Patent Application
20240358735
Secondary metabolites derived from plants, fungi and microbes are among the richest sources of therapeutically useful chemical compounds. For example, in the decade between 2000 and 2010, approximately 50% of all NCEs (new chemical entities) approved by the US FDA for use as human drugs were natural products or derivatives of natural products (J Nat Prod. 2012 Mar. 23; 75(3): 311-335).
Recent investigations by the inventors have demonstrated that nematodes are an unexpected and rich source of molecules with diverse biological activities. Meanwhile, as the underlying mechanisms of aging, and a wide range of human health disorders becomes better understood, the need for more selective and efficacious therapeutic and pharmaceutical treatments has never been greater.
The present invention addresses these and other related needs.
This invention pertains to the field of small molecule therapeutics and provides therapeutic compositions and pharmacologically active analogs of compounds first identified in nematodes as well as methods of using the same therapeutically.
Among other things, the present invention encompasses the inventors' discovery of a family of novel small molecule metabolites produced by nematodes including C. elegans. The inventors have made important additional observations regarding the production and function of these metabolites including: the biosynthetic processes by which the novel metabolites are produced (and the similarity of those biosynthetic pathways to those known to operate in other more complex animals including humans); the distribution of the new metabolites within the producing organisms' bodies; the levels of excretion (or lack thereof) of the metabolites into the producing organisms' environment; the different absolute and/or relative abundances of the metabolites among different species of producing organism; changes in such abundances at different life stages of the producing organisms; and the changing levels of absolute and/or relative production, accumulation or consumption of these metabolites in response to diverse metabolic and/or environmental stimuli. Based on these insights, the inventors have recognized that administering compositions containing the identified metabolites (or analogs thereof) provides a useful strategy to treat certain diseases and/or improve the health of animals including humans.
The nematode C. elegans has become an important model system for metabolomics and small molecule signaling in animals. These efforts have led to the identification of a large, structurally diverse library of signaling molecules derived from glycosides of the dideoxysugar ascarylose (
The biosynthesis of most cest-dependent ascarosides further depends on the activity of Cel-GLO-1, a Rab GTPase that is required for the formation of lysosome-related organelles (LROs), cellular compartments similar to mammalian melanosomes.7,12 Recent comparative metabolomic studies of Cel-glo-1 mutants and wildtype C. elegans led to the discovery of a previously undescribed class of metabolites, a large library of over one hundred modular glucosides (MOGLs).7 The MOGLs are derived from combinatorial attachment of a wide range of metabolic building blocks to several different core scaffolds, e.g. indole glucoside (iglu #1 (4), iglu #2 (5)), anthranilic acid glucoside (angl #1 (6), angl #2 (7)), or tyramine glucoside (tyglu #3 (8), tyglu #1 (9),
In the MOGLs, other building blocks are linked to the core scaffolds via ester bonds, suggesting that MOGL biosynthesis may also be mediated by cest homologs. Comparative metabolomic analysis of Cel-cest-4 mutants recently showed that Cel-CEST-4 is required for 6-O-attachment of anthranilic acid in two MOGLs, iglu #3 (10) and iglu #4 (11) (
Therefore, in one aspect, the present invention encompasses therapeutic compositions comprising a therapeutically effective amount of one or more such metabolites or derivatives or analogs of such metabolites. In certain embodiments, such therapeutic compositions comprise compounds known as Modular Glucosides or MOGLs—a family of small molecules newly identified in nematodes. MOGLs all contain a glucose moiety decorated with specific substituents present in a variety of substitution patterns. The substitution patterns are described herein with reference to the carbon atom of the glucose ring to which such substituents are attached. For reference, the numbering convention used herein to describe these glucose substitution patterns is shown below.
In most cases, the substituents described herein are attached via covalent bonds to one of the hydroxyl oxygen atoms of the glucose molecule (e.g. through ester, or ether linkages) however, for substituents attached at the 1-position (also referred to as the anomeric position), substituents may either be attached via the oxygen atom, or may be attached via another heteroatom covalently bound to the C1 position—an example of the latter would be an N-linked heterocycle attached to the 1-position.
In certain embodiments, the present invention provides therapeutic compositions comprising one or more MOGLs featuring a glucose molecule having a phosphate group (or a derivative of a phosphate group) at the 3-position of the glucose. In certain embodiments, such MOGLs have additional substitution at one or more of the 1-, 2-, and 6-positions. In certain embodiments, such MOGLs have a phosphate group (or a derivative of a phosphate group) at the 3-position of the glucose and a free —OH group at the 4-position.
In certain embodiments, the present invention provides therapeutic compositions comprising one or more MOGLs featuring a glucose molecule substituted at the 1-, 2-, 3-, and 6-positions, wherein:
In certain embodiments, the provided compositions are further characterized in that the glucose derivative is not substituted at the 4-position.
In certain embodiments, such therapeutic compositions comprise MOGLs featuring a glucose derivative substituted at the 1-, 2-, and 3-positions, wherein:
In certain embodiments, the provided compositions are further characterized in that the glucose derivative is not substituted at the 4- or 6-position.
In certain embodiments, the present invention provides therapeutic compositions comprising MOGLs featuring a glucose derivative substituted at the 1-, 3-, and 6-positions, wherein:
In certain embodiments, the provided compositions are further characterized in that the glucose derivative is not substituted at the 2- or 4-position
In certain embodiments, the present invention provides therapeutic compositions comprising MOGLs featuring a glucose derivative substituted at the 1- and 3-positions, wherein:
In certain embodiments, the provided compositions are further characterized in that the glucose derivative is not substituted at the 2-, 4- or 6-position.
In certain embodiments, the present invention provides therapeutic compositions comprising MOGLs featuring a glucose derivative substituted at the 1-, 2-, and 6-positions, wherein:
In certain embodiments, the provided compositions are further characterized in that the glucose derivative is not substituted at the 3- or 4-position.
