarbonyl groups such as benzyloxycarbonyl (Cbz) and 9-fluorenylmethoxycarbonyl (Fmoc); arylmethyl groups such as benzyl (Bn), trityl (Tr), and 1,1-di-(4'-methoxyphenyl)methyl; silyl groups such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBDMS); and the like.

[0043] A "hydroxyl protecting group" is a protecting group suitable for preventing undesired reactions at hydroxyl oxygen. Exemplary hydroxyl protecting groups include, but are not limited to, acyl groups such as acetyl; arylmethyl groups such as benzyl (Bn), trityl (Tr) and 1,1-di-(4'-methoxyphenyl)methyl; silyl groups such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBDMS); ethers such as methoxymethyl (MOM), tetrahydropyranyl (THP) and benzyl (Bn); and the like.

[0044] The "yield" for each reaction described herein is given as a percentage of the theoretical yield.

[0045] "Subject" and "patient" are used interchangeably. A "subject" or "patient" for the purposes of this invention includes humans and other animals, particularly mammals, and other organisms. Thus, the methods are applicable to both human therapy and veterinary applications. In a specific embodiment, the patient is a mammal, and in a more specific embodiment, the patient is human.

[0046] A "pharmaceutically acceptable salt" of a compound means a salt that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound. It is understood that pharmaceutically acceptable salts are non-toxic. Further information on suitable pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 17, incorporated herein by reference. th ed., Mack Publishing Company, Easton, PA, 1985, or S. M. Berge, et al., "Pharmaceutical Salts," J. Pharm. Sci., 1977;66:1-19, both of which are incorporated herein by reference.

[0047] Examples of pharmaceutically acceptable acid addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; as well as acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, 3-(4-hydroxybenzoyl)benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzoic ... These include those formed with organic acids such as benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, p-toluenesulfonic acid, and salicylic acid.

[0048] Examples of pharmaceutically acceptable base addition salts include those formed when an acidic proton present in the parent compound is replaced by a metal ion, such as, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum salts. Specific salts are ammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins. Examples of organic bases include, but are not limited to, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, tromethamine, N-methylglucamine, and polyamine resins, etc. Exemplary organic salts are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0049] A "therapeutically effective amount" is an amount of a compound of the present invention that, when administered to a patient, ameliorates the symptoms of a disease. The amount of a compound of the present invention that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the age of the patient being treated, and the like. One of ordinary skill in the art, having regard to their knowledge and this disclosure, can routinely determine a therapeutically effective amount.

[0050] The term "genetic disease" as used herein means a genetic disorder, genetic disease, genetic condition or genetic syndrome.

[0051] "Prevention" of a disease, disorder, or syndrome includes inhibiting the occurrence of the disease in humans, i.e., preventing the clinical symptoms of the disease, disorder, or syndrome from developing in animals that may be exposed to or predisposed to the disease, disorder, or syndrome but have not yet experienced or exhibited the disease, disorder, or syndrome.

[0052] "Treating" a disease, disorder, or syndrome, as used herein, includes (i) inhibiting the disease, disorder, or syndrome, i.e., arresting its progression; and (ii) alleviating the disease, disorder, or syndrome, i.e., causing regression of the disease, disorder, or syndrome. As known in the art, adjustments for systemic versus local delivery, age, weight, general health, sex, diet, time of administration, drug interactions, and severity of the condition may be necessary and ascertainable with routine experimentation by one of ordinary skill in the art.

[0053] "FAP" refers to an autosomal dominant condition characterized by the formation of numerous adenomatous polyps, primarily in the epithelium of the large intestine. These polyps are initially benign, but if left untreated, they can transform into colon cancer. Three variants are known to exist: FAP, attenuated FAP (originally called hereditary flat adenomatous polyposis syndrome), and autosomal recessive FAP (originally called MUTYH-associated polyposis). FAP and attenuated FAP are caused by defects in the APC gene on chromosome 5, while autosomal recessive FAP is caused by defects in the MUTYH gene on chromosome 1. Of the three, FAP itself is the most severe and common; however, in all three, the resulting colon polyps and cancer are initially confined to the colon wall. Detection and removal before they spread outside the colon can significantly reduce and often eliminate the cancer's spread.

[0054] Implementation In one embodiment, there is provided a method for treating FAP in a patient in need thereof, comprising administering to said patient a compound of Formula I: [ka] [During the ceremony, R 9a is H or optionally substituted C 1-6 alkyl; and R 10a is an optionally substituted heteroaryl. or a pharmaceutically acceptable salt thereof, A method is provided.

[0055] In one embodiment, R 9a is H.

[0056] In another embodiment, R 9a is an optionally substituted C 1-6 It is alkyl.

[0057] In another embodiment, R 9a is selected from optionally substituted methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, and hexyl.

[0058] In another embodiment, R 9a is selected from methyl, ethyl, propyl, butyl, and isobutyl.

[0059] In another embodiment, R 10a teeth, [ka] and optionally substituted heteroaryl selected from the group consisting of:

[0060] In a further embodiment, R 9a is selected from H, methyl, ethyl, propyl, butyl, and isobutyl, and R 10a teeth, [ka] and a bicyclic heteroaryl selected from the group consisting of:

[0061] In another embodiment, there is provided a method for treating FAP in a patient in need thereof, comprising administering to said patient a compound of Formula II: [ka] [During the ceremony, R 9a is H or optionally substituted C 1-6 alkyl; and [ka] is an optionally substituted 6-membered aromatic or heteroaromatic ring. or a pharmaceutically acceptable salt thereof, A method is provided.

[0062] In one embodiment, R 9a is H.

[0063] In another embodiment, R 9a is an optionally substituted C 1-6 It is alkyl.

[0064] In another embodiment, R 9a is selected from optionally substituted methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, and hexyl.

[0065] In another embodiment, R 9a is selected from methyl, ethyl, propyl, butyl, and isobutyl.

[0066] In another embodiment, [ka] is an optionally substituted aromatic ring.

[0067] In another embodiment, [ka] is optionally substituted [ka] is.

[0068] In another embodiment, [ka] is an optionally substituted heteroaromatic ring.

[0069] In another embodiment, [ka] is optionally substituted [ka] is.

[0070] In a further embodiment, R 9a is selected from H, methyl, ethyl, propyl, butyl, and isobutyl; and [ka] teeth, [ka] (All of which may be optionally substituted.) is selected from the group consisting of:

[0071] Compounds of the present invention include those shown in Table A or a pharmaceutically acceptable salt thereof. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0072] Methods for preparing compounds The compounds disclosed herein can be prepared as provided in WO2020 / 106627, the entire contents of which are incorporated herein by reference, or as described herein.

[0073] The compound is prepared via two intermediates. The eastern intermediate is of formula P-1: [ka] In the compound of formula P-1, R 3 , R 4a , R 4b , R 5 , R 6a , R 6b , R 8a , and R 8b is as defined herein; G 4 is the expression: [ka] and; R 15is independently at each occurrence silyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl, or two R 15 groups are joined to form an optionally substituted heterocyclyl or heteroaryl ring; and R 16a is independently at each occurrence hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0074] The uncyclized eastern half intermediate has formula P-2: [ka] [During the ceremony, PG is a hydroxyl protecting group; R 4a , R 4b , R5, R 6a , R 6b , R 8a , and R 8b is as defined herein; G 4 is the expression: [ka] and; R 15is independently at each occurrence silyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl, or two R 15 groups are joined to form an optionally substituted heterocyclyl or heteroaryl ring; and R 16a is independently at each occurrence hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl. or a salt thereof.

[0075] In some embodiments, -OPG is -OBz.

[0076] Formula P-3: [ka] wherein the variables are as defined herein. or a salt thereof is also disclosed.

[0077] Coupling and macrolactonization In certain embodiments, compounds of the present disclosure are those having R s is a sugar residue: [ka] [During the ceremony, PG is a hydroxyl protecting group, [ka] indicates the point of attachment. The compound is prepared by coupling a compound of formula P1 (east half), where: with a compound of formula P-4 (west half) to provide the uncyclized compound precursor of formula P-5. [ka]

[0078] As shown in the scheme below, formula P-5 is cyclized to give compounds of formula I after deprotection of the sugar residue. [ka]

[0079] Alternatively, as shown in the following scheme, R 9a is hydrogen to provide a compound of formula I, which can be subjected to reductive amination to afford a compound of formula I. 9a Compounds of formula I, wherein is other than H, can be provided. [ka]

[0080] R in later stages 2b Introduction of the group can be achieved by treating the compound of formula P-6 provided above with a base and an appropriate electrophilic group (e.g., a halogenating agent or R2-LG, where LG is a leaving group), as shown in the following scheme: In this method, the sugar residue in P-6 is protected and R 9a is H or alkyl. [ka]

[0081] For all intermediates, the variables are as defined herein for compounds of Formula I.

[0082] Other variables shown for the intermediates and precursors are defined as follows: LG is a leaving group; G 4 is the expression: [ka] and; R 15 is independently at each occurrence silyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl, or two R 15 groups join to form an optionally substituted heterocyclyl or heteroaryl ring; and R 16a is independently at each occurrence hydrogen, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl.

[0083] As mentioned above, R s is the sugar part: [ka] The sugar moiety is generally attached to the compound structure during synthesis of the eastern half, but can also be attached at other stages of production. The sugar moiety can be attached by a chemical or enzymatic glycosylation reaction between the hydroxyl group at the C5 position and a glycosyl donor. In certain embodiments, the sugar moiety is attached to the compound structure as a thioglycoside. In certain embodiments, the substituent of the sugar moiety is modified after glycosylation of the compound or compound precursor (e.g., the eastern half).

[0084] method In certain embodiments, the present invention provides methods for treating FAP, attenuated FAP (originally called hereditary flat adenomatous polyposis syndrome), and autosomal recessive FAP (originally called MUTYH-associated polyposis), comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound of formula I.

[0085] In one embodiment, the FAP is a FAP.

[0086] In another embodiment, the FAP is attenuated FAP.

[0087] In another embodiment, the FAP is autosomal recessive FAP.

[0088] In a further aspect, the present invention provides a method for treating a genetic disease or disorder caused by a defect in the APC gene on chromosome 5, comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound of formula I.

[0089] In a further aspect, the present invention provides a method for treating a genetic disease or disorder caused by a mutation in the APC gene on chromosome 5, comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound of formula I.

[0090] In these and other embodiments, the APC mutation is at codon 1309. In another embodiment, the APC mutation is at codons 1250 to 1464. In a further embodiment, the mutation is at codons 168 to 1580. In a further embodiment, the mutation is 5' of codon 168 and 3' of 1580.

[0091] In these and other embodiments, the mutation is in the 5' portion of the APC gene (codons 1-177) and the terminal 3' portion of the gene. In further embodiments, the mutation is an interstitial deletion of chromosome 5q22. In further embodiments, the mutation is in the 5' end of the region spanning exon 4 and exon 5. In further embodiments, the mutation is in the 5' end of exon 9.

[0092] In a further aspect, the present invention provides a method for treating a genetic disease or disorder caused by a mutation in the MUTYH gene on chromosome 1, comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound of formula I.

[0093] In a further aspect, the present invention provides a method for treating a genetic disease or disorder caused by a genetic defect in the MUTYH gene on chromosome 1, comprising administering to a subject in need of such treatment a therapeutically effective amount of a compound of formula I.

[0094] Pharmaceutical Compositions and Administration The present invention provides a pharmaceutical composition comprising the compound of the present invention and a pharmaceutically acceptable additive.In certain embodiments, the compound of the present invention is provided in an effective amount in the pharmaceutical composition.In certain embodiments, the effective amount is a therapeutically effective amount.In certain embodiments, the effective amount is a prophylactically effective amount.

[0095] Pharmaceutically acceptable excipients include any and all solvents, diluents, or other liquid vehicles, dispersants, suspending aids, surfactants, tonicity agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc., appropriate for the particular dosage form desired. General considerations in the formulation and / or manufacture of pharmaceutical compositions can be found, for example, in Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) and Remington: The Science and Practice of Pharmacy, 21st Edition (Lippincott Williams & Wilkins, 2005).

[0096] The pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology. Generally, such preparations include combining a compound of the present invention (the "active ingredient") with a carrier and / or one or more other accessory ingredients, and, if necessary and / or desired, shaping and / or packaging the product into a desired single- or multi-dosage unit. Pharmaceutical compositions may be prepared, packaged, and / or sold in bulk, as a single unit dose and / or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient will generally be equal to the dosage of the active ingredient that would be administered to a subject and / or a convenient fraction of such a dosage, such as, for example, one-half or one-third of such a dosage.

[0097] The relative amounts of the active ingredient, pharmaceutically acceptable additives, and / or any additional ingredients in a pharmaceutical composition of the present invention will vary depending on the identity, size, and / or condition of the subject being treated, and further depending on the route by which the composition is administered. By way of example, the composition may contain 0.1% to 100% (w / w) of the active ingredient.

[0098] Pharmaceutically acceptable additives used in the preparation of the provided pharmaceutical compositions include inert diluents, dispersing and / or granulating agents, surfactants and / or emulsifying agents, disintegrating agents, binders, preservatives, buffers, lubricants, and / or oils. Additives such as cocoa butter and suppository waxes, coloring agents, coating agents, sweeteners, flavoring agents, and perfuming agents may also be present in the compositions.

[0099] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and mixtures thereof.

[0100] Exemplary granulating and / or dispersing agents include potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation exchange resins, calcium carbonate, silicic acid, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, and mixtures thereof.

[0101] Exemplary surfactants and / or emulsifiers include natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and Veegum [magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylhexyl hydroxybenzoate ... ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulose derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60], polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate [Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span 65], glyceryl monooleate, sorbitan monooleate [Span 80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [Myrj 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., Cremophor), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [Brij30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or mixtures thereof.

[0102] Exemplary binders include starch (e.g., corn starch and starch paste), gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.), natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum), and larch arabinogalactan (larch arabogalactan), alginic acid, polyethylene oxide, polyethylene glycol, inorganic calcium salts, silicic acid, polymethacrylic acid, wax, water, alcohol, and / or mixtures thereof.

[0103] Exemplary preservatives include antioxidants, chelating agents, antibacterial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and other preservatives.