In certain embodiments, the present invention provides therapeutic compositions comprising MOGLs featuring a glucose derivative substituted at the 1- and 2-, positions, wherein:
In certain embodiments, the provided compositions are further characterized in that the glucose derivative is not substituted at the 3-, 4- or 6-position.
In certain embodiments, the present invention provides therapeutic compositions comprising MOGLs featuring a glucose derivative substituted at the 1- and 6-positions, wherein:
In certain embodiments, the provided compositions are further characterized in that the glucose derivative is not substituted at the 2-, 3- or 4-position.
In certain embodiments, the present invention provides therapeutic compositions comprising a gluconucleoside. In certain embodiments, the MOGL-containing therapeutic compositions described above are characterized in that the substituent at the 1-position comprises a nucleobase. In certain embodiments, the nucleobase is N-linked to the 1-position of the glucose scaffold. In certain embodiments, the N-linked nucleobase comprises a pyrimidine base. In certain embodiments, the N-linked nucleobase comprises a purine base. In certain embodiments, the N-linked nucleobase comprises a primary nucleobase. In certain embodiments, the N-linked nucleobase is other than a primary nucleobase, or is an analog or adduct of a primary nucleobase. In certain embodiments, the N-linked nucleobase is a methylated nucleobase. In certain embodiments, the N-linked nucleobase is selected from the group consisting of adenine, cytosine, guanine, thymine, and uracil. In certain embodiments, the nucleobase comprises guanine. In certain embodiments, the nucleobase comprises a methylated analog of guanine. In certain embodiments, the nucleobase comprises 6-O-methyl guanine.
In certain embodiments, the MOGL-containing therapeutic compositions described above are characterized in that the substituent at the 1-position comprises an optionally substituted N-linked heterocycle. In certain embodiments, the N-linked heterocycle comprises a 5- or 6-membered ring containing at least one nitrogen atom. In certain embodiments, the N-linked heterocycle contains one or more sites of unsaturation. In certain embodiments, the N-linked heterocycle comprises indole. In certain embodiments, the N-linked heterocycle comprises a substituted indole. In certain embodiments, the N-linked heterocycle comprises a hydroxy indole. In certain embodiments, the N-linked heterocycle comprises serotonin.
In certain embodiments, the MOGL-containing therapeutic compositions described above are characterized in that the substituent at the 1-position comprises an optionally unsaturated acyl group. In certain embodiments, the substituent at the 1-position comprises an alpha beta unsaturated acyl group. In certain embodiments, an acyl substituent at the 1-position comprises a C3-8 aliphatic group with alpha beta unsaturation. In certain embodiments, the substituent at the 1-position comprises crotonate. In certain embodiments, the substituent at the 1-position comprises tiglate. In certain embodiments, the substituent at the 1-position comprises angelate. In certain embodiments, the substituent at the 1-position comprises valerate. In certain embodiments, the substituent at the 1-position comprises acrylate, methacrylate, or cinnamate. In certain embodiments, the substituent at the 1-position comprises 2-imidazoleacrylate. In certain embodiments, the substituent at the 1-position comprises urocanate.
In certain embodiments, the MOGL-containing therapeutic compositions described above are characterized in that the substituent at the 1-position comprises an acyl- or ether-linked aromatic moiety substituted with an amine or an aminoalkyl group. In certain embodiments, the provided compositions are characterized in that the substituent at the 1-position comprises an acyl-linked aromatic moiety substituted with an amine. In certain embodiments, the acyl-linked aromatic moiety comprises a phenyl ring. In certain embodiments, the acyl-linked aromatic moiety comprises a substituted benzoyl group. In certain embodiments, the acyl-linked aromatic moiety comprises an optionally substituted aminobenzoyl group. In certain embodiments, the acyl-linked aromatic moiety comprises anthranilic acid.
In certain embodiments, substituent at the 1-position comprises an ether-linked aromatic moiety substituted with an aminoalkyl group. In certain embodiments, the ether-linked aromatic moiety comprises a phenyl ring. In certain embodiments, substituent at the 1-position comprises a phenolic ether where the phenyl ring of the phenol is substituted with an aminoalkyl group. In certain embodiments, the ether-linked aromatic moiety comprises a phenol substituted with an optionally substituted 2-aminoethyl group. In certain embodiments, the ether-linked aromatic moiety comprises tyramine. In certain embodiments, the ether-linked aromatic moiety comprises octopamine. In certain embodiments, the substituent at the 1-position comprises O-linked serotonin. In certain embodiments, the substituent at the 1-position comprises O-linked N-acetylserotonin (normelatonin). In certain embodiments, the substituent at the 1-position comprises O-linked dopamine. In certain embodiments, the substituent at the 1-position comprises 3-O-linked dopamine. In certain embodiments, the substituent at the 1-position comprises 4-O-linked dopamine. In certain embodiments, the substituent at the 1-position comprises 3-O-linked norepinepherine. In certain embodiments, the substituent at the 1-position comprises 4-O-linked norepinepherine. In certain embodiments, the substituent at the 1-position comprises 3-O-linked epinepherine. In certain embodiments, the substituent at the 1-position comprises 4-O-linked epinepherine.
In certain embodiments, the MOGL-containing therapeutic compositions described above are characterized in that the substituent at the 2-position comprises an optionally substituted aromatic or heteroaromatic acyl group. In certain embodiments, the substituent at the 2-position comprises an optionally substituted benzoate. In certain embodiments, the optionally substituted benzoate is selected from the group consisting of benzoate, anthranilate, and p-hydroxybenzoate. In certain embodiments, the substituent at the 2-position comprises an optionally substituted heteroaromatic acyl group. In certain embodiments, the substituent at the 2-position comprises a heteroaromatic acyl group with a 6-membered heteroaromatic moiety. In certain embodiments, the substituent at the 2-position comprises a pyridine or pyrimidine carboxylate ester. In certain embodiments, the 2-substituent comprises nicotinate. In certain embodiments, the 2-substituent comprises picolinate. In certain embodiments, the 2-substituent comprises isonicotinate. In certain embodiments, the substituent at the 2-position comprises a heteroaromatic acyl group with a 5-membered heteroaromatic moiety. In certain embodiments, the substituent at the 2-position comprises the ester of a pyrrole or imidazole carboxylic acid. In certain embodiments, the substituent at the 2-position is pyrrole-2-carboxylate.