[0104] Exemplary antioxidants include alpha tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and sodium sulfite.

[0105] Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA) and its salts and hydrates (e.g., edetate sodium, edetate disodium, edetate trisodium, edetate calcium disodium, and edetate dipotassium), citric acid and its salts and hydrates (e.g., citric acid monohydrate), fumaric acid and its salts and hydrates; malic acid and its salts and hydrates; phosphoric acid and its salts and hydrates; and tartaric acid and its salts and hydrates. Exemplary antimicrobial preservatives include benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.

[0106] Exemplary antifungal preservatives include butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and sorbic acid.

[0107] Exemplary alcohol preservatives include ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoic acid, and phenylethyl alcohol.

[0108] Exemplary acidic preservatives include vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and phytic acid.

[0109] Other preservatives include tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben, Germall 115, Germaben II, Neolone, Kathon, and Euxyl.In certain embodiments, the preservative is an antioxidant.In other embodiments, the preservative is a chelating agent.

[0110] Exemplary buffering agents include citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.

[0111] Exemplary lubricants include magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and mixtures thereof.

[0112] Exemplary natural oils include almond, apricot kernel, avocado, babassu, bergamot, blackcurrant seed, borage, cade, chamomile, canola, caraway, carnauba, castor bean, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cottonseed oil, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazelnut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, and lavender. Examples of suitable synthetic oils include, but are not limited to, butyl stearate, caprylic / capric triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and mixtures thereof.

[0113] Liquid dosage forms for oral and parenteral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (such as cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitan fatty acid esters, and mixtures thereof.In addition to inert diluents, oral compositions may contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavorings and fragrances. In certain embodiments for parenteral administration, the conjugates of the invention are mixed with a solubilizing agent such as Cremophor, alcohol, oil, modified oil, glycol, polysorbate, cyclodextrin, polymer, and mixtures thereof.

[0114] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated using suitable dispersing or wetting agents and suspending agents according to techniques known in the art. Sterile injectable preparations can be sterile injectable solutions, suspensions, or emulsions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, USP, and isotonic sodium chloride solution. Additionally, sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, bland fixed oils, including synthetic monoglycerides or diglycerides, can be used. Additionally, fatty acids such as oleic acid are also used in the preparation of injectables. Injectable preparations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents into the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable media prior to use.

[0115] Sterile injectable compositions, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated according to techniques known in the art using suitable dispersing or wetting agents (e.g., Tween 80) and suspending agents. Sterile injectable preparations can also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as solvents or suspending media (e.g., synthetic monoglycerides or diglycerides). Fatty acids, such as oleic acid and its glyceride derivatives, are useful in the preparation of injectables, as are pharmaceutically acceptable natural oils, such as olive oil or castor oil, especially their polyoxyethylated versions. These oil solutions or suspensions can also contain long-chain alcohol diluents or dispersants, or carboxymethylcellulose or similar dispersing agents. Other commonly used surfactants such as Tween or Span, or other similar emulsifiers or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms, may also be used for formulation purposes.

[0116] To prolong the effect of a drug, it is often desirable to delay the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of crystalline or amorphous substances with poor water solubility. The rate of drug absorption depends on the dissolution rate, which may depend on the size and crystalline form of the crystals. Alternatively, delayed absorption of parenterally administered drug forms can be achieved by dissolving or suspending the drug in an oil vehicle.

[0117] Compositions for rectal or vaginal administration are generally suppositories, which can be prepared by mixing a conjugate of the invention with a suitable non-irritating excipient or carrier, such as cocoa butter, polyethylene glycol, or a suppository wax, which is solid at ambient temperature but liquid at body temperature, and therefore will melt in the rectum or vaginal cavity and release the active ingredient.

[0118] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; b) binders such as, for example, carboxymethylcellulose, alginic acid, gelatin, polyvinylpyrrolidinone, sucrose, and acacia; c) humectants such as glycerol; d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicic acids, and sodium carbonate; e) solution retarding agents such as paraffin; f) absorption accelerators such as quaternary ammonium compounds; g) humectants such as, for example, cetyl alcohol and glycerol monostearate; h) absorbents such as kaolin and bentonite clay; and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may comprise buffering agents.

[0119] Solid compositions of a similar type can be used as fillers in soft- and hard-filled gelatin capsules using additives such as lactose or milk sugar and high molecular weight polyethylene glycols. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. These can optionally contain opacifying agents and can be of a composition that releases the active ingredient only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed release manner. Examples of embedding compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type can be used as fillers in soft- and hard-filled gelatin capsules using additives such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0120] Orally available pharmaceutical compositions include push-fit capsules made of gelatin and soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol. Push-fit capsules may contain the active ingredient mixed with a filler such as lactose, a binder such as starch, a lubricant such as talc or magnesium stearate, and optionally a stabilizer. In soft capsules, the compounds described herein may be dissolved or suspended in a suitable liquid such as fatty oils, liquid paraffin, or liquid polyethylene glycol. Additionally, stabilizers may be added. All formulations for oral administration are in dosages appropriate for the selected administration route.

[0121] The sugar-coated core is provided with a suitable coating. For this purpose, concentrated sugar solutions, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures, can be used. Dyes or pigments can be added to the tablets or sugar-coated coatings for identification or to characterize various combinations of aminoglycoside compound effective doses.

[0122] The active ingredient may be in microencapsulated form with one or more additives, as described above. Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. In such solid dosage forms, the active ingredient may be mixed with at least one inert diluent, such as sucrose, lactose, or starch. These dosage forms may, as is customary, contain additional substances other than inert diluents, such as tableting lubricants and other tableting aids, such as magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets, and pills, the dosage forms may contain buffering agents. These may optionally contain opacifying agents and may be of a composition that releases the active ingredient only or preferentially in a certain part of the intestinal tract, optionally with delayed release. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0123] Dosage forms for topical and / or transdermal administration of the compounds of the present invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and / or patches. Generally, the active ingredient is mixed under sterile conditions with a pharmaceutically acceptable carrier and / or any necessary preservatives and / or required buffers. In addition, the use of transdermal patches is contemplated, which often has the additional advantage of providing controlled delivery of the active ingredient to the body. Such dosage forms can be prepared, for example, by dissolving and / or dispersing the active ingredient in a suitable medium. Alternatively or additionally, the rate can be controlled by providing a rate-controlling membrane and / or dispersing the active ingredient in a polymer matrix and / or gel.

[0124] Devices suitable for use in delivering the intradermal pharmaceutical compositions described herein include short needle devices such as those described in U.S. Patent Nos. 4,886,499, 5,190,521, 5,328,483, 5,527,288, 4,270,537, 5,015,235, 5,141,496, and 5,417,662. Intradermal compositions can be administered using devices that limit the effective needle penetration length into the skin, such as the devices described in PCT Publication WO 99 / 34850 and their functional equivalents. Jet injection devices that deliver liquid vaccines to the dermis via a liquid jet injector and / or via a needle that provides a jet that penetrates the stratum corneum and reaches the dermis are suitable. Jet injection devices are described, for example, in U.S. Pat. Nos. 5,480,381, 5,599,302, 5,334,144, 5,993,412, 5,649,912, 5,569,189, 5,704,911, 5,383,851, 5,893,397, 5,466,220, and U.S. Pat. Nos. 5,339,163, 5,312,335, 5,503,627, 5,064,413, 5,520,639, 4,596,556, 4,790,824, 4,941,880, 4,940,460, and PCT Publications WO 97 / 37705 and WO 97 / 13537. A suitable ballistic powder / particle delivery device uses compressed gas to accelerate a powdered vaccine through the outer layer of the skin to the dermis. Alternatively or additionally, a conventional syringe can be used for the classic Mantoux technique of intradermal administration.

[0125] Pharmaceutical compositions of the invention can be prepared, packaged, and / or sold as formulations suitable for pulmonary administration via the buccal cavity. Such formulations can comprise dry particles containing the active ingredient and having diameters ranging from about 0.5 to about 7 nanometers or from about 1 to about 6 nanometers. Such compositions are conveniently in the form of a dry powder for administration using a device containing a dry powder reservoir capable of directing a stream of propellant to disperse the powder and / or for administration using a self-propelled solvent / powder dispensing container, such as a device containing the active ingredient dissolved and / or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles in which at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions can include a solid fine powder diluent, such as sugar, and are conveniently provided in a unit dose form.

[0126] Low-boiling propellants generally include liquid propellants having a boiling point below 65°F at atmospheric pressure. Generally, the propellant may comprise 50-99.9% (w / w) of the composition, and the active ingredient may comprise 0.1-20% (w / w) of the composition. The propellant may further comprise additional components such as a liquid nonionic surfactant and / or a solid anionic surfactant and / or a solid diluent (which may have a particle size on the same order as the particles containing the active ingredient).

[0127] Pharmaceutical compositions of the present invention formulated for pulmonary delivery may provide the active ingredient in the form of droplets of a solution and / or suspension. Such formulations may be prepared, packaged, and / or sold as, optionally sterile, aqueous and / or dilute alcoholic solutions and / or suspensions containing the active ingredient, and may be conveniently administered using any nebulizer and / or atomizer device. Such formulations may further contain one or more additional ingredients, including, but not limited to, flavorings such as sodium saccharin, volatile oils, buffers, surfactants, and / or preservatives such as methyl hydroxybenzoate. The droplets provided by this route of administration may have an average diameter in the range of about 0.1 to about 200 nanometers.

[0128] Formulations described herein as useful for pulmonary delivery are also useful for intranasal delivery of the pharmaceutical compositions of the invention. Another formulation suitable for intranasal administration is a coarse powder containing the active ingredient and having an average particle size of about 0.2 to 500 micrometers. Such formulations are administered by rapid inhalation through the nasal passages from a powder container held close to the nostrils.

[0129] Formulations for nasal administration may contain, for example, from as little as about 0.1% (w / w) to as much as 100% (w / w) of the active ingredient, and may include one or more additional ingredients described herein. Pharmaceutical compositions of the invention may be prepared, packaged, and / or sold as formulations for buccal administration. Such formulations may be, for example, in the form of tablets and / or lozenges prepared using conventional methods and may contain, for example, 0.1 to 20% (w / w) of the active ingredient, with the remainder comprising an orally dissolvable and / or orally disintegrable composition, and optionally one or more additional ingredients described herein. Alternatively, formulations for buccal administration may comprise a powder and / or aerosolized and / or atomized solution and / or suspension comprising the active ingredient. Such powdered, aerosolized, and / or aerosolized formulations, when dispersed, may have an average particle and / or droplet size in the range of about 0.1 to about 200 nanometers and may further comprise one or more additional ingredients described herein.

[0130] Although the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to animals of all kinds. Modifications of pharmaceutical compositions suitable for administration to humans to make them suitable for administration to a variety of animals are well understood, and those skilled in the art of veterinary pharmacology can design and / or implement such modifications with routine experimentation.

[0131] The compound provided herein is generally formulated in unit dosage form for ease of administration and uniform dosage.However, it is understood that the total daily use amount of the composition of the present invention is determined by attending physician within the scope of sound medical judgment.The specific therapeutically effective dose level for any specific subject or organism depends on various factors, including the disease, disorder or symptom and severity of disorder being treated, the activity of the specific active ingredient used, the specific composition used, the age, weight, general health condition, sex and diet of subject, the administration time, administration route and excretion rate of the specific active ingredient used, treatment period, the drug used in combination with or simultaneously with the specific active ingredient used, and similar factors well known in the medical field.

[0132] To carry out the method of the present invention, the compound or its pharmaceutical composition can be administered intravenously, intravitreally, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intraosseously, periprosthetically, topically, intramuscularly, subcutaneously, mucosally, intraosseously, periprosthetically, intrauterinely, orally, topically, locally, via inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by local perfusion of direct immersion in target cells, via catheter, via lavage, in cream, in lipid composition (e.g., liposome), or by other methods known to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences, 2003, incorporated herein by reference), or by any combination of the above. Generally, the most suitable administration route depends on various factors, including the nature of the drug (e.g., its stability in the gastrointestinal environment) and / or the condition of the subject (e.g., whether the subject can tolerate oral administration).

[0133] In certain embodiments, pharmaceutical compositions and / or additional agents are formulated to be administered via digestive route.Digestive route includes all possible administration routes that allow compositions to directly contact with the digestive tract.Specifically, pharmaceutical compositions disclosed herein can be administered orally, bucally, rectally or sublingually.Therefore, these compositions can be formulated with inert diluents or assimilable edible carriers, or can be enclosed in hard or soft gelatin capsules, compressed into tablets, or can be directly incorporated into food.

[0134] In another embodiment, the compositions described herein can be administered via parenteral route.As used herein, the term "parenteral" includes a route that bypasses the digestive tract.Specifically, the pharmaceutical compositions disclosed herein can be administered, for example, but not limited to, intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneously, or intraperitoneally.

[0135] In some embodiments, administration is oral. For oral administration, the compounds described herein can be easily formulated by combining the compounds with pharmaceutically acceptable carriers known in the art. Such carriers allow the compounds described herein to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like for oral ingestion by patients. Pharmaceutical formulations for oral use can be prepared using solid additives, optionally milling the resulting mixture, and processing the granular mixture into tablets or dragee cores, after adding suitable additives as needed. Suitable additives include, inter alia, fillers such as sugars including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carbomethylcellulose; and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0136] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0137] For administration by inhalation, the compounds described herein are conveniently delivered in aerosol spray presentation form (generally containing powder, liquefied and / or gaseous carrier) from pressurized pack or nebulizer, using suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide.In the case of pressurized aerosol, dosage unit can be determined by providing a valve to deliver a metered amount.Capsules and cartridges such as gelatin for use in inhaler or insufflator can be formulated to contain a powder mixture of the compounds described herein and a suitable powder base, for example, but not limited to, lactose or starch.

[0138] For administration by injection, the compounds described herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer, with or without organic solvents such as propylene glycol, polyethylene glycol, and the like.

[0139] Pharmaceutical compositions for topical administration may include compositions formulated for medicinal use, such as ointments, pastes, creams, or powders. Ointments include all oil-based, adsorbent, emulsion, and water-soluble base compositions for topical application, while creams and lotions are compositions containing only an emulsion base. Topically administered drugs may contain a penetration enhancer to promote absorption of the active ingredient through the skin. Suitable penetration enhancers include glycerin, alcohol, alkyl methyl sulfoxides, pyrrolidone, and laurocapram. Possible bases for topical compositions include polyethylene glycol, lanolin, cold cream, and petrolatum, as well as any other suitable absorption base, emulsion base, or water-soluble ointment base. Topical formulations may contain emulsifiers, gelling agents, and antimicrobial preservatives as needed to preserve the composition and provide a uniform mixture. Transdermal administration of a composition may also involve the use of a "patch." For example, a patch may deliver one or more compositions continuously at a predetermined rate over a period of time.

[0140] In certain embodiments, composition can be delivered by eye drops, nasal spray, inhalation, and / or other aerosol delivery vehicles.The method for directly delivering composition to lungs via nasal aerosol spray is described in US Patent No. 5,756,353 and US Patent No. 5,804,212 (each of which is specifically incorporated herein by reference in its entirety).Similarly, drug delivery using intranasal microparticle resin (Takenaga et al., 1998) and lysophosphatidylglycerol compound (US Patent No. 5,725,871, specifically incorporated herein by reference in its entirety) is also well known in the pharmaceutical field, and can be used to deliver the compositions described herein.Similarly, transmucosal drug delivery in the form of polytetrafluoroethylene support matrix is ​​described in US Patent No. 5,780,045 (specifically incorporated herein by reference in its entirety), and can be used to deliver the compositions described herein.

[0141] Furthermore, it is envisioned that the compositions disclosed herein can be delivered via aerosol. The term aerosol refers to a colloidal system of finely divided solid or liquid particles dispersed in a liquefied or pressurized gas propellant. A typical inhalation aerosol consists of a suspension of the active ingredient in a liquid propellant, or a mixture of a liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. The appropriate container varies depending on the propellant pressure requirements. The administration of aerosols varies depending on the subject's age, weight, symptom severity, and symptom response.

[0142] For transmucosal administration, penetrants are used in the formulation. Such penetrants are generally known in the art.

[0143] The compounds described herein can be formulated for parenteral administration, for example, by bolus injection or continuous infusion.Injectable preparations can be provided in unit dosage form, for example, in ampoules or multi-dose containers, optionally containing preservatives.The composition can be a suspension, solution or emulsion in an oily or aqueous medium, and can contain formulating agents such as suspending agents, stabilizing agents and / or dispersing agents.

[0144] Alternatively, the compounds described herein may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use.

[0145] The exact amount of compound required to achieve an effective dose varies from subject to subject, depending on, for example, the species, age and general condition of subject, the severity of side effects or disorders, the identity of specific compound, and administration method.Desired dosage can be delivered 3 times a day, 2 times a day, once a day, every other day, every 3 days, every week, every 2 weeks, every 3 weeks, or every 4 weeks.In certain embodiments, desired dosage can be delivered by multiple administrations (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more administrations).

[0146] In certain embodiments, an effective amount of compound for administration to a 70 kg adult once or more times per day can be in the range of about 0.0001 mg to about 3000 mg of compound, about 0.0001 mg to about 2000 mg, about 0.0001 mg to about 1000 mg, about 0.001 mg to about 1000 mg, about 0.01 mg to about 1000 mg, about 0.1 mg to about 1000 mg, about 1 mg to about 1000 mg, about 1 mg to about 100 mg, about 10 mg to about 1000 mg, or about 100 mg to about 1000 mg of compound per unit dosage form.

[0147] In certain embodiments, the compounds of the invention may be administered orally or parenterally, one or more times daily, at a dosage level sufficient to deliver about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 50 mg / kg, preferably about 0.1 mg / kg to about 40 mg / kg, preferably about 0.5 mg / kg to about 30 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, and more preferably about 1 mg / kg to about 25 mg / kg of subject body weight per day to obtain the desired therapeutic effect.

[0148] It is understood that the dosage ranges described herein provide guidance for administering the pharmaceutical compositions provided to adults. The amount administered to children, adolescents, etc. can be determined by a physician or person skilled in the art and may be less than or the same as the amount administered to adults.

[0149] It is also understood that the compound or composition described herein can be administered in combination with one or more additional therapeutically active agents.Compound or composition can be administered in combination with additional therapeutically active agents to improve their bioavailability, reduce and / or modify their metabolism, inhibit their excretion, and / or modify their biodistribution.It is also understood that the treatments used can achieve the desired effect for the same disorder and / or achieve different effects.

[0150] The compound or composition may be administered simultaneously with, before, or after one or more additional therapeutically active agents. Generally, each agent is administered at a dose and / or time schedule determined for that agent. It is further understood that the additional therapeutically active agents utilized in this combination may be administered together in a single composition or separately in different compositions. The specific combination used in a regimen will take into account the compatibility of the compound of the present invention with the additional therapeutically active agents and / or the desired therapeutic effect to be achieved. Generally, it is expected that the additional therapeutically active agents utilized in combination will be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than the levels utilized individually. Additional therapeutically active agents include antibiotics, such as antibiotics useful for treating tuberculosis. Exemplary antibiotics include, but are not limited to, isoniazid, rifampin, pyrazinamide, ethambutol, and streptomycin.

[0151] Kits (e.g., pharmaceutical packs) are also encompassed by the present invention. The provided kits may include a pharmaceutical composition or compound of the present invention and a container (e.g., a vial, an ampoule, a bottle, a syringe, and / or a dispenser package, or other suitable container). In some embodiments, the provided kits may optionally further include a second container containing pharmaceutical additives for diluting or suspending the pharmaceutical composition or compound of the present invention. In some embodiments, the pharmaceutical composition or compound of the present invention provided in the container and the second container are combined to form a single-unit dosage form. [Example]

[0152] In order to more fully understand the invention described herein, the following examples are provided. The synthetic and biological examples described herein are provided to illustrate the compounds, pharmaceutical compositions, and methods provided herein, and should not be construed as limiting the scope thereof in any way. The compounds were prepared as described in WO2020 / 106627 and as described below.

[0153] Intermediate Scheme 6 [ka]

[0154] [ka] tert-Butyl (R)-(1,5-dihydroxypentan-2-yl)carbamate (IS6-1) A suspension of LiAlH4 (1.0 M in THF, 800 mL, 800 mmol, 4.2 equiv.) was added dropwise to a solution of Boc-D-Glu-OBzl (64.1 g, 190 mmol, 1.0 equiv.) in anhydrous tetrahydrofuran (474 ​​mL) at 0 °C. The internal temperature was maintained below 10 °C. After the addition was complete, the reaction mixture was stirred at 0 °C for 0.5 h and then warmed to room temperature for 1 h. The mixture was cooled to 0 °C and quenched by the careful addition of water (30.4 mL), 3N NaOH solution (38.4 mL), and water (84 mL). The mixture was dried over Na2SO4, and the precipitate was removed by filtration. The precipitate was washed with EtOAc, and the combined filtrate was concentrated under reduced pressure to give IS6-1. This material was used in the next step without further purification.

[0155] [ka] tert-Butyl (R)-4-(3-hydroxypropyl)-2,2-dimethyloxazolidine-3-carboxylate (IS6-2) To a solution of compound IS6-1 (41.6 g, 189 mmol, 1.0 equiv) in anhydrous methylene chloride (240 mL) was added 2,2-dimethoxypropane (231 mL, 1.89 mol, 10 equiv) at 25 °C. Then, TsOH·HO (3.59 g, 18.9 mmol, 0.1 equiv) was added in one portion. The reaction mixture was stirred at 25 °C for 4 h. The mixture was partitioned between EtOAc and saturated aqueous NaHCO. The organic layer was washed with brine, dried over NaSO, and concentrated. The mixture was purified by silica gel chromatography (40% EtOAc in heptane) to give 20.13 g (41% over two steps) of compound IS6-2. 1 H NMR (400 MHz, chloroform-d) δ 4.04–3.91 (m, 2H), 3.79–3.63 (m, 4H), 1.68–1.54 (m, 4H), 1.49 (s, 15H).

[0156] [ka] tert-Butyl (R)-2,2-dimethyl-4-(3-oxopropyl)oxazolidine-3-carboxylate (IS6-3) To a solution of compound IS6-2 (20.13 g, 77.5 mmol, 1.0 equiv.) in DCM (155 mL) was added DMSO (44.0 mL, 620 mmol, 8.0 equiv.), followed by Hunig's base (53.9 mL, 310 mmol, 4.0 equiv.), and the mixture was cooled to 0 °C. To this mixture, SO₃·Pyr (24.6 g, 155 mmol, 2.0 equiv.) was added portionwise while maintaining the internal temperature below 5 °C. The reaction mixture was stirred at 0–5 °C for 1 h. MTBE and brine (500 mL + 500 mL) were added in batches and stirred for approximately 10–15 min. The organic layer was separated and washed with brine (4 times). The final organic layer was dried over sodium sulfate and concentrated to give crude product IS6-3, which was used in the next step without further purification.

[0157] [ka] tert-Butyl (R)-4-(3-(7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propyl)-2,2-dimethyloxazolidine-3-carboxylate (IS6-4) To a solution of compound IS6-3 (19.9 g, 77.3 mmol, 1.0 equiv) in DCM (154 mL) was added compound A (12.5 g, 92.7 mmol, 1.2 equiv), followed by AcOH (4.85 mL, 85.0 mmol, 1.1 equiv) and cooled to 0 °C. To this mixture was added NaBH(OAc) (24.3 g, 115 mmol, 1.5 equiv) in portions, maintaining the internal temperature below 5 °C. The reaction mixture was stirred at 25 °C for 16 h. Saturated aqueous NaHCO was added to the reaction mixture. The organic layer was separated, and the aqueous layer was extracted with DCM (3 times). The combined organic layers were dried over sodium sulfate and concentrated under reduced pressure. The mixture was purified by silica gel chromatography (0–3–5% MeOH in DCM containing NHOH) to give 28.32 g (97.2% yield) of compound IS6-4.

[0158] [ka] (R)-2-Amino-5-(7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)pentan-1-ol hydrochloride (IS6-5) Compound IS6-4 (28.32 g, 75.1 mmol, 1.0 equiv.) was dissolved in MeOH (150 mL) and HCl (4 M in dioxane, 93.7 mL, 375 mmol, 5.0 equiv.) was added at room temperature. The reaction mixture was stirred at room temperature for 4 h, at which point UPLC showed complete conversion. The reaction mixture was concentrated under reduced pressure to give 22.9 g (100% crude yield) of compound IS6-5.

[0159] [ka] (R)-2-Amino-5-(7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)pentan-1-ol (I-12) Amberlyst A26(OH) (200 g, >0.8 eq / L) was added to MeOH (500 mL) under mechanical stirring, and the mixture was stirred for 30 minutes. The solvent was removed by filtration, and the same procedure was repeated four times. Compound 5 (22.9 g, 1.0 eq) was dissolved in MeOH (500 mL) and added to the washed resin at room temperature. The reaction mixture was stirred at room temperature for 30 minutes. After filtration, the solution was collected, and the resin was washed three times with MeOH (500 mL) until UPLC showed that the desired product was not present in the solution. The combined organic solution was concentrated under reduced pressure to give the free base (16.97 g, 96.0% yield). 1 H NMR (400 MHz, CDCl3) δ 8.99 (s, 1H), 8.41 (s, 1H), 3.66 - 3.57 (m, 3H), 3.51 (s, 2H), 3.32 (dd, 1H), 3.03 (t, 2H), 2.93 - 2.81 (m, 3H), 2.64 - 2.54 (m, 2H), 1.80 - 1.43 (m, 4H).

[0160] Scheme 6 [ka]

[0161] [ka] (2S,3R,4S,6R)-4-(Dimethylamino)-2-(((3S,6R,8R,9R,10R)-3-(3-hydroxypropyl)-8-methoxy-4,6,8,10,12,12-hexamethyl-11,13-dioxo-1-oxa-4-azacyclotridecan-9-yl)oxy)-6-methyltetrahydro-2H-pyran-3-yl benzoate (S6-1-I1-1) To S2-1-I1-1 (240 mg, 0.372 mmol) in dry THF (3.71 mL) was added 9-BBN (0.5 M THF solution, 2.22 mL, 1.11 mmol). After 30 min at room temperature, the mixture was cooled to 0 °C, and NaOH (6 N aqueous solution, 371 μL, 2.23 mmol) and HO (30% aqueous solution, 252 μL, 2.23 mmol) were added. After 15 min, the mixture was extracted three times with t-butyl methyl ether / EtOAc (2:1). The organic layer was washed once with water and once with brine and dried over NaSO. After removal of the solvent, the residue was purified on 4 g of silica gel (eluted with a 0-20% MeOH-dichloromethane / 0.5% NHOH gradient) to give the title compound (145 mg, 59%). MS (ESI+) m / z: 663.37 [M + H]+; 1H NMR (400 MHz, chloroform-d) δ 8.08 - 7.94 (m, 2H), 7.55 (dd, 1H), 7.44 (t, 2H), 5.03 (dd, 1H), 4.57 (d, 1H), 4.10 (dd, 1H), 4.01 (d, 1H), 3.95 (dd, 1H), 3.72 - 3.50 (m, 3H), 3.41 (dt, 1H), 3.04 (s, 1H), 2.87 - 2.81 (m, 1H), 2.80 (s, 3H), 2.32 (dd, 1H), 2.26 (s, 6H), 2.10 (t, 1H), 1.93 (d, 1H), 1.83 - 1.47 (m, 10H), 1.40 (s, 4H), 1.31 - 1.22 (m, 9H), 1.16 - 1.07 (m, 1H), 1.03 (d, 3H), 0.91 (d, 3H).

[0162] [ka] (2S,3R,4S,6R)-4-(Dimethylamino)-2-(((3S,6R,8R,9R,10R)-8-methoxy-4,6,8,10,12,12-hexamethyl-11,13-dioxo-3-(3-oxopropyl)-1-oxa-4-azacyclotridecan-9-yl)oxy)-6-methyltetrahydro-2H-pyran-3-yl benzoate (S6-2-I1-1) To S6-1-I1-1 (145 mg, 218 mmol) in dry dichloromethane / CH3CN (9:1, 2.9 mL) was added activated 4A molecular sieves (100 mg, powder), N-methylmorpholine N-oxide (33 mg, 283 mmol), and tetrapropylammonium perruthenate (4 mg, 10.9 mmol). After 1 h at room temperature, the solvent was removed. The dry residue was dissolved in t-butyl methyl ether / hexane (1:1) and filtered through Celite® (3 times). After removing the solvent, the residue was dried under vacuum to give the aldehyde as a white foam. MS (ESI+) m / z: 661.35 [M + H]+. This was used directly in the next step.