In certain embodiments, the MOGL-containing therapeutic compositions described above are characterized in that the substituent at the 2-position comprises an optionally substituted aliphatic acyl group. In certain embodiments, the acyl group at the 2-position comprises an optionally substituted aliphatic group. In certain embodiments, the acyl group at the 2-position comprises an optionally substituted C1-40 aliphatic group, an optionally substituted C2-24 aliphatic group, an optionally substituted C12-24 aliphatic group, an optionally substituted C2-18 aliphatic group, an optionally substituted C2-12 aliphatic group, an optionally substituted C2-8 aliphatic group, or an optionally substituted C1-6 aliphatic group. In certain embodiments, the optionally substituted acyl group at the 2-position comprises a hydroxylated C1-40 aliphatic group. In certain embodiments, the optionally substituted acyl group at the 2-position comprises an epoxidized C1-40 aliphatic group. In certain embodiments, such optionally substituted aliphatic groups are saturated. In certain embodiments, such optionally substituted aliphatic groups have one or more sites of unsaturation. In certain embodiments, such unsaturated aliphatic groups have unsaturation adjacent to the carbonyl of the acyl linkage (e.g. they are alpha-beta unsaturated esters). In certain embodiments, an acyl substituent at the 2-position comprises a C2-8 aliphatic group with alpha beta unsaturation. In certain embodiments, the substituent at the 2-position comprises crotonate. In certain embodiments, the substituent at the 2-position comprises tiglate.
In certain embodiments, the substituent at the 2-position comprises angelate. In certain embodiments, the substituent at the 2-position comprises acrylate, methacrylate, 3-methylcrotonate, isocrotonate, or optionally substituted cinnamate.
In certain embodiments, the MOGL-containing therapeutic compositions described above are characterized in that the substituent at the 3-position of the glucose comprises a phosphate—this may be a simple phosphate (e.g. —OPO3H2) or may comprise a di-, tri- or higher phosphate (e.g. —O—(P(O3H)n—H, where n is an integer greater than 1, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10), or a phosphate derivative. In certain embodiments, the 3-substituent is phosphate. In certain embodiments, the 3-substituent is diphosphate. In certain embodiments, the 3-substituent is triphosphate. For the synthesis of di- and triphosphate MOGLs, the corresponding mono-phosphates can be synthesized and subsequently converted into diphosphates and triphosphates using, for example, the strategy outlined in Angewandte Chemie-International Edition, 2022, vol. 61, Issue 22 (May 23, 2022, E202201731).
In certain embodiments, the composition is provided in a form wherein the phosphate moiety at the 3-position is protonated. In certain embodiments, the composition is provided in a form wherein the phosphate moiety at the 3-position comprises a salt (e.g. where one or more of —H groups on the phosphate are replaced by a metal cation, organic ‘onium’ or inorganic ‘onium’ group).
In certain embodiments, the MOGL-containing therapeutic compositions described above are characterized in that the substituent at the 3-position of the glucose comprises a sulfate. In certain embodiments, the composition is provided in a form wherein the sulfate moiety at the 3-position comprises a salt (e.g. where one or more of —H groups on the sulfate are replaced by a metal cation, organic ‘onium’ or inorganic ‘onium’ group).
In certain embodiments, the MOGL-containing therapeutic compositions described above are characterized in that the substituent at the 6-position comprises an optionally substituted moiety selected from the group consisting of: an acyl-linked amino acid, an aromatic acyl group and an aliphatic acyl group. In certain embodiments, the substituent at the 6-position comprises an acyl linked amino acid. In certain embodiments, the amino acid is an alpha amino acid. In certain embodiments, the amino acid comprises a proteinogenic amino acid. In certain embodiments, the amino acid comprises one of the 20 encoded proteogenic amino acids. In certain embodiments, the amino acid is phenylalanine. In certain embodiments, the substituent at the 6-position comprises a peptide linked to the glucose via an ester bond.
In certain embodiments, the substituent at the 6-position comprises an aromatic acyl group. In certain embodiments, the substituent at the 6-position comprises an optionally substituted benzoate. In certain embodiments, the optionally substituted benzoate is selected from the group consisting of: benzoate, anthranilate, and p-hydroxybenzoate. In certain embodiments, the substituent at the 6-position comprises anthranilate.
In certain embodiments, the substituent at the 6-position comprises a heteroaromatic acyl group. In certain embodiments, the substituent at the 6-position comprises an optionally substituted heteroaromatic acyl group with a 6-membered heteroaromatic moiety. In certain embodiments, the substituent at the 6-position comprises a pyridine or pyrimidine carboxylate ester. In certain embodiments, the 6-substituent comprises nicotinate. In certain embodiments, the 6-substituent comprises picolinate. In certain embodiments, the 6-substituent comprises isonicotinate. In certain embodiments, the substituent at the 6-position comprises a heteroaromatic acyl group with a 5-membered heteroaromatic moiety. In certain embodiments, the substituent at the 6-position comprises the ester of a pyrrole or imidazole carboxylic acid. In certain embodiments, the substituent at the 6-position is pyrrole-2-carboxylate
In certain embodiments, the MOGL-containing therapeutic compositions described above are characterized in that the substituent at the 6-position comprises an optionally substituted aliphatic acyl group. In certain embodiments, the acyl group at the 6-position comprises an optionally substituted aliphatic group. In certain embodiments, the acyl group at the 6-position comprises an optionally substituted C1-30 aliphatic group, an optionally substituted C2-24 aliphatic group, an optionally substituted C12-24 aliphatic group, an optionally substituted C2-18 aliphatic group, an optionally substituted C2-12 aliphatic group, an optionally substituted C2-8 aliphatic group, or an optionally substituted C1-6 aliphatic group. In certain embodiments, the acyl group at the 6-position comprises phenylacetate. In certain embodiments, such optionally substituted aliphatic groups are saturated. In certain embodiments, acyl groups at the 6-position have one or more sites of unsaturation. In certain embodiments, the 6-substituent comprises an unsaturated aliphatic group having unsaturation adjacent to the carbonyl of the acyl linkage (e.g. they are alpha-beta unsaturated esters). In certain embodiments, an acyl substituent at the 6-position comprises a C3-8 aliphatic group with alpha beta unsaturation. In certain embodiments, the substituent at the 6-position comprises crotonate. In certain embodiments, the substituent at the 6-position comprises crotonate. In certain embodiments, the substituent at the 6-position comprises tiglate. In certain embodiments, the substituent at the 6-position comprises angelate. In certain embodiments, the substituent at the 6-position comprises acrylate, methacrylate, or cinnamate. In certain embodiments, the substituent at the 6-position comprises 2-imidazoleacrylate. In certain embodiments, the substituent at the 6-position comprises urocanate.