[0163] compound 143 [ka] (3S,6R,8R,9R,10R)-9-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-3-(3-(isopropyl(methyl)amino)propyl)-8-methoxy-4,6,8,10,12,12-hexamethyl-1-oxa-4-azacyclotridecane-11,13-dione (S6-3-I1-1-1) A mixture of S6-2-I1-1 (25 mg, 37.8 mmol) and methylisopropylamine (8 mg, 113 mmol) in dichloromethane (2 mL) was stirred for 30 min, after which NaBH(OAc) (12 mg, 56.7 mmol) was added. After 20 min, the solvent was removed, and the residue was dissolved in MeOH (2 mL) and heated at 50 °C overnight. The reaction was allowed to cool to room temperature and concentrated. The residue was purified by HPLC (Atlantis T3 column, 5-50% MeCN-water-0.1% HCOH) to give 8.6 mg of the title compound as the formate salt. MS (ESI+) m / z: 614.48 [M + H]+; 1H NMR (400 MHz, methanol-d4) δ 4.45 (d, 1H), 4.28 (d, 1H), 4.20 (d, 1H), 3.88 - 3.65 (m, 2H), 3.56 (hept, 1H), 3.51 - 3.23 (m, 4H), 3.05 (t, 7H), 2.79 (s, 8H), 2.72 (s, 3H), 2.17 (s, 1H), 2.02 (ddd, 1H), 1.84 (d, 4H), 1.67 - 1.41 (m, 7H), 1.45 - 1.19 (m, 19H), 1.05 (d, 3H).

[0164] compound 158 [ka] (3S,6R,8R,9R,10R)-9-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-3-(3-(isoindolin-2-yl)propyl)-8-methoxy-4,6,8,10,12,12-hexamethyl-1-oxa-4-azacyclotridecane-11,13-dione (S6-3-I1-1-16) Prepared from isoindoline according to method S6-3-I1-1-1, the title compound was obtained as the formate salt. (ESI+) m / z: 660.29 [M + H]+; 1H NMR (400 MHz, methanol-d4) δ 7.27 (p, 4H), 4.46 (d, 1H), 4.26 (dd, 2H), 4.11 (s, 4H), 3.89 - 3.64 (m, 2H), 3.53 - 3.27 (m, 5H), 3.18 - 2.91 (m, 9H), 2.80 (s, 7H), 2.20 (s, 1H), 2.10 - 1.92 (m, 2H), 1.75 (ddd, 3H), 1.47 (d, 6H), 1.44 - 1.29 (m, 12H), 1.06 (d, 3H).

[0165] compound 166 [ka] (3S,6R,8R,9R,10R)-3-(3-(3,4-dihydroisoquinolin-2(1H)-yl)propyl)-9-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-8-methoxy-4,6,8,10,12,12-hexamethyl-1-oxa-4-azacyclotridecane-11,13-dione (S6-3-I1-1-24) Prepared from 1,2,3,4-tetrahydroisoquinoline according to the method in S6-3-I1-1-1, the title compound was obtained as the formate salt. MS (ESI+) m / z: 674.33 [M + H]+; 1H NMR (400 MHz, methanol-d4) δ 7.28 - 6.98 (m, 4H), 4.45 (d, 1H), 4.27 (dd, 2H), 3.98 - 3.62 (m, 4H), 3.55 - 3.34 (m, 4H), 3.19 - 2.85 (m, 11H), 2.77 (s, 10H), 2.18 (d, 1H), 2.01 (ddd, 2H), 1.91 - 1.65 (m, 3H), 1.65 - 1.44 (m, 6H), 1.44 - 1.25 (m, 12H), 1.05 (d, 3H).

[0166] compound 167 [ka] (3S,6R,8R,9R,10R)-3-(3-(3,4-dihydro-2,7-naphthyridin-2(1H)-yl)propyl)-9-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-8-methoxy-4,6,8,10,12,12-hexamethyl-1-oxa-4-azacyclotridecane-11,13-dione (S6-3-I1-1-25) Prepared from 1,2,3,4-tetrahydro-2,7-naphthyridine according to the method in S6-3-I1-1-1, the title compound was obtained as the formate salt. MS (ESI+) m / z: 675.28 [M + H]+; 1H NMR (400 MHz, methanol-d4) δ 8.32 (s, 1H), 8.26 (d, 1H), 7.16 (s, 1H), 4.45 (d, 1H), 4.27 (dd, 2H), 3.89 - 3.64 (m, 4H), 3.52 - 3.32 (m, 4H), 2.99 (dd, 9H), 2.89 - 2.75 (m, 9H), 2.66 (t, 2H), 2.20 (s, 1H), 2.03 (ddd, 2H), 1.89 - 1.64 (m, 3H), 1.63 - 1.45 (m, 6H), 1.45 - 1.26 (m, 12H), 1.05 (d, 3H).

[0167] compound 173 [ka] (3S,6R,8R,9R,10R)-3-(3-(7,8-dihydro-1,6-naphthyridin-6(5H)-yl)propyl)-9-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-ethyl-8-methoxy-6,8,10,12,12-pentamethyl-1-oxa-4-azacyclotridecane-11,13-dione (S6-3-I1-2-5) Prepared from S2-1-I1-2 and 5,6,7,8-tetrahydro-1,6-naphthyridine according to the method of S6-3-I1-1-1, the title compound was obtained as the formate salt. MS (ESI+) m / z: 689.36 [M + H]+; 1H NMR (400 MHz, MeOD-d4): δ 8.42 (s, 3H), 8.33 (dd, 1H), 7.67 - 7.46 (m, 1H), 7.30 - 7.06 (m, 1H), 4.59 (t, 1H), 4.51 - 4.37 (m, 2H), 4.25 (d, 1H), 4.00 (s, 1H), 3.77 (s, 2H), 3.75 - 3.65 (m, 1H), 3.55 (s, 1H), 3.47 - 3.32 (m, 3H), 3.12 (d, 2H), 2.99 (td, 7H), 2.89 - 2.82 (m, 1H), 2.81 (d, 6H), 2.76 - 2.66 (m, 2H), 2.14 (s, 1H), 2.07 - 1.98 (m, 1H), 1.94 (s, 1H), 1.87 - 1.69 (m, 3H), 1.56 - 1.47 (m, 1H), 1.44 (s, 3H), 1.40 - 1.23 (m, 16H), 1.04 (d, 3H).

[0168] compound 174 [ka] (3S,8R,9R,10R)-3-(3-(7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propyl)-9-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-ethyl-8-methoxy-8,10,12,12-tetramethyl-1-oxa-4-azacyclotridecane-11,13-dione (S6-3-I1-2-6) Prepared from S2-1-I1-2 and 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine according to the method of S6-3-I1-1-1, the title compound was obtained as the formate salt. MS (ESI+) m / z: 690.33 [M + H]+; 1H NMR (400 MHz, MeOD-d4): δ 8.90 (s, 1H), 8.51 (s, 1H), 8.46 (s, 2H), 4.60 (t, 1H), 4.45 (dd, 2H), 4.26 (d, 1H), 3.99 (s, 1H), 3.71 (s, 3H), 3.55 (s, 1H), 3.50 - 3.33 (m, 3H), 3.25 - 3.06 (m, 2H), 3.00 (d, 5H), 2.95 - 2.83 (m, 3H), 2.81 (s, 6H), 2.68 (tt, 2H), 2.18 (d, 1H), 2.08 - 1.99 (m, 1H), 1.95 (s, 1H), 1.88 - 1.68 (m, 3H), 1.58 - 1.49 (m, 1H), 1.45 (s, 3H), 1.42 - 1.20 (m, 16H), 1.04 (d, 3H).

[0169] compound 183 [ka] (3S,6R,8R,9R,10R)-9-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-3-(3-(isoindolin-2-yl)propyl)-8-methoxy-6,8,10,12,12-pentamethyl-4-propyl-1-oxa-4-azacyclotridecane-11,13-dione (S6-3-I1-3-3) Prepared from S2-1-I1-3 and isoindoline according to the method of S6-3-I1-1-1 to give 11.9 mg of the title compound as the formate salt. MS (ESI+) m / z: 230.1 [M + 3H]3+, 344.7 [M + 2H]2+, 688.3 [M + H]+; 1H NMR (400 MHz, methanol-d4) δ 8.54 (s, 2H), 7.26 (s, 4H), 4.69 - 4.20 (m, 3H), 4.20 - 3.86 (m, 6H), 3.69 (dd, 1H), 3.65 - 3.51 (m, 1H), 3.48 - 3.34 (m, 2H), 3.28 - 3.09 (m, 2H), 3.00 (s, 2H), 2.96 - 2.78 (m, 4H), 2.78 - 2.51 (m, 7H), 2.51 - 2.10 (m, 2H), 2.00 - 1.89 (m, 2H), 1.87 - 1.58 (m, 5H), 1.58 - 1.19 (m, 19H), 1.14 - 0.82 (m, 7H).

[0170] compound 184 [ka] (3S,8R,9R,10R)-3-(3-(cyclopropyl(methyl)amino)propyl)-9-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-8-methoxy-8,10,12,12-tetramethyl-4-propyl-1-oxa-4-azacyclotridecane-11,13-dione (S6-3-I1-3-4) Prepared from S2-1-I1-3 and N-methylcyclopropanamine according to the method of S6-3-I1-1-1 to give the title compound as the formate salt. MS (ESI+) m / z: 640.03 [M + H]+; 1H NMR (400 MHz, methanol-d4): δ 4.57 (s, 1H), 4.42 (dd, 2H), 4.25 (s, 1H), 3.90 (s, 1H), 3.78 - 3.64 (m, 1H), 3.53 - 3.32 (m, 3H), 3.22 (d, 2H), 3.08 - 2.91 (m, 3H), 2.81 (s, 9H), 2.53 (s, 4H), 2.18 (s, 1H), 2.11 - 1.88 (m, 3H), 1.73 (s, 4H), 1.61 - 1.20 (m, 19H), 1.06 (t, 6H), 0.78 - 0.46 (m, 4H).

[0171] compound 185 [ka] (3S,6R,8R,9R,10R)-3-(3-(7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propyl)-9-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-8-methoxy-6,8,10,12,12-pentamethyl-4-propyl-1-oxa-4-azacyclotridecane-11,13-dione (S6-3-I1-3-5) Prepared from S2-1-I1-3 and 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine according to the method of S6-3-I1-1-1 to give the title compound as the formate salt. MS (ESI+) m / z: 703.98 [M + H]+; 1H NMR (400 MHz, methanol-d4): δ 8.91 (s, 1H), 8.52 (s, 1H), 4.57 (d, 1H), 4.46 (dd, 2H), 4.25 (s, 1H), 3.98 (s, 1H), 3.81 - 3.64 (m, 3H), 3.51 - 3.33 (m, 3H), 3.17 (s, 2H), 3.01 (d, 5H), 2.94 - 2.85 (m, 3H), 2.82 (s, 7H), 2.69 (hept, 2H), 2.18 (s, 1H), 2.09 - 1.89 (m, 3H), 1.76 (ddt, 4H), 1.57 - 1.25 (m, 19H), 1.09 - 0.92 (m, 6H).

[0172] compound 189 [ka] (3S,6R,8R,9R,10R)-3-(3-(3,4-dihydroisoquinolin-2(1H)-yl)propyl)-9-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-isobutyl-8-methoxy-6,8,10,12,12-pentamethyl-1-oxa-4-azacyclotridecane-11,13-dione (S6-3-I1-4-3) Prepared from S2-1-I1-4 and 1,2,3,4-tetrahydroisoquinoline according to the method of S6-3-I1-1-1, the title compound was obtained as the formate salt. MS (ESI+) m / z: 716.32 [M + H]+; 1H NMR (400 MHz, methanol-d4) δ 7.41 - 6.89 (m, 4H), 4.66 - 4.40 (m, 2H), 4.27 - 4.01 (m, 3H), 4.01 - 3.62 (m, 3H), 3.62 - 3.30 (m, 3H), 3.15 (dd, 5H), 3.05 - 2.67 (m, 11H), 2.66 - 2.01 (m, 6H), 2.01 - 1.49 (m, 10H), 1.49 - 1.25 (m, 12H), 1.25 - 1.02 (m, 5H), 1.02 - 0.77 (m, 5H).

[0173] Compound 198 [ka] (3R,6R,8R,9R,10R)-3-(3-(7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propyl)-9-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-8-methoxy-6,8,10,12,12-pentamethyl-4-propyl-1-oxa-4-azacyclotridecane-11,13-dione (S6-3-I2-3-1) Prepared according to the method of S6-3-I1-1-1 from (2S,3R,4S,6R)-4-(dimethylamino)-2-(((3R,6R,8R,9R,10R)-8-methoxy-6,8,10,12,12-pentamethyl-11,13-dioxo-3-(3-oxopropyl)-4-propyl-1-oxa-4-azacyclotridecan-9-yl)oxy)-6-methyltetrahydro-2H-pyran-3-yl benzoate and 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine to provide the title compound as the formate salt. MS (ESI+) m / z: 704.04 [M + H]+; 1H NMR (400 MHz, methanol-d4) δ 8.93 (s, 1H), 8.54 (s, 1H), 4.45 (d, 1H), 4.41 - 4.10 (m, 2H), 3.73 (q, 4H), 3.57 - 3.16 (m, 6H), 3.08 - 2.87 (m, 9H), 2.84 (s, 6H), 2.80 - 2.63 (m, 2H), 2.18 - 1.60 (m, 8H), 1.61 - 1.44 (m, 6H), 1.42 - 1.22 (m, 13H), 1.01 - 0.85 (m, 6H).

[0174] compound 200 [ka] (3R,6R,8R,9R,10R)-9-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-3-(3-(isoindolin-2-yl)propyl)-8-methoxy-6,8,10,12,12-pentamethyl-4-propyl-1-oxa-4-azacyclotridecane-11,13-dione (S6-3-I2-3-3) Prepared according to the method of S6-3-I1-1-1 from (2S,3R,4S,6R)-4-(dimethylamino)-2-(((3R,6R,8R,9R,10R)-8-methoxy-6,8,10,12,12-pentamethyl-11,13-dioxo-3-(3-oxopropyl)-4-propyl-1-oxa-4-azacyclotridecan-9-yl)oxy)-6-methyltetrahydro-2H-pyran-3-yl benzoate and isoindoline to provide the title compound as the formate salt. MS (ESI+) m / z: 688.27 [M + H]+; 1H NMR (400 MHz, methanol-d4) δ 7.29 (t, 4H), 4.46 (d, 1H), 4.18 (s, 6H), 3.72 (ddt, 1H), 3.61 - 3.31 (m, 5H), 2.98 (d, 8H), 2.79 (s, 7H), 2.02 (ddd, 2H), 1.82 (s, 7H), 1.51 (d, 5H), 1.44 - 1.23 (m, 13H), 1.00 (t, 6H).

[0175] Compound 202 [ka] (3R,6R,8R,9R,10R)-3-(3-(3,4-dihydroisoquinolin-2(1H)-yl)propyl)-9-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-8-methoxy-6,8,10,12,12-pentamethyl-4-propyl-1-oxa-4-azacyclotridecane-11,13-dione (S6-3-I2-3-5) Prepared according to the method of S6-3-I1-1-1 from (2S,3R,4S,6R)-4-(dimethylamino)-2-(((3R,6R,8R,9R,10R)-8-methoxy-6,8,10,12,12-pentamethyl-11,13-dioxo-3-(3-oxopropyl)-4-propyl-1-oxa-4-azacyclotridecan-9-yl)oxy)-6-methyltetrahydro-2H-pyran-3-yl benzoate and 1,2,3,4-tetrahydroisoquinoline to give the title compound as the formate salt. MS (ESI+) m / z: 234.8 [M + 3H]3+, 351.8 [M + 2H]2+, 702.5 [M + H]+; 1 H NMR (400 MHz, methanol-d4) δ 6.91 - 6.82 (m, 1H), 6.76 (dd, 1H), 6.48 (d, 1H), 6.38 (td, 1H), 4.25 (d, 1H), 4.08 (dd, 1H), 3.87 (d, 1H), 3.77 (t, 1H), 3.55 (ddt, 1H), 3.46 (dtt, 1H), 3.16 (dt, 5H), 2.88 (d, 1H), 2.65 (d, 3H), 2.62 (s, 1H), 2.62 - 2.41 (m, 3H), 2.24 (s, 6H), 2.17 (d, 1H), 1.95 (dd, 1H), 1.88 - 1.78 (m, 2H), 1.65 (ddd, 1H), 1.55 (s, 1H), 1.54 - 1.47 (m, 3H), 1.41 (s, 3H), 1.39 - 1.31 (m, 1H), 1.23 (s, 3H), 1.21 - 1.09 (m, 10H), 0.93 - 0.77 (m, 4H), 0.72 (d, 3H).

[0176] compound 205 [ka] (3R,6R,8R,9R,10R)-3-(3-(5,7-dihydro-6H-pyrrolo[3,4-d]pyrimidin-6-yl)propyl)-9-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-8-methoxy-6,8,10,12,12-pentamethyl-4-propyl-1-oxa-4-azacyclotridecane-11,13-dione (S6-3-I2-3-8) Prepared according to the method of S6-3-I1-1-1 from (2S,3R,4S,6R)-4-(dimethylamino)-2-(((3R,6R,8R,9R,10R)-8-methoxy-6,8,10,12,12-pentamethyl-11,13-dioxo-3-(3-oxopropyl)-4-propyl-1-oxa-4-azacyclotridecan-9-yl)oxy)-6-methyltetrahydro-2H-pyran-3-yl benzoate and 6,7-dihydro-5H-pyrrolo[3,4-d]pyrimidine to give the title compound as the formate salt. MS (ESI+) m / z: 230.8 [M + 3H]3+, 345.8 [M + 2H]2+, 690.5 [M + H]+; 1 H NMR (400 MHz, methanol-d4) δ 9.03 (s, 1H), 8.66 (s, 1H), 8.50 (s, 2H), 4.46 (d, 1H), 4.21 (dd, 2H), 4.06 - 3.98 (m, 3H), 3.74 (ddt, 1H), 3.51 (dd, 1H), 3.42 (ddd, 2H), 3.00 (s, 2H), 2.95 (t, 2H), 2.84 (s, 3H), 2.10 - 2.01 (m, 1H), 1.61 - 1.47 (m, 5H), 1.44 - 1.30 (m, 12H), 1.02 (t, 5H).

[0177] compound 208 [ka] (3R,6R,8R,9R,10R)-3-(3-(5,8-dihydropyrido[3,4-d]pyrimidin-7(6H)-yl)propyl)-9-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-8-methoxy-6,8,10,12,12-pentamethyl-4-propyl-1-oxa-4-azacyclotridecane-11,13-dione (S6-3-I2-3-11) Prepared according to the method of S6-3-I1-1-1 from (2S,3R,4S,6R)-4-(dimethylamino)-2-(((3R,6R,8R,9R,10R)-8-methoxy-6,8,10,12,12-pentamethyl-11,13-dioxo-3-(3-oxopropyl)-4-propyl-1-oxa-4-azacyclotridecan-9-yl)oxy)-6-methyltetrahydro-2H-pyran-3-yl benzoate and 5,6,7,8-tetrahydropyrido[3,4-d]pyrimidine to give the title compound as the formate salt. MS (ESI+) m / z: 235.5 [M + 3H]3+, 352.8 [M + 2H]2+, 704.5 [M + H]+; 1 H NMR (400 MHz, methanol-d4) δ 8.93 (s, 1H), 8.61 (s, 1H), 8.47 (s, 2H), 4.46 (d, 1H), 4.35 (s, 1H), 4.25 (d, 1H), 3.74 (s, 3H), 3.51 (dd, 1H), 3.42 (ddd, 1H), 3.29 (s, 2H), 3.00 (d, 5H), 2.92 (q, 1H), 2.85 (s, 7H), 2.74 (qd, 2H), 2.06 (ddd, 1H), 1.96 (s, 1H), 1.87 - 1.77 (m, 2H), 1.77 - 1.72 (m, 1H), 1.61 - 1.55 (m, 1H), 1.53 (s, 4H), 1.41 - 1.28 (m, 13H), 1.04 - 0.96 (m, 6H).

[0178] compound 209 [ka] (3R,6R,8R,9R,10R)-3-(3-(3,4-dihydro-2,6-naphthyridin-2(1H)-yl)propyl)-9-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-8-methoxy-6,8,10,12,12-pentamethyl-4-propyl-1-oxa-4-azacyclotridecane-11,13-dione (S6-3-I2-3-12) Prepared according to the method of S6-3-I1-1-1 from (2S,3R,4S,6R)-4-(dimethylamino)-2-(((3R,6R,8R,9R,10R)-8-methoxy-6,8,10,12,12-pentamethyl-11,13-dioxo-3-(3-oxopropyl)-4-propyl-1-oxa-4-azacyclotridecan-9-yl)oxy)-6-methyltetrahydro-2H-pyran-3-yl benzoate and 1,2,3,4-tetrahydro-2,6-naphthyridine to give the title compound as the formate salt. MS (ESI+) m / z: 235.2 [M + 3H]3+, 352.3 [M + 2H]2+, 703.5 [M + H]+; 1 H NMR (400 MHz, methanol-d4) δ 8.48 (s, 2H), 8.36 - 8.17 (m, 2H), 7.26 (d, 1H), 4.46 (d, 1H), 4.35 (s, 1H), 4.25 (d, 1H), 3.87 - 3.66 (m, 4H), 3.55 - 3.38 (m, 2H), 3.28 (s, 1H), 3.01 (s, 6H), 2.85 (s, 6H), 2.06 (ddd, 2H), 1.95 (s, 1H), 1.75 (s, 1H), 1.54 (s, 3H), 1.37 (dt, 14H), 1.00 (t, 3H), 0.91 (d, 3H).

[0179] compound 210 [ka] (3R,6R,8R,9R,10R)-3-(3-(5,8-dihydro-1,7-naphthyridin-7(6H)-yl)propyl)-9-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-8-methoxy-6,8,10,12,12-pentamethyl-4-propyl-1-oxa-4-azacyclotridecane-11,13-dione (S6-3-I2-3-13) Prepared according to the method of S6-3-I1-1-1 from (2S,3R,4S,6R)-4-(dimethylamino)-2-(((3R,6R,8R,9R,10R)-8-methoxy-6,8,10,12,12-pentamethyl-11,13-dioxo-3-(3-oxopropyl)-4-propyl-1-oxa-4-azacyclotridecan-9-yl)oxy)-6-methyltetrahydro-2H-pyran-3-yl benzoate and 5,6,7,8-tetrahydro-1,7-naphthyridine to give the title compound as the formate salt. MS (ESI+) m / z: 235.2 [M + 3H]3+, 352.3 [M + 2H]2+, 703.5 [M + H]+; 1 H NMR (400 MHz, methanol-d4) δ 8.46 (s, 2H), 8.36 (dd, 1H), 7.62 (dd, 1H), 7.27 (dd, 1H), 4.46 (d, 1H), 4.36 (s, 1H), 4.27 (d, 1H), 3.86 - 3.75 (m, 3H), 3.75 - 3.71 (m, 1H), 3.55 - 3.39 (m, 2H), 3.11 - 2.97 (m, 8H), 2.92 (d, 1H), 2.85 (s, 6H), 2.77 (t, 2H), 2.06 (dt, 1H), 1.95 (s, 1H), 1.84 (dt, 2H), 1.77 (s, 1H), 1.62 - 1.49 (m, 6H), 1.42 - 1.31 (m, 13H), 1.00 (t, 3H), 0.92 (d, 3H).

[0180] compound 213 [ka] (3R,6R,8R,9R,10R)-9-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-8-methoxy-6,8,10,12,12-pentamethyl-4-propyl-3-(3-(quinazolin-6-ylamino)propyl)-1-oxa-4-azacyclotridecane-11,13-dione (S6-3-I2-3-16) Prepared according to the method of S6-3-I1-1-1 from (2S,3R,4S,6R)-4-(dimethylamino)-2-(((3R,6R,8R,9R,10R)-8-methoxy-6,8,10,12,12-pentamethyl-11,13-dioxo-3-(3-oxopropyl)-4-propyl-1-oxa-4-azacyclotridecan-9-yl)oxy)-6-methyltetrahydro-2H-pyran-3-yl benzoate and quinazolin-6-amine to give the title compound as the formate salt. MS (ESI+) m / z: 238.8 [M + 3H]3+, 357.8 [M + 2H]2+, 714.5 [M + H]+; 1 H NMR (400 MHz, methanol-d4) δ 9.09 (s, 1H), 8.75 (s, 1H), 8.45 (s, 1H), 7.64 (d, 1H), 7.39 (dd, 1H), 6.78 (d, 1H), 4.30 (d, 1H), 4.18 (d, 1H), 3.86 (d, 1H), 3.78 (t, 1H), 3.52 (ddt, 1H), 3.30 - 3.23 (m, 2H), 3.17 (s, 1H), 2.98 (s, 1H), 2.86 (s, 1H), 2.67 (s, 2H), 2.45 (s, 4H), 2.42 (s, 3H), 1.86 (t, 1H), 1.76 (d, 1H), 1.64 (s, 2H), 1.50 (s, 1H), 1.41 (s, 3H), 1.37 - 1.25 (m, 4H), 1.25 - 1.10 (m, 13H), 0.84 - 0.66 (m, 4H), 0.60 (d, 2H).

[0181] compound 214 [ka] (3R,6R,8R,9R,10R)-9-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-8-methoxy-6,8,10,12,12-pentamethyl-4-propyl-3-(3-((quinolin-3-ylmethyl)amino)propyl)-1-oxa-4-azacyclotridecane-11,13-dione (S6-3-I2-3-17) Prepared according to the method of S6-3-I1-1-1 from (2S,3R,4S,6R)-4-(dimethylamino)-2-(((3R,6R,8R,9R,10R)-8-methoxy-6,8,10,12,12-pentamethyl-11,13-dioxo-3-(3-oxopropyl)-4-propyl-1-oxa-4-azacyclotridecan-9-yl)oxy)-6-methyltetrahydro-2H-pyran-3-yl benzoate and quinolin-3-ylmethanamine to give the title compound as the formate salt. MS (ESI+) m / z: 243.2 [M + 3H]3+, 364.3 [M + 2H]2+, 727.5 [M + H]+; 1 H NMR (400 MHz, methanol-d4) δ 8.95 (d, 1H), 8.53 (s, 2H), 8.48 (d, 1H), 8.08 (d, 1H), 8.00 (dd, 1H), 7.84 (ddd, 1H), 7.69 (ddd, 1H), 4.46 (d, 1H), 4.31 (s, 1H), 4.14 (s, 1H), 3.77 - 3.68 (m, 1H), 3.51 - 3.34 (m, 2H), 3.01 (q, 3H), 2.91 (s, 2H), 2.81 (s, 6H), 2.03 (ddd, 1H), 1.83 - 1.77 (m, 2H), 1.59 - 1.45 (m, 5H), 1.39 - 1.29 (m, 11H), 0.99 (d, 2H), 0.96 (s, 2H).