In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula I:
wherein:
In certain embodiments, a compound of Formula I comprises any one or more of the modular glucosides encompassed by the formula:
For avoidance of the doubt, the depiction above represents the combinatorial range of unique molecules resulting from independently choosing any one of the depicted moieties for attachment to each of the indicated positions by replacement of a dashed line in the figure with a covalent bond.
In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula I:
wherein each of G1, G2 and X is as defined above and in the genera and subgenera herein.
In certain embodiments, a compound of Formula II comprises any of the modular glucosides encompassed by the formula:
In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula III:
wherein each of G1, X and G6 is as defined above and in the genera and subgenera herein.
In certain embodiments, a compound of Formula III comprises any of the modular glucosides encompassed by the formula:
In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula IV:
wherein each of G1 and X is as defined above and in the genera and subgenera herein.
In certain embodiments, a compound of Formula IV comprises any of the modular glucosides encompassed by the formula:
In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula V:
wherein each of G1, G2 and G6 is as defined above and in the genera and subgenera herein.
In certain embodiments, a compound of Formula V comprises any of the modular glucosides encompassed by the formula:
In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula VI:
wherein each of G1 and G2 is as defined above and in the genera and subgenera herein.
In certain embodiments, a compound of Formula VI comprises any of the modular glucosides encompassed by the formula:
In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula VII:
wherein each of G1, and G6 is as defined above and in the genera and subgenera herein.
In certain embodiments, a compound of Formula VII comprises any of the modular glucosides encompassed by the formula:
In another aspect, the present invention encompasses methods of improving the health of an animal or of treating or ameliorating a health disorder in an animal by administering to the animal an effective amount of any one or more of the therapeutic compositions described above. In certain embodiments, the method comprises administering such a composition to a mammal. In certain embodiments, the method comprises administering such a composition to a human.
In another aspect, the present invention comprises methods of making therapeutic compositions comprising formulating an effective amount of one or more purified or synthetically-produced MOGLs (or a pharmaceutically-acceptable salt, prodrug or derivative thereof) into a therapeutic composition. In certain embodiments, such therapeutic compositions are selected from the group consisting of: an injectible liquid, a tablet, a capsule, a pill, a solution or suspension for oral administration, a solid dosage form for suspension or dissolution into a drinkable- or injectible liquid, a dermal patch, an eye drop, a cream, an ointment, a gel, a powder, a spray, an inhalable composition, and a nasal spray.
Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Organic Chemistry, Thomas Sorrell, University Science Books, Sausalito, 1999; Smith and March March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987; the entire contents of each of which are incorporated herein by reference.
Certain compounds of the present invention can comprise one or more asymmetric centers, and thus can exist in various stereoisomeric forms, e.g., enantiomers and/or diastereomers. Thus, inventive compounds and compositions thereof may be in the form of an individual enantiomer, diastereomer or geometric isomer, or may be in the form of a mixture of stereoisomers. Unless otherwise stated, all tautomeric forms of the compounds of the disclosure are within the scope of the invention. In certain embodiments, the compounds of the invention are enantiopure compounds. In certain other embodiments, mixtures of enantiomers or diastereomers are provided.
Furthermore, certain compounds, as described herein may have one or more double bonds that can exist as either a Z or E isomer, unless otherwise indicated. The invention additionally encompasses the compounds as individual isomers substantially free of other isomers and alternatively, as mixtures of various isomers, e.g., racemic mixtures of enantiomers.
In addition to the above-mentioned compounds per se, this invention also encompasses compositions comprising one or more compounds.
Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13C- or 14C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present invention.
As used herein, the term “isomers” includes any and all geometric isomers and stereoisomers. For example, “isomers” include cis- and trans-isomers, E- and Z-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention. For instance, a compound may, in some embodiments, be provided substantially free of one or more corresponding stereoisomers, and may also be referred to as “stereochemically enriched.”
Where a particular enantiomer is preferred, it may, in some embodiments be provided substantially free of the opposite enantiomer, and may also be referred to as “optically enriched.” “Optically enriched,” as used herein, means that the compound is made up of a significantly greater proportion of one enantiomer. In certain embodiments the compound is made up of at least about 90% by weight of an enantiomer. In some embodiments the compound is made up of at least about 95%, 97%, 98%, 99%, 99.5%, 99.7%, 99.8%, or 99.9% by weight of an enantiomer. In some embodiments the enantiomeric excess of provided compounds is at least about 90%, 95%, 97%, 98%, 99%, 99.5%, 99.7%, 99.8%, or 99.9%. In some embodiments, enantiomers may be isolated from racemic mixtures by any method known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts or prepared by asymmetric syntheses. See, for example, Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, S. H., et al., Tetrahedron 33:2725 (1977); Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, S. H. Tables of Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972).
The terms “halo” and “halogen” as used herein refer to an atom selected from fluorine (fluoro, —F), chlorine (chloro, —Cl), bromine (bromo, —Br), and iodine (iodo, —I).