[0182] Scheme 8 [ka]

[0183] [ka] (2S,3R,4S,6R)-4-(Dimethylamino)-2-(((2R,3R,4R,6R)-7-(((R)-1-hydroxy-5-(pyrrolidin-1-yl)pentan-2-yl)amino)-4-methoxy-4,6-dimethyl-2-(2,2,5-trimethyl-4-oxo-4H-1,3-dioxin-6-yl)heptan-3-yl)oxy)-6-methyltetrahydro-2H-pyran-3-yl benzoate (S1-2-I10) S1-1 (0.675 g, 1.14 mmol) and (R)-2-amino-5-(pyrrolidin-1-yl)pentan-1-ol (I10, 234 mg, 1.36 mmol) were dissolved in EtOH (5 mL), and Ti(OEt) (0.72 mL, 2.96 mmol) was added. After 30 min, a small aliquot was removed from the reaction mixture and added to a suspension of NaBH in MeOH. LC / MS analysis indicated complete conversion. NaBH (107 mg, 3.42 mmol) was added. When gas evolution ceased, 30% aqueous NHOH (3 mL) was added, and the mixture was filtered through a pad of Celite® and washed with EtOAc. The filtrate was washed with brine, dried over NaSO, filtered, and concentrated to give S1-2-I10. This material was used without further purification. MS (ESI+) m / z: 746.49 [M + H] + , 374.00 [M + 2H] 2+ , 249.66 [M + 3H] 3+ .

[0184] [ka] (2S,3R,4S,6R)-2-(((2R,3R,4R,6R)-7-((tert-butoxycarbonyl)((R)-1-hydroxy-5-(pyrrolidin-1-yl)pentan-2-yl)amino)-4-methoxy-4,6-dimethyl-2-(2,2,5-trimethyl-4-oxo-4H-1,3-dioxin-6-yl)heptan-3-yl)oxy)-4-(dimethylamino)-6-methyltetrahydro-2H-pyran-3-yl benzoate (S8-1-I10) A 40 mL vial was charged with S1-2-I10 (850 mg, 1.13 mmol) in dichloromethane (5 mL) and stirred at room temperature to give a yellow solution. BocO (0.33 mL, 1.46 mmol) was added in one portion and stirred at room temperature for 2 h. The reaction was diluted with dichloromethane and poured into saturated aqueous NaHCO3. The aqueous phase was extracted with dichloromethane, and the combined organic phases were dried over MgSO4, filtered, and concentrated. The residue was purified on 24 g of silica gel (eluted with 0–6% MeOH-dichloromethane) to give S8-1-I10 (520 mg, 54% over two steps). MS (ESI+) m / z: 846.5 [M + H]+, 423.8 [M + 2H]2+.

[0185] [ka] tert-Butyl (3R,6R,8R,9R,10R)-9-(((2S,3R,4S,6R)-3-(benzoyloxy)-4-(dimethylamino)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-8-methoxy-6,8,10,12-tetramethyl-11,13-dioxo-3-(3-(pyrrolidin-1-yl)propyl)-1-oxa-4-azacyclotridecane-4-carboxylate (S8-2-I10) The flask was fitted with a reflux condenser, which was flame-dried under vacuum, allowed to cool, and backfilled with nitrogen. A solution of S8-2-I10 (520 mg, 0.614 mmol) in chlorobenzene (150 mL) was added via cannula, and the flask was placed under low vacuum, sonicated for 2 minutes, and then backfilled with nitrogen. After repeated degassing, the mixture was heated at a bath temperature of 155 °C for 16 hours. The reaction was allowed to cool to room temperature and concentrated. The residue was purified on 24 g of silica gel (eluted with 0–10% MeOH-dichloromethane + 0.5% 30% NH4OH in water) to give S8-2-I10 (395 mg, 82%). MS (ESI+) m / z: 394.8 [M + 2H] 2+ , 788.5 [M + H] + .

[0186] [ka] tert-Butyl (3R,6R,8R,9R,10R)-9-(((2S,3R,4S,6R)-3-(benzoyloxy)-4-(dimethylamino)-6-methyltetrahydro-2H-pyran-2-yl)oxy)-8-methoxy-6,8,10,12,12-pentamethyl-11,13-dioxo-3-(3-(pyrrolidin-1-yl)propyl)-1-oxa-4-azacyclotridecane-4-carboxylate (S8-3-I10) A 20 mL vial was charged with a solution of S8-2-I10 (360 mg, 0.46 mmol) in 1,2-dimethoxyethane (5 mL) precooled to -60 °C. KHMDS (0.68 mL, 0.68 mmol) was added dropwise. The reaction mixture was stirred at -60 °C for 20 minutes. Then, Me2SO4 (65 μL, 0.68 mmol) was added. The reaction mixture was warmed to -15 °C. LC / MS showed complete conversion. The reaction was quenched by adding trimethylamine (0.88 mL), and the resulting mixture was diluted with dichloromethane and saturated NaHCO3 was added. The aqueous layer was extracted with dichloromethane, and the combined organic layers were dried over MgSO4, filtered, and concentrated. The residue was purified on 4 g of silica gel (eluted with 0–10% MeOH-dichloromethane + 0.5% 30% aqueous NH4OH) to give S8-3-I10 (145 mg, 40%). MS (ESI+) m / z: 401.8 [M + 2H]2+, 802.5 [M + H]+.

[0187] [ka] (2S,3R,4S,6R)-4-(Dimethylamino)-2-(((3R,6R,8R,9R,10R)-8-methoxy-6,8,10,12,12-pentamethyl-11,13-dioxo-3-(3-(pyrrolidin-1-yl)propyl)-1-oxa-4-azacyclotridecan-9-yl)oxy)-6-methyltetrahydro-2H-pyran-3-yl benzoate (S8-4-I10) In a 20 mL flask at room temperature, S8-3-I10 (135 mg, 0.17 mmol) was dissolved in 1.5 mL of DCM. TFA (0.52 mL, 6.74 mmol) was added, and the mixture was stirred at room temperature. The reaction was complete by UPLC. The mixture was diluted with 30 mL of DCM, and 30 mL of saturated aqueous NaHCO3 was added. The aqueous phase was extracted three times with DCM, and the combined organic phases were dried over MgSO4, filtered, and concentrated to give S8-4-I10. This product was used without further purification. MS (ESI+) m / z: 234.8 [M + 3H]3+, 351.8 [M + 2H]2+, 702.5 [M + H]+.

[0188] [ka] (2S,3R,4S,6R)-2-(((3R,6R,8R,9R,10R)-3-(3-(7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propyl)-8-methoxy-6,8,10,12,12-pentamethyl-11,13-dioxo-1-oxa-4-azacyclotridecan-9-yl)oxy)-4-(dimethylamino)-6-methyltetrahydro-2H-pyran-3-yl benzoate (S8-4-I12) Prepared according to the method for S8-4-I10, substituting intermediate I12 to give S8-4-I12. MS (ESI+) m / z: 383.58 [M + 2H]2+, 766.00 [M + H]+.

[0189] [ka] (2S,3R,4S,6R)-2-(((3R,6R,8R,9R,10R)-4-(2-((tert-butyldimethylsilyl)oxy)ethyl)-8-methoxy-6,8,10,12,12-pentamethyl-11,13-dioxo-3-(3-(pyrrolidin-1-yl)propyl)-1-oxa-4-azacyclotridecan-9-yl)oxy)-4-(dimethylamino)-6-methyltetrahydro-2H-pyran-3-yl benzoate (S8-5-TBS-I10) Compound S8-4-I10 (105 mg, 0.15 mmol) was dissolved in dry methylene chloride (1 mL), and 2-((tert-butyldimethylsilyl)oxy)acetaldehyde (0.042 mL, 0.223 mmol) and AcOH (0.026 mL, 0.45 mmol) were added. NaBH(OAc)3 (63 mg, 0.30 mmol) was then added in one portion to the reaction mixture. The reaction was stirred at room temperature for 2 hours, and LC / MS showed complete conversion. The reaction was quenched by adding saturated NaHCO3 (5 mL), and the aqueous layer was extracted three times with methylene chloride (10 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated. The residue was purified on 4 g of silica gel (eluted with 0–10% MeOH-dichloromethane + 0.5% 30% aqueous NH4OH) to give 61 mg of S8-5-TBS-I10 (48% yield). MS (ESI+) m / z: 287.5 [M + 3H]3+, 430.8 [M + 2H]2+, 860.6 [M + H]+.

[0190] [ka] (2S,3R,4S,6R)-4-(Dimethylamino)-2-(((3R,6R,8R,9R,10R)-4-(2-hydroxyethyl)-8-methoxy-6,8,10,12,12-pentamethyl-11,13-dioxo-3-(3-(pyrrolidin-1-yl)propyl)-1-oxa-4-azacyclotridecan-9-yl)oxy)-6-methyltetrahydro-2H-pyran-3-yl benzoate (S8-5-I10) S8-5-TBS-I10 (61 mg, 0.071 mmol) was dissolved in dry THF (2 mL) and TBAF (1 M in THF, 0.21 mL, 0.021 mmol) was added at room temperature. The reaction mixture was stirred at room temperature for 2 h and concentrated. The residue was purified on 4 g of silica gel (eluted with 0–20% MeOH-dichloromethane + 0.5% 30% aqueous NH4OH) to give S8-5-I10 (46 mg, 87%). MS (ESI+) m / z: 249.5 [M + 3H]3+, 373.8 [M + 2H]2+, 746.5 [M + H]+.

[0191] compound 229 [ka] (3R,6R,8R,9R,10R)-9-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-8-methoxy-6,8,10,12,12-pentamethyl-3-(3-(pyrrolidin-1-yl)propyl)-1-oxa-4-azacyclotridecane-11,13-dione (S8-6-I10) S8-4-I10 (25 mg, 0.036 mmol) was dissolved in MeOH (1 mL), and the reaction mixture was heated to 40 °C (external temperature) overnight. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. This material was purified by HPLC (Atlantis T3 column, 5-30% MeCN-water-0.1% HCOH) to give 6.52 mg of S8-6-I10 as the formate salt. MS (ESI+) m / z: 200.1 [M + 3H]3+, 299.7 [M +2H]2+, 598.4 [M + H]+; 1H NMR (400 MHz, methanol-d) δ 8.56 (s, 1H), 4.39 (d, 1H), 4.13 (d, 1H), 3.87 (dd, 1H), 3.63 (ddt, 1H), 3.49 (dt, 1H), 3.35 - 3.27 (m, 3H), 3.11 (d, 1H), 3.10 - 2.96 (m, 3H), 2.94 (d, 5H), 2.86 (s, 1H), 2.69 (dd, 1H), 2.50 (s, 4H), 2.44 (t, 3H), 2.04 - 1.96 (m, 4H), 1.91 - 1.78 (m, 4H), 1.71 - 1.57 (m, 3H), 1.48 (s, 3H), 1.44 (d, 1H), 1.41 - 1.32 (m, 8H), 1.27 (d, 5H), 1.02 (d, 3H).

[0192] compound 230 [ka] (3R,6R,8R,9R,10R)-9-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-(2-hydroxyethyl)-8-methoxy-6,8,10,12,12-pentamethyl-3-(3-(pyrrolidin-1-yl)propyl)-1-oxa-4-azacyclotridecane-11,13-dione (S7-7-I10) Prepared from S8-5-I10 according to the method of S8-6-I10 to give the title compound as the formate salt. MS (ESI+) m / z: 214.8 [M + 3H]3+, 321.8 [M +2H]2+, 642.5 [M + H]+; 1H NMR (400 MHz, methanol-d) δ 8.49 (s, 2H), 4.52 (s, 1H), 4.44 (d, 1H), 4.04 (d, 1H), 3.84 (s, 1H), 3.70 (ddd, 1H), 3.55 (d, 3H), 3.49 - 3.33 (m, 2H), 3.33 (s, 3H), 3.30 (p, 1H), 3.22 - 3.08 (m, 2H), 3.03 (s, 1H), 2.81 (s, 6H), 2.55 (s, 2H), 2.26 (s, 1H), 2.08 (d, 1H), 2.09 - 2.00 (m, 3H), 2.03 - 1.91 (m, 1H), 1.83 - 1.74 (m, 2H), 1.58 - 1.45 (m, 1H), 1.45 (s, 2H), 1.39 (s, 1H), 1.35 (s, 2H), 1.33 - 1.24 (m, 8H), 1.15 (s, 1H), 0.84 (d, 3H).

[0193] compound 232 [ka] (3R,6R,8R,9R,10R)-3-(3-(7,8-dihydropyrido[4,3-d]pyrimidin-6(5H)-yl)propyl)-9-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-isopentyl-8-methoxy-6,8,10,12,12-pentamethyl-1-oxa-4-azacyclotridecane-11,13-dione (S8-7-I12-2) Prepared from S8-4-I12 according to the method for S8-7-I10, substituting 3-methylbutanal to give 16.47 mg of S8-7-I12-2 as the formate salt. MS (ESI+) m / z: 366.51 [M + 2H]2+, 731.94 [M + H]+; 1H NMR (400 MHz, methanol-d4) δ 8.94 (s, 1H), 8.55 (s, 1H), 8.51 (s, 2H), 4.47 (d, 1H), 4.22 (s, 1H), 3.74 (q, 4H), 3.51 (dd, 1H), 3.41 (ddd, 2H), 3.04 (t, 3H), 3.01 - 2.89 (m, 6H), 2.84 (s, 7H), 2.78 - 2.61 (m, 3H), 2.05 (d, 3H), 1.87 - 1.62 (m, 5H), 1.60 - 1.54 (m, 2H), 1.52 (s, 6H), 1.38 (d, 5H), 1.34 (t, 8H), 0.94 (t, 9H).

[0194] compound 280 [ka] (3S,6R,8R,9R,10R)-3-(3-(3,4-dihydro-2,6-naphthyridin-2(1H)-yl)propyl)-9-(((2S,3R,4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-4-ethyl-8-methoxy-6,8,10,12,12-pentamethyl-1-oxa-4-azacyclotridecane-11,13-dione Prepared from S2-1-I1-2 and 1,2,3,4-tetrahydro-2,6-naphthyridine according to the method of S6-3-I1-1 to give the title compound as the formate salt. MS (ESI+) m / z: 689.3 [M + H]+; 1H NMR (400 MHz, methanol-d4) δ 8.40 (s, 2.6H), 8.33 (s, 1H), 8.26 (d, 1H), 7.17 (d, 1H), 4.66 - 4.57 (m, 1H), 4.52 - 4.40 (m, 2H), 4.27 (br d, 1H), 4.03 (br d, 1H), 3.81 - 3.67 (m, 3H), 3.63 - 3.50 (m, 1H), 3.49 - 3.34 (m, 3H), 3.25 - 3.07 (m, 2H), 3.07 - 2.94 (m, 5H), 2.92 - 2.77 (m, 9H), 2.69 (br t, 2H), 2.17 (br d, 1H), 2.08 - 2.01 (m, 1H), 1.95 (br d, 1H), 1.88 - 1.70 (m, 3H), 1.63 (br d, 1H), 1.58 - 1.50 (m, 1H), 1.49 - 1.44 (m, 3H), 1.44 - 1.27 (m, 16H), 1.06 (br d, 3H).

[0195] compound 282 [ka] (3R,6R,8R,9R,10R)-9-(((2S,3R,4S,6R)-4-(Dimethylamino)-3-hydroxy-6-methyltetrahydro-2H-pyran-2-yl)oxy)-3-(3-(2,4-dioxo-1,3,4,5,7,8-hexahydropyrido[4,3-d]pyrimidin-6(2H)-yl)propyl)-8-methoxy-6,8,10,12,12-pentamethyl-4-propyl-1-oxa-4-azacyclotridecane-11,13-dione Prepared according to the method of S6-3-I1-1-1 from (2S,3R,4S,6R)-4-(dimethylamino)-2-(((3R,6R,8R,9R,10R)-8-methoxy-6,8,10,12,12-pentamethyl-11,13-dioxo-3-(3-oxopropyl)-4-propyl-1-oxa-4-azacyclotridecan-9-yl)oxy)-6-methyltetrahydro-2H-pyran-3-yl benzoate and hexahydropyrido[4,3-d]pyrimidine-2,4(1H,3H)-dione.

[0196] Biological Examples A series of macrolide compounds was prepared to target mammalian ribosomes and regulate the readthrough of premature stop codons. In this study, we explored the possibility that 13-membered macrolides could induce readthrough of the premature stop codon in the APC gene.

[0197] Human colorectal cancer SW403 and SW1417 cells harboring PTC mutations in the APC gene treated with the 13-membered macrolides disclosed herein exhibited reduced levels of nuclear b-catenin and c-myc, indicating that the macrolides mediated readthrough of premature termination codons and produced bioactive APC protein that suppressed the b-catenin / Wnt pathway. In a mouse model of adenomatous polyposis coli, APC min Treatment of mice with the macrolides disclosed herein significantly reduced intestinal polyps, adenomas, and associated anemia, resulting in prolonged survival. min Decreased nuclear b-catenin staining in epithelial cells of mouse polyps was demonstrated, confirming an effect on the b-catenin / Wnt pathway. These results indicate that the macrolides disclosed herein may have therapeutic potential for the treatment of FAP caused by nonsense mutations in the APC gene.

[0198] material and method cell line Human colon cancer cells SW1417 and SW403 were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM high glucose with GlutaMAX, Gibco, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco) and maintained at 37°C in a humidified incubator with 5% CO2. Cells were passaged weekly with trypsin.

[0199] Cell proliferation inhibition Western blot Cells, 4x10 6 Cells were plated at a density of 100 cells / 100 mm dish. After 48 hours, cells were treated with either DMSO or a compound of the present invention (50 mM) listed in Table A. After 48 hours of treatment, cell pellets were harvested and divided into two aliquots. One aliquot was used to prepare whole-cell lysates (RIPA lysis and extraction buffer, Thermo Fisher Scientific), and the other was used to prepare nuclear and cytoplasmic protein extracts (NE-PER Nuclear and Cytoplasmic Extraction Reagents, Thermo Fisher Scientific). Proteins were separated on a 4-12% gradient gel (NuPAGE Bis-Tris Protein Gel, Invitrogen, Thermo Fisher Scientific) using SDS running buffer (NuPAGE MES SDS running buffer, Invitrogen). Primary antibodies used included non-phosphorylated b-catenin (Ser33 / 37 / Thr41) (1:1000, catalog 8814, Cell Signaling Technology, Danvers, MA): c-myc (1:1000, catalog ab32072, Abcam, Waltham, MA), MEK1 / 2 (1:1000, catalog 8727, Cell Signaling Technology), histone H3 (1:1000, catalog 4499, Thermo Fisher Scientific), and a-tubulin (1:10,000, catalog T6199, MilliporeSigma, Burlington, MA).

[0200] Animal experiments Pharmacokinetic studies were performed at WuXi AppTec Laboratory Testing Division (Cranbury, NJ) on male CD-1 mice aged 6-8 weeks obtained from Hilltop Lab Animals, Inc. (Scottdale, PA). Mice had free access to water and rodent chow pellets (LabDiet, Certified Rodent Diet #5002). Animals received a single oral dose of a compound of the present invention (100 mg / kg body weight) listed in Table A from a sterile stock solution in saline. Blood samples were collected from a peripheral vein into EDTA tubes and processed for plasma at 0.083, 0.250, 0.500, 1.00, 2.00, 4.00, 8.00, and 24.0 hours post-dose. After the final blood draw, mice were euthanized, and the hair on their backs was shaved and 1 cm 2 Skin samples were excised and weighed. 2 cm sections of jejunum were harvested, rinsed with saline, and weighed. Tissue samples and plasma were stored at -70°C. Thawed skin samples were minced, extracted with acetonitrile, centrifuged at 3000 x g, and the supernatant was collected for LC-MS / MS analysis. Water was added to the thawed intestinal tissue at a ratio of 6:1 (ml water / mg tissue), homogenized, and processed for LC-MS / MS analysis. Plasma, jejunal homogenates, and skin / acetonitrile samples were analyzed using a SCIEX Triple Quad 6500+ LC-MS / MS system (SCIEX, Framingham, MA). Briefly, proteins were precipitated using IS solution (100 ng / mL labetanol and 100 ng / mL tolbutamide in acetonitrile) and centrifuged at 3900 x g. The supernatant was mixed with an equal volume of water, and 2.00–4.00 mL was injected for LC-MS / MS analysis. A calibration curve ranging from 1.00–3000 ng / mL was generated using corresponding tissue preparations from naive CD-1 mice. Data analysis was performed using Phoenix WinNonlin software (Certara, Princeton, NJ).

[0201] C57BL / 6 system APC min Mice were obtained from the Jackson Laboratory (Bar Harbor, ME) and were female APCs aged 10 weeks and weighing more than 15 g. min Mice were used. Mice were housed in individually ventilated cage systems and had free access to sterile rodent chow (Teklad 2919) and water. Clinical observations and body weights were recorded daily, and mice with weight loss exceeding 20% ​​were excluded from the study. The study was conducted in compliance with the UK Animals Scientific Procedures Act 1986 and carried out at Crown Biosciences UK Ltd (Loughborough, UK). Compounds were prepared in sterile saline at a working concentration of 5 mg / ml. Mice were orally administered compound (50 mg, 25 mg, or 12.5 mg / kg body weight) or saline daily for 8 weeks. Drugs were well tolerated, and no adverse events were observed. When animals became moribund or at the end of the study, mice were euthanized, a complete necropsy was performed, spleens were weighed, and the complete intestinal tract was removed for analysis. Whole blood was collected into EDTA tubes and complete blood counts (CBC) were performed on a Woodley InSight 5 Diff Retic Haematology Analyzer and compared with normal mouse CBC ranges (Pinmoore Animal Laboratory Services Limited, Cheshire, UK).

[0202] Polyp scoring, histopathology, and immunohistochemistry. The excised intestinal tracts (small and large intestines) were washed with PBS and sliced ​​longitudinally. 10-cm sections were visually inspected and polyps were counted. Midline sections of the small intestine were fixed in 10% formalin, formed into Swiss rolls, and processed into FFPE blocks. 4-mm sections were used for analysis. Hematoxylin and eosin (H&E) staining was performed using a Tissue-Tek Prisma Plus autostainer (Sakura Finetek USA, Torrance, CA). Sections were scanned at 40x magnification using Pannoramic Digital Slide Scanners (3DHISTECH, Pannoramic SCAN, Budapest, Hungary). Analysis and quantification of H&E-stained slides were performed using HALO Image Analysis software (Indica Labs, Albuquerque, NM, USA). Lesions were identified based on the nuclear-to-cytoplasmic staining ratio, and lesion area was calculated (percent lesion area = lesion area / small intestine area × 100) [31, 32]. Adenomatous areas were identified by a board-certified pathologist. Immunohistochemistry (IHC) was performed on sections from FFPE blocks using the Leica BOND-RXm Automated Stainer (Deer Park, IL, USA) with the following antigen retrieval conditions: citric acid, pH 6.0; 100°C; 20 min; primary antibody at a 1:100 dilution. Rabbit IgG mAb against b-catenin was obtained from Cell Signaling Technology. Polyclonal anti-rabbit IgG conjugated to horseradish peroxidase was used for detection (Leica Biosystems, Buffalo Grove, IL, USA). Nonspecific rabbit IgG (Abcam) was used as an isotype control. Sections were rinsed and counterstained with H&E.

[0203] statistical analysis GraphPad Prism software version 9 (GraphPad Software, La Jolla, CA) was used for data analysis and graphing. Mean values ​​were plotted, with standard deviations (SD) and standard errors of the mean (SEM) indicated by bars. An unpaired student t-test was used to compare means between treatment and control groups. For survival analysis, the log-rank test was used to determine significance between control and treatment groups. A p-value of less than 0.05 was considered significant.

[0204] result Compound-induced growth inhibition of human colon cancer cells. Ribosome-targeting antibiotics that promote readthrough of premature stop codons can suppress the growth of tumor cells harboring nonsense mutations in the APC gene via downregulation of b-catenin / c-myc signaling [23, 24, 33]. SW1417 and SW403 human colon cancer cells harbor nonsense mutations in the APC gene (R1450* and S1278*, respectively) that result in nonfunctional truncated APC proteins, while HCT116 human colon cancer cells harbor wild-type APC genes

[17] . Growth inhibition assays were performed on these three tumor cell lines to evaluate the antiproliferative activity of the compounds disclosed herein. Tumor cells were cultured in the presence of titrated concentrations of the compounds disclosed herein, and the drug concentrations that inhibited cell growth by 50% (GI50) compared to vehicle-treated cells are shown in Table 1 (Figure 1). This compound did not affect the proliferation of HCT116 cells at all concentrations tested (up to 100 mM), and the GI50 of SW1417 and SW403 cells was 34.8 mM and 39.8 mM, respectively.

[0205] Treatment of human colon cancer cells with the compounds disclosed herein results in a decrease in the levels of nuclear b-catenin and c-myc. Adenomatous polyposis coli is a large, complex protein (312 kDa) that directly mediates the ubiquitin degradation of phosphorylated b-catenin in the cytoplasm

[34] . In the absence of APC activity, nuclear translocation of b-catenin increases. Therefore, the amount of nuclear b-catenin relative to the amount of cytoplasmic b-catenin is used as an indicator of the level of functional APC, especially since there are no reliable commercially available antibodies for detecting APC [35, 36]. SW403 and SW1417 cells were treated with 50 mM compounds for 48 hours, and the amount of b-catenin in nuclear and cytoplasmic extracts was measured by Western blot. As shown in Figure 1 (Figure 3), colon cancer cells showed a significant decrease in nuclear b-catenin accumulation in response to compound exposure. Nuclear levels of b-catenin were reduced by 40% in SW1417 and SW403-treated cells compared to DMSO-treated cells. This indicates that compound-mediated readthrough of the APC nonsense mutation resulted in increased levels of functional APC. Cytoplasmic levels of b-catenin were similar in cells treated with DMSO or the compounds.

[0206] c-myc gene expression is transcriptionally co-regulated by b-catenin [8, 37]. As shown in Figure 2 (Figure 4), treatment with the compounds of the present invention listed in Table A (50 mM, 48 hours) reduced c-myc protein levels by 25% in SW1417 cells and by 30% in SW403 cells compared to cells treated with DMSO. This provides further evidence that compound treatment increases the level of activated APC, promotes the cytoplasmic degradation of b-catenin, reduces the nuclear level of b-catenin, and consequently reduces c-myc expression.

[0207] Treatment with the compounds disclosed herein reduces APC min Improves mouse survival and reduces the anemic phenotype. A single-dose mouse study was conducted to determine the pharmacokinetics and exposure of the compounds disclosed herein in intestinal tissues after oral administration. Male CD-1 mice (n = 3) were orally administered the compound at 100 mg / kg body weight, and drug plasma levels were measured at several time points over a 24-hour period. The results are summarized in Table 2 (Figure 2). The mean plasma C-max was 1307 ng / ml (±430 SD), peaking approximately 15 minutes after administration. At the end of the 24-hour study, the mean plasma level of the compound was 44.7 ng / ml, and the mean drug concentration in the jejunum was 24,383 ng / g tissue. This data indicates that the compound can achieve an intestinal exposure of approximately 35 mM following a single oral dose, potentially impacting disease progression. The observed plasma half-life, effective tissue penetration, and high tissue-to-plasma concentration ratio are consistent with the pharmacokinetics of macrolide drugs

[38] .

[0208] A well-established model of FAP is the APC gene, which contains a nonsense mutation in the human APC homolog (min) gene, generating a nonfunctional truncated protein. min APC min Mice develop numerous small intestinal adenomas as a primary phenotype, anemia as a secondary phenotype, and are lethal with an average lifespan of 120 days [18-20]. min In mice, the majority of adenomas are present, and the number of adenomas does not increase significantly thereafter.

[39] In an 8-week efficacy study, APC min Mice were orally administered a compound disclosed herein (50 mg / kg body weight) or vehicle saline daily. Three weeks into the study, mice in the vehicle control group began to show severe clinical signs of disease progression, forcing the study to be discontinued. Only five vehicle-treated mice (50%) remained in the study at the end of the study. In contrast, all compound-treated mice remained in the study for the entire duration. Similar efficacy results were obtained in replicate studies using daily oral doses of 50, 25, and 12.5 mg / kg body weight of the compound, with 100% survival achieved at the lowest dose used in the 8-week study. APC minAnemia resulting from chronic blood loss due to numerous intestinal lesions in mice is believed to be the main cause of animal death

[18] . Compound treatment reduced APC in mice at all three dose levels tested, as indicated by a reduction of splenomegaly by approximately 35%, an increase in hemoglobin levels (approximately 45%), an increase in packed cell volume (approximately 55%), and an increase in red blood cell count (approximately 60%). min The anemia associated with the model was significantly improved (Figure 3B) (Figure 5B). min Clinical observations consistent with the model were reported in both groups, and weight gain was comparable between vehicle and treatment groups throughout the study period.