The term “acyl” as used herein refers to a group having a formula —C(O)R where R is hydrogen or an optionally substituted aliphatic, carbocyclic, heteroaliphatic, aryl, heteroaryl, or heterocyclic group. In some embodiments, a carbon atom of R is attached to the carbonyl carbon of an acyl group.
The term “aliphatic” or “aliphatic group”, as used herein, denotes a hydrocarbon moiety that may be straight-chain (i.e., unbranched), branched, or cyclic (including fused, bridging, and spiro-fused polycyclic) and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. Unless otherwise specified, aliphatic groups contain 1-30 carbon atoms. In certain embodiments, aliphatic groups contain 1-40 carbon atoms. In certain embodiments, aliphatic groups contain 1-24 carbon atoms. In certain embodiments, aliphatic groups contain 1-12 carbon atoms. In certain embodiments, aliphatic groups contain 1-8 carbon atoms. In certain embodiments, aliphatic groups contain 1-6 carbon atoms. In some embodiments, aliphatic groups contain 1-5 carbon atoms, in some embodiments, aliphatic groups contain 1-4 carbon atoms, in yet other embodiments aliphatic groups contain 1-3 carbon atoms, and in yet other embodiments aliphatic groups contain 1-2 carbon atoms. Suitable aliphatic groups include, but are not limited to, linear or branched, alkyl, alkenyl, and alkynyl groups, and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.
The term “unsaturated”, as used herein, means that a moiety has one or more double or triple bonds.
The terms “cycloaliphatic”, used alone or as part of a larger moiety, refer to a saturated or partially unsaturated cyclic aliphatic monocyclic, bicyclic, or polycyclic ring systems, as described herein, having from 3 to 12 members, wherein the aliphatic ring system is optionally substituted as defined above and described herein. Cycloaliphatic groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cyclooctenyl, and cyclooctadienyl. In some embodiments, the cycloaliphatic group has 3-6 carbons. The terms “cycloaliphatic”, also include aliphatic rings that are fused to one or more aromatic or nonaromatic rings, such as decahydronaphthyl or tetrahydronaphthyl, where the radical or point of attachment is on the aliphatic ring. In some embodiments, a cycloaliphatic group is bicyclic. In some embodiments, a cycloaliphatic group is tricyclic. In some embodiments, a cycloaliphatic group is polycyclic.
In some embodiments, a cycloaliphatic group is 3- to 7-membered saturated or partially unsaturated monocyclic carbocyclyl. In some embodiments, a cycloaliphatic group is 4- to 12-membered saturated or partially unsaturated bicyclic carbocyclyl.
The term “alkyl,” as used herein, refers to saturated, straight- or branched-chain hydrocarbon radicals derived from an aliphatic moiety containing between one and six carbon atoms by removal of a single hydrogen atom. Unless otherwise specified, alkyl groups contain 1-12 carbon atoms. In certain embodiments, alkyl groups contain 1-8 carbon atoms. In certain embodiments, alkyl groups contain 1-6 carbon atoms. In some embodiments, alkyl groups contain 1-5 carbon atoms, in some embodiments, alkyl groups contain 1-4 carbon atoms, in yet other embodiments alkyl groups contain 1-3 carbon atoms, and in yet other embodiments alkyl groups contain 1-2 carbon atoms. Examples of alkyl radicals include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, sec-pentyl, iso-pentyl, tert-butyl, n-pentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, dodecyl, and the like.
The term “alkenyl,” as used herein, denotes a monovalent group derived from a straight- or branched-chain aliphatic moiety having at least one carbon-carbon double bond by the removal of a single hydrogen atom. Unless otherwise specified, alkenyl groups contain 2-12 carbon atoms. In certain embodiments, alkenyl groups contain 2-8 carbon atoms. In certain embodiments, alkenyl groups contain 2-6 carbon atoms. In some embodiments, alkenyl groups contain 2-5 carbon atoms, in some embodiments, alkenyl groups contain 2-4 carbon atoms, in yet other embodiments alkenyl groups contain 2-3 carbon atoms, and in yet other embodiments alkenyl groups contain 2 carbon atoms. Alkenyl groups include, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.
The term “alkynyl,” as used herein, refers to a monovalent group derived from a straight- or branched-chain aliphatic moiety having at least one carbon-carbon triple bond by the removal of a single hydrogen atom. Unless otherwise specified, alkynyl groups contain 2-12 carbon atoms. In certain embodiments, alkynyl groups contain 2-8 carbon atoms. In certain embodiments, alkynyl groups contain 2-6 carbon atoms. In some embodiments, alkynyl groups contain 2-5 carbon atoms, in some embodiments, alkynyl groups contain 2-4 carbon atoms, in yet other embodiments alkynyl groups contain 2-3 carbon atoms, and in yet other embodiments alkynyl groups contain 2 carbon atoms. Representative alkynyl groups include, but are not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.
The term “carbocycle” and “carbocyclic ring” as used herein, refers to monocyclic and polycyclic moieties wherein the rings contain only carbon atoms. Unless otherwise specified, carbocycles may be saturated, partially unsaturated or aromatic, and contain 3 to 20 carbon atoms. Representative carbocyles include cyclopropane, cyclobutane, cyclopentane, cyclohexane, bicyclo[2,2,1]heptane, norbomene, phenyl, cyclohexene, naphthalene, spiro[4.5]decane.
The term “aryl” used alone or as part of a larger moiety as in “aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic and polycyclic ring systems having a total of five to 20 ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to twelve ring members. The term “aryl” may be used interchangeably with the term “aryl ring”. In certain embodiments of the present invention, “aryl” refers to an aromatic ring system which includes, but is not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Also included within the scope of the term “aryl”, as it is used herein, is a group in which an aromatic ring is fused to one or more additional rings, such as benzofuranyl, indanyl, phthalimidyl, naphthimidyl, phenantriidinyl, or tetrahydronaphthyl, and the like. The term “aromatic” is not limited to only carbocyclic ring and also encompasses heteroaryl rings as well.
The term “heteroaliphatic,” as used herein, refers to aliphatic groups wherein one or more carbon atoms are independently replaced by one or more atoms selected from the group consisting of oxygen, sulfur, nitrogen, or phosphorus. In certain embodiments, one to six carbon atoms are independently replaced by one or more of oxygen, sulfur, nitrogen, or phosphorus.
Heteroaliphatic groups may be substituted or unsubstituted, branched or unbranched, cyclic or acyclic, and include saturated, unsaturated, or partially unsaturated groups. In some embodiments, a heteroaliphatic group is an aliphatic group having 1-32 (e.g., 1-24, 1-12, 1-8, or 1-6) carbons where 1-6 (e.g., 1-4, 1-3, or 1-2) carbons are independently replaced by a heteroatom selected from oxygen, sulfur, nitrogen, and phosphorus
The terms “heteroaryl” and “heteroar-”, used alone or as part of a larger moiety, e.g., “heteroaralkyl”, or “heteroaralkoxy”, refer to groups having 5 to 14 ring atoms, preferably 5, 6, or 9 ring atoms; having 6, 10, or 14 π electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. The term “heteroatom” refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, benzofuranyl and pteridinyl. The terms “heteroaryl” and “heteroar-”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or saturated or partially unsaturated heterocyclyl rings. Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one. A heteroaryl group may be mono- or bicyclic. The term “heteroaryl” may be used interchangeably with the terms “heteroaryl ring”, “heteroaryl group”, or “heteroaromatic”, any of which terms include rings that are optionally substituted. The term “heteroaralkyl” refers to an alkyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently are optionally substituted. In some embodiments, a heteroaryl ring is 5- to 6-membered monocyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, a heteroaryl ring is 8- to 10-membered bicyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
As used herein, the terms “heterocycle”, “heterocyclyl”, “heterocyclic radical”, and “heterocyclic ring” are used interchangeably and refer to a stable 5- to 7-membered monocyclic or 7-14-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, or aromatic (i.e., heteroaryl), and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term “nitrogen” includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or +NR (as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and quinuclidinyl. The terms “heterocycle”, “heterocyclyl”, “heterocyclyl ring”, “heterocyclic group”, “heterocyclic moiety”, and “heterocyclic radical”, are used interchangeably herein, and also include groups in which a saturated or partically unsaturated heterocyclyl ring is fused to one or more aryl, heteroaryl, or cycloaliphatic rings, such as indolinyl, 3H-indolyl, chromanyl, phenanthridinyl, or tetrahydroquinolinyl, where the radical or point of attachment is on the heterocyclyl ring. A heterocyclyl group may be mono- or bicyclic. The term “heterocyclylalkyl” refers to an alkyl group substituted by a heterocyclyl, wherein the alkyl and heterocyclyl portions independently are optionally substituted. In some embodiments, a heterocylic ring is 3- to 7-membered saturated or partially unsaturated monocyclic heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In some embodiments, a heterocylic ring is 7- to 10-membered saturated or partially unsaturated bicyclic heterocyclyl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
As used herein, the term “partially unsaturated” refers to a ring moiety that includes at least one double or triple bond. The term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.
As described herein, compounds of the invention may contain “optionally substituted” moieties. In general, the term “substituted”, whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term “stable”, as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein.
In some chemical structures herein, substituents are shown attached to a bond which crosses a bond in a ring of the depicted molecule. This means that one or more of the substituents may be attached to the ring at any available position (usually in place of a hydrogen atom of the parent ring structure). In cases where an atom of a ring so substituted has two substitutable positions, two groups may be present on the same ring atom. When more than one substituent is present, each is defined independently of the others, and each may have a different structure. In certain cases where the substituent shown crossing a bond of the ring is —R, this has the same meaning as if the ring were said to be “optionally substituted” as described in the preceding paragraph.
Suitable monovalent substituents on a substitutable carbon atom of an “optionally substituted” group are independently halogen; —(CH2)0-4R∘; —(CH2)0-4OR∘; —O—(CH2)0-4C(O)OR∘; —(CH2)0-4CH(OR∘)2; —(CH2)0-4SR∘; —(CH2)0-4Ph, which may be substituted with R∘; —(CH2)0-4O(CH2)0-1Ph which may be substituted with R∘; —CH═CHPh, which may be substituted with R∘; —NO2; —CN; —N3; —(CH2)0-4N(R∘)2; —(CH2)0-4N(R∘)C(O)R∘; —N(R∘)C(S)R∘; —(CH2)0-4N(R∘)C(O)NR∘2; —N(R∘)C(S)NR∘2; —(CH2)0-4N(R∘)C(O)OR∘; —N(R∘)N(R∘)C(O)R∘; —N(R∘)N(R∘)C(O)NR∘2; —N(R∘)N(R∘)C(O)OR∘; —(CH2)0-4C(O)R∘; —C(S)R∘; —(CH2)0-4C(O)OR∘; —(CH2)0-4C(O)N(R∘)2; —(CH2)0-4C(O)SR∘; —(CH2)0-4C(O)OSiR∘3; —(CH2)0-4OC(O)R∘; —OC(O)(CH2)0-4SR—, SC(S)SR∘; —(CH2)0-4SC(O)R∘; —(CH2)0-4C(O)NR∘2: —C(S)NR∘2; —C(S)SR∘; —SC(S)SR∘, —(CH2)0-4OC(O)NR∘2; —C(O)N(OR∘)R∘; —C(O)C(O)R∘; —C(O)CH2C(O)R∘; —C(NOR∘)R∘; —(CH2)0-4SSR∘; —(CH2)0-4S(O)2R∘; —(CH2)0-4S(O)2OR∘; —(CH2)0-4OS(O)2R∘; —S(O)2NR∘2; —(CH2)0-4S(O)R∘; —N(R∘)S(O)2NR∘2; —N(R∘)S(O)2R∘; —N(OR∘)R∘; —C(NH)NR∘2; —P(O)2R∘; —P(O)R∘2; —OP(O)R∘2; —OP(O)(OR∘)2; SiR∘3; —(C1-4 straight or branched alkylene)O—N(R∘)2; or —(C1-4 straight or branched alkylene)C(O)O—N(R∘)2, wherein each R∘ may be substituted as defined below and is independently hydrogen, C1-8 aliphatic, —CH2Ph, —O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R∘, taken together with their intervening atom(s), form a 3-12-membered saturated, partially unsaturated, or aryl mono- or polycyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.
Suitable monovalent substituents on R∘ (or the ring formed by taking two independent occurrences of R∘ together with their intervening atoms), are independently halogen, —(CH2)0-2R●, -(haloR●), —(CH2)0-2OH, —(CH2)0-2OR●, —(CH2)0-2CH(OR●)2; —O(haloR●), —CN, —N3, —(CH2)0-2C(O)R●, —(CH2)0-2C(O)OH, —(CH2)0-2C(O)OR●, —(CH2)0-4C(O)N(R∘)2; —(CH2)0-2SR●, —(CH2)0-2SH, —(CH2)0-2NH2, —(CH2)0-2NHR●, —(CH2)0-2NR●2, —NO2, —SiR●3, —OSiR●3, —C(O)SR●, —(C1-4 straight or branched alkylene)C(O)OR●, or —SSR● wherein each R● is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently selected from C1-4 aliphatic, —CH2Ph, —O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R∘ include ═O and ═S.
Suitable divalent substituents on a saturated carbon atom of an “optionally substituted” group include the following: ═O, ═S, ═NNR*2, ═NNHC(O)R*, ═NNHC(O)OR*, ═NNHS(O)2R*, ═NR*, ═NOR*, —O(C(R*2))2-3—O—, or —S(C(R*2))2-3S—, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an “optionally substituted” group include: —O(CR*2)2-3O—, wherein each independent occurrence of R* is selected from hydrogen, C1-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
Suitable substituents on the aliphatic group of R● include halogen, —R●, -(haloR●), —OH, —OR●, —O(haloR●), —CN, —C(O)OH, —C(O)OR●, —NH2, —NHR●, —NR●2, or —NO2, wherein each R● is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, —CH2Ph, —O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
Suitable substituents on a substitutable nitrogen of an “optionally substituted” group include —R†, —NR†2, —C(O)R†, —C(O)OR†, —C(O)C(O)R†, —C(O)CH2C(O)R†, —S(O)2R, —S(O)2NR2, —C(S)NR2, —C(NH)NR†2, or —N(R†)S(O)2R†; wherein each R† is independently hydrogen, C1-6 aliphatic which may be substituted as defined below, unsubstituted —OPh, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of RT, taken together with their intervening atom(s) form an unsubstituted 3-12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
Suitable substituents on the aliphatic group of RT are independently halogen, —R●, -(haloR●), —OH, —OR●, —O(haloR●), —CN, —C(O)OH, —C(O)OR●, —NH2, —NHR●, —NR●2, or —NO2, wherein each R● is unsubstituted or where preceded by “halo” is substituted only with one or more halogens, and is independently C1-4 aliphatic, —CH2Ph, —O(CH2)0-1Ph, or a 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
As used herein, the term “isolated” refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting) and/or otherwise previously associated, and/or (2) designed, produced, prepared, and/or manufactured by the hand of man. In some embodiments, a substance may be considered to be “isolated” if it is (or has been caused to be) free of or separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of other components (e.g., components with which it was previously associated). In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% free of other components). Techniques useful to quantify isolation or purity are known in the art and include standard techniques such as nuclear magnetic resonance or high-performance liquid chromatography. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered “isolated” or even “pure”, after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients.
To give but one example, in some embodiments, a chemical compound that occurs in nature is considered to be “isolated” when, a) by virtue of its origin or source of derivation is not associated with some or all of the components that accompany it in its native state in nature; b) it is substantially free of other chemical compounds, polypeptides, or nucleic acids of the same species from the species that produces it in nature; c) is expressed by or is otherwise in association with components from a cell or other expression system that is not of the species that produces it in nature. Thus, for instance, in some embodiments, a chemical compound that is chemically synthesized or is synthesized in a cellular system different from that which produces it in nature is considered to be an “isolated” compound. Alternatively or additionally, in some embodiments, a compound that has been subjected to one or more purification techniques may be considered to be an “isolated” compound to the extent that it has been separated from other components a) with which it is associated in nature; and/or b) with which it was associated when initially produced.
As used herein, the term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference.
In certain embodiments, the neutral forms of the compounds are regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. In some embodiments, the parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents.
As used herein, the term “therapeutically effective amount” refers to an amount of a therapeutic agent that confers a therapeutic effect on the treated subject, at a reasonable benefit/risk ratio applicable to any medical treatment. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect). In particular, the “therapeutically effective amount” refers to an amount of a therapeutic agent effective to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and/or also lessening the severity or frequency of symptoms of the disease. A therapeutically effective amount is commonly administered in a dosing regimen that may comprise multiple unit doses. For any particular therapeutic agent, a therapeutically effective amount (and/or an appropriate unit dose within an effective dosing regimen) may vary, for example, depending on route of administration, on combination with other pharmaceutical agents. Also, the specific therapeutically effective amount (and/or unit dose) for any particular subject may depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific therapeutic agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and/or rate of excretion or metabolism of the specific therapeutic agent employed; the duration of the treatment; and like factors as is well known in the medical arts.
As used herein, the term “treatment” (also “treat” or “treating”) refers to any administration of a substance (e.g., provided compositions) that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition. Such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition.
In one aspect, the present invention encompasses therapeutic compositions comprising a therapeutically effective amount of one or more Modular Glucosides (MOGLs). In certain embodiments, the provided therapeutic compositions comprise one or more MOGLs having a phosphate group (or a derivative of a phosphate group) at the 3-position of the glucose. In certain embodiments, such MOGLs have additional substitutents at one or more of the 1-, 2-, and 6-positions. In certain embodiments, the provided compositions comprise MOGLs having a phosphate group (or a derivative of a phosphate group) at the 3-position of the glucose and a free —OH group at the 4-position.
In certain embodiments, provided therapeutic compositions comprise an effective amount one or more MOGLs featuring a glucose molecule substituted at each of the 1-, 2-, 3-, and 6-positions, wherein:
In certain embodiments, the provided compositions are further characterized in that the glucose derivative is not substituted at the 4-position.
In certain embodiments, such compositions comprise one or more molecules of Formula I:
wherein:
It will be appreciated that throughout the disclosure, references such as “aliphatic, aromatic, heteroaromatic, or aliphatic acyl group” and the like at the 2- and 6-positions of the glucose ring (e.g., G2 and G6) has the meaning “aliphatic acyl, aromatic acyl, heteroaromatic acyl, or aliphatic acyl group.”
In certain embodiments, a compound of Formula I comprises any one or more of the modular glucosides encompassed by the formula:
For avoidance of the doubt, the depiction above represents the combinatorial range of unique molecules resulting from independently choosing any one of the depicted moieties at each of the indicated positions by replacement of a dashed line in the figure with a covalent bond. The family of molecules represented by this depiction (and other similar depictions herein) includes all combinatorial permutations resulting from independent selection of each moiety at each position.
In certain embodiments, provided therapeutic compositions comprise MOGLs featuring a glucose derivative substituted at the 1-, 2-, and 3-positions, wherein:
In certain embodiments, such compositions comprise a therapeutically effective amount of one or more compounds of Formula II:
In certain embodiments, a compound of Formula II comprises any of the modular glucosides encompassed by the formula:
In certain embodiments, the present invention provides therapeutic compositions comprising MOGLs featuring a glucose derivative substituted at the 1-, 3-, and 6-positions, wherein:
In certain embodiments, the provided compositions are further characterized in that the glucose derivative is not substituted at the 2- or 4-position
In certain embodiments, such therapeutic compositions comprise a therapeutically effective amount of one or more compounds of Formula III:
In certain embodiments, a compound of Formula III comprises any of the modular glucosides encompassed by the formula:
In certain embodiments, such therapeutic compositions comprise MOGLs featuring a glucose derivative substituted at the 1- and 3-positions, wherein:
In certain embodiments, the provided compositions are further characterized in that the glucose derivative is not substituted at the 2-, 4- or 6-position.
In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula IV:
wherein each of G1 and X is as defined above and in the genera and subgenera herein.
In certain embodiments, a compound of Formula IV comprises any of the modular glucosides encompassed by the formula:
In certain embodiments, the present invention provides therapeutic compositions comprising MOGLs featuring a glucose derivative substituted at the 1-, 2-, and 6-positions, wherein:
In certain embodiments, the provided compositions are further characterized in that the glucose derivative is not substituted at the 3- or 4-position.
In certain embodiments, such compositions comprise a therapeutically effective amount of one or more compounds of Formula V:
In certain embodiments, a compound of Formula V comprises any of the modular glucosides encompassed by the formula:
In certain embodiments, the present invention provides therapeutic compositions comprising MOGLs featuring a glucose derivative substituted at the 1- and 2-, positions, wherein:
In certain embodiments, the provided compositions are further characterized in that the glucose derivative is not substituted at the 3-, 4- or 6-position.
In certain embodiments, such compositions comprise a therapeutically effective amount of one or more compounds of Formula VI:
In certain embodiments, a compound of Formula VI comprises any of the modular glucosides encompassed by the formula:
In certain embodiments, the present invention provides therapeutic compositions comprising MOGLs featuring a glucose derivative substituted at the 1- and 6-positions, wherein:
In certain embodiments, the provided compositions are further characterized in that the glucose derivative is not substituted at the 2-, 3- or 4-position.
In certain embodiments, such compositions comprise a therapeutically effective amount of one or more compounds of Formula VII:
In certain embodiments, a compound of Formula VII comprises any of the modular glucosides encompassed by the formula:
In certain embodiments, the MOGL-containing therapeutic compositions described above are characterized in that the substituent at the 1-position comprises a nucleobase. In certain embodiments, the nucleobase is N-linked to the 1-position of the glucose scaffold. In certain embodiments, the N-linked nucleobase comprises a pyrimidine base. In certain embodiments, the N-linked nucleobase comprises a purine base. In certain embodiments, the N-linked nucleobase comprises a primary nucleobase. In certain embodiments, the N-linked nucleobase is other than a primary nucleobase, or is an analog or adduct of a primary nucleobase. In certain embodiments, the N-linked nucleobase is a methylated nucleobase. In certain embodiments, the N-linked nucleobase is selected from the group consisting of adenine, cytosine, guanine, thymine, and uracil. In certain embodiments, the nucleobase comprises guanine. In certain embodiments, the nucleobase comprises a methylated analog of guanine. In certain embodiments, the nucleobase comprises 6-O-methyl guanine.
In certain embodiments, the MOGL-containing therapeutic compositions described above are characterized in that the substituent at the 1-position comprises an optionally substituted N-linked heterocycle. In certain embodiments, the N-linked heterocycle comprises a 5- or 6-membered ring containing at least one nitrogen atom. In certain embodiments, the N-linked heterocycle is optionally substituted 5- to 6-membered monocyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 8- to 10-membered bicyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur. In certain embodiments, the N-linked heterocycle contains one or more sites of unsaturation. In certain embodiments, the N-linked heterocycle comprises indole. In certain embodiments, the N-linked heterocycle comprises a substituted indole. In certain embodiments, the N-linked heterocycle comprises a hydroxy indole. In certain embodiments, the N-linked heterocycle comprises serotonin.
In certain embodiments, the MOGL-containing therapeutic compositions described above are characterized in that the substituent at the 1-position comprises an optionally unsaturated acyl group. In certain embodiments, the substituent at the 1-position comprises an alpha beta unsaturated acyl group. In certain embodiments, an acyl substituent at the 1-position comprises a C3-8 aliphatic group with alpha beta unsaturation. In certain embodiments, the substituent at the 1-position comprises crotonate