[0209] Compound treatment increased APC min Dysplastic intestinal polyps and lesion area were reduced in mice. When mice became moribund or at the end of the study in which mice were treated with 50 mg / kg bw of a compound disclosed herein, the entire gastrointestinal track was removed and polyps were manually counted. The total number of visible polyps in the small intestines of mice treated with the compound was significantly lower than that of control mice, with a 39% reduction in polyp counts (Figure 4A) (Figure 6A). Few polyps were detected in the large intestines of either group of mice, which is characteristic of this model (data not shown). Hematoxylin-eosin-stained sections from the small intestines of control and compound-treated mice are shown in Figure 4B and were used for histopathological analysis. In compound-treated mice, the total lesion area (polyps, adenomas, and carcinomas) was reduced by 52%, and the adenoma area was also reduced by 60% (Figure 4B) (Figure 6B). Immunohistochemistry, shown in Figure 5 (Figure 7), revealed prominent nuclear b-catenin staining in numerous epithelial cells in the lesions of saline-treated mice. In the lesions of compound-treated mice, significantly fewer epithelial cells displayed nuclear b-catenin staining; many epithelial cells in these lesions displayed membrane-associated b-catenin staining and retained their columnar morphology (Figure 5) (Figure 7).

[0210] Consideration Recent studies have shown that nonsense mutation readthrough can be induced in human cancer cells by several macrolide and aminoglycoside antibiotics, partially restoring the synthesis of functional full-length proteins such as p53 and APC [23, 24, 40, 41]. The primary role of APC as a tumor suppressor is to form a core complex with Axin, the Ser / Thr kinase glycogen synthase kinase 3, and casein kinase 1 for ubiquitination and proteasomal degradation of cytoplasmic b-catenin

[34] . This results in decreased nuclear translocation of b-catenin and reduced transcription of b-catenin / WNT pathway genes, including oncogenic c-myc [16, 34]. The APC protein is composed of multiple functional domains. The regions between amino acids 1020–1169 and 1342–2075 are essential for b-catenin binding and degradation [8–10]. Nonsense mutations often result in the synthesis of truncated APC proteins lacking these domains that are unable to mediate the degradation of b-catenin.

[0211] Aminoglycoside- and macrolide-induced readthrough generally results in a recovery of 2–10% of normal protein levels. It has been reported that this amount of full-length protein is sufficient to restore APC function

[40] . However, traditional antibody-based methods have difficulty detecting small amounts of large proteins such as APC. Recently, the sensitivity and specificity of commercially available anti-APC antibodies have been questioned. Therefore, we quantified the relative levels of cytoplasmic and nuclear b-catenin as an indicator of functional activity of the APC protein

[36] . Western blot analysis and immunohistochemistry showed that human colon cancer cells containing nonsense mutations in the APC gene and APC minWe found evidence that treatment of mouse adenomatous epithelial cells with the compounds of the present invention listed in Table A resulted in a decrease in nuclear b-catenin. Furthermore, in colon cancer cells, this decrease in nuclear b-catenin subsequently led to a decrease in c-myc protein levels and inhibition of cell proliferation. This is consistent with other studies in which treatment of cultured colorectal cancer cells containing APC nonsense mutations with macrolide antibiotics and aminoglycosides resulted in a decrease in cell proliferation and nuclear b-catenin levels [23, 24, 33]. In these studies, treatment of SW1417 colon cancer cells with tylosin promoted some readthrough of the nonsense mutation, leading to the synthesis of low levels of full-length, active APC protein

[23] . In a study aimed at assessing aminoglycoside-mediated readthrough of several APC nonsense mutations, Floquet et al. utilized a luciferase-based reporter vector to measure the interaction of active APC with b-catenin and showed that the L360X stop codon was most susceptible to gentamicin-mediated readthrough

[40] . This indirect approach alleviated the potential difficulty of detecting low levels of APC protein that may be sufficient for biological activity but cannot be detected by Western blotting

[40] . Restoring even 1% of normal protein function may be sufficient to reduce the severity of diseases associated with APC dysfunction.

[0212] APC min In certain animal models of disease associated with inactive APC, such as mouse or human colon cancer tumor xenografts, inhibition of premature translation termination of APC mRNA has been shown to ameliorate the disease in terms of reducing intestinal polyps and inhibiting tumor growth [23, 24]. min Similar results were obtained from in vivo studies using models, where we demonstrate that treatment with the novel macrolide compounds of the present invention as set forth in Table A reduced the number of intestinal polyps and inhibited the neoplastic development of intestinal lesions. Furthermore, APCs treated with the compounds of the present invention as set forth in Table A min100% of the mice survived the 8-week study without overt clinical signs of disease. In the control group, adverse clinical findings were observed by the third week of the study, with only 50% of the vehicle-treated mice remaining at the end of the study. Furthermore, compound treatment reduced the number of visible polyps in the small intestine by 39%, which was consistent with anemia and treatment APC. min It is the main factor in mouse survival.

[20] Subsequent dose-response studies showed that APC min All doses of the compound tested in mice were effective. Even the lowest daily dose of 12.5 mg / kg bw reduced anemia and contributed to long-term survival, as indicated by a 43% increase in hemoglobin levels and a 41% reduction in splenomegaly. Our in vivo studies demonstrated that older APCs, at an age where intestinal adenomas are pre-existing, min It is important to note that this study utilized mice; thus, providing reasonable support for the therapeutic use of the compounds of the present invention described in Table A for the treatment of FAP. In addition to its nuclear function, b-catenin is also an important part of membrane adherens junctions, where it associates with E-cadherin (PMID: 26240067). Dysregulation of the Wnt pathway, leading to increased nuclear localization of b-catenin, also results in decreased cell-cell adhesion and cell loss from villi (PMID: 26240067). As shown in Figure 5, many epithelial cells in adenomas from compound-treated mice have a conventional columnar morphology and exhibit b-catenin staining aligned along the cell membrane. We also observed increased levels of e-cadherin associated with the epithelial cell membranes in adenomas from compound-treated mice (data not shown). Taken together, compound treatment can enhance differentiation, as suggested by increased levels of membrane-bound b-catenin and e-cadherin, which form adherens junctions, and may slow or reverse disease progression

[42] .

[0213] In eukaryotic cells, macrolide antibiotics target the proximal region of the nascent peptide exit tunnel of the large ribosomal subunit, disrupting the peptidyl transferase center and slowing nascent polypeptide synthesis. This stall allows a near-cognate tRNA amino acid to be inserted at the site of a premature termination codon, allowing translation to continue [43, 44]. Conventional macrolide antibiotics that exhibit readthrough activity are unsuitable for the chronic administration required to treat FAP patients. Long-term use of these conventional macrolide antibiotics can cause cardiotoxicity and hepatotoxicity. These toxicities are related to macrolide-mediated inhibition of the hERG potassium channel in cardiomyocytes and the bile salt export pump (BSEP) in hepatocytes [28-30, 45-47]. We have developed and optimized the novel macrolides disclosed herein with enhanced specificity for mammalian ribosomes and minimal off-target inhibition of hERG or BSEP activity (data not shown). Furthermore, our pharmacokinetic data demonstrate that the compounds disclosed herein can penetrate and effectively accumulate in intestinal tissues after a single oral dose. This pharmacological profile, with improved efficacy, minimal off-target activity, and appropriate target tissue exposure, supports the application of the compounds disclosed herein for the chronic treatment of FAP patients.

[0214] Our findings demonstrate that the macrolides disclosed herein can suppress premature protein translation termination induced by nonsense mutations in the APC gene, resulting in the restoration of active APC protein. In vitro, treatment with the compounds disclosed herein restored APC function in human colon cancer cells harboring known nonsense mutations in the APC gene. This was demonstrated by reduced b-catenin levels in the nuclei of SW403 and SW1417 colon cancer cells treated with the compounds disclosed herein, indicating APC-mediated degradation of cytoplasmic b-catenin. Furthermore, protein levels of c-myc, a downstream target of the b-catenin / Wnt pathway, were reduced in SW1417 and SW403 cells treated as disclosed herein. These results provide strong evidence that the compounds disclosed herein promote nonsense codon readthrough and restore functional APC protein. The in vivo efficacy of the compounds disclosed herein was demonstrated in APC cells treated at an age when polyps are already present. min This has been demonstrated in a FAP model. Treatment with the compounds disclosed herein significantly reduced the number of intestinal polyps and adenomatous tissues in these mice, alleviating the anemia associated with the model and leading to improved survival rates. Furthermore, immunopathology showed that many epithelial cells in the lesions of mice treated with the compounds disclosed herein had reduced nuclear b-catenin levels, increased membrane-bound b-catenin, and a more differentiated morphology. Taken together, our findings provide preclinical support for evaluating the therapeutic value of long-term use of the compounds disclosed herein for the treatment of FAP patients.

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Moser AR, Luongo C, Gould KA, McNeley MK, Shoemaker AR, Dove WF. ApcMin: a mouse model for intestinal and mammary tumorigenesis. Eur J Cancer 1995; 31A: 1061-1064. 21. Heyer J, Yang K, Lipkin M, Edelmann W, Kucherlapati R. Mouse models for colorectal cancer. Oncogene 1999; 18: 5325-5333. 22. Zhang J, Cao H, Zhang B, Cao H, Xu X, Ruan H et al. Berberine potently attenuates intestinal polyps growth in ApcMin mice and familial adenomatous polyposis patients through inhibition of Wnt signalling. J Cell Mol Med 2013; 17: 1484-1493. 23. Zilberberg A, Lahav L, Rosin-Arbesfeld R. Restoration of APC gene function in colorectal cancer cells by aminoglycoside- and macrolide-induced read-through of premature termination codons. Gut 2010; 59: 496-507. 24. Caspi M, Firsow A, Rajkumar R, Skalka N, Moshkovitz I, Munitz A et al. A flow cytometry-based reporter assay identifies macrolide antibiotics as nonsense mutation read-through agents. J Mol Med (Berl) 2016; 94: 469-482. 25. Kariv R, Caspi M, Fliss-Isakov N, Shorer Y, Shor Y, Rosner G et al. Resorting the function of the colorectal cancer gatekeeper adenomatous polyposis coli. Int J Cancer 2020; 146: 1064-1074. 26. Keeling KM, Lanier J, Du M, Salas-Marco J, Gao L, Kaenjak-Angeletti A et al. Leaky termination at premature stop codons antagonizes nonsense-mediated mRNA decay in S. cerevisiae. RNA 2004; 10: 691-703. 27. McCaughan KK, Brown CM, Dalphin ME, Berry MJ, Tate WP. Translational termination efficiency in mammals is influenced by the base following the stop codon. Proc Natl Acad Sci U S A 1995; 92: 5431-5435. 28. Guo D, Cai Y, Chai D, Liang B, Bai N, Wang R. The cardiotoxicity of macrolides: a systematic review. Pharmazie 2010; 65: 631-640. 29. Li X, Wang M, Liu G, Zhou L, Wang Z, Li C. Macrolides use and the risk of sudden cardiac death. Expert Rev Anti Infect Ther 2016; 14: 535-537. 30. Brinker AD, Wassel RT, Lyndly J, Serrano J, Avigan M, Lee WM et al. 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[0216] Equivalents and ranges Furthermore, the present invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, any claim that depends on another claim can be amended to include one or more limitations found in any other claim that depends from the same base claim. When elements are presented as a list, e.g., in Markush group format, each subgroup of elements is also disclosed, and any element can be deleted from the group. In general, when the invention or aspects of the invention are referred to as comprising particular elements and / or features, it is understood that a particular embodiment of the invention or aspect of the invention consists of, or consists essentially of, such elements and / or features. For the sake of brevity, these embodiments have not been specifically described verbatim herein. It should also be noted that the terms "comprising" and "having" are intended to be open and allow for the inclusion of additional elements or steps. When ranges are specified, endpoints are included. Furthermore, unless otherwise indicated or apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges can assume any specific value or subrange within the stated range in various embodiments of the invention, down to one-tenth of the unit of the lower limit of the range.

[0217] This application references various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of a conflict between any of the incorporated references and this specification, this specification will control. Furthermore, certain embodiments of the present invention that fall within the prior art may be expressly excluded from any one or more of the claims. Such embodiments are deemed known to those of ordinary skill in the art and may therefore be excluded even if the exclusion is not expressly set forth herein. Any particular embodiment of the present invention may be excluded from any claim for any reason, whether or not related to the existence of prior art.

[0218] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above, but rather is as set forth in the appended claims. Those skilled in the art will appreciate that various changes and modifications can be made to the specification without departing from the spirit or scope of the invention as defined in the claims.

Claims

1. Formula I for treating familial adenomatous polyposis (FAP): 【Chemistry 1】 [During the ceremony, R 9a H and C which may be substituted 1-6 Selected from the group consisting of alkyl groups; and R 10a This is a heteroaryl compound that may be substituted. A pharmaceutical composition comprising a compound or a pharmaceutically acceptable salt thereof as an active ingredient.

2. The pharmaceutical composition according to claim 1, wherein R 9a is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, or isopentyl.

3. R 10a is: 【Chemistry 2】 The pharmaceutical composition according to claim 1.

4. The compound of formula I is formula II: 【Chemistry 2】 [During the ceremony, R9a is selected from the group consisting of H and optionally substituted C1-6 alkyl groups; and 【Transformation 3】 This is a six-membered aromatic or heteroaromatic ring that may be optionally substituted. The pharmaceutical composition according to claim 1, wherein the compound is or a pharmaceutically acceptable salt thereof.

5. The pharmaceutical composition according to claim 4, wherein R 9a is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, or isopentyl. 【Request Item 6】 【Chemistry 4】 but 【Transformation 5】 The pharmaceutical composition according to claim 4.

7. The pharmaceutical composition according to claim 1, wherein the compound of formula I or a pharmaceutically acceptable salt thereof is selected from the compounds shown below. Table 1 Table 2 Table 3

8. The pharmaceutical composition according to claim 1, wherein FAP is FAP, mild FA, or autosomal recessive FAP.

9. The pharmaceutical composition according to claim 8, wherein FAP is FAP.

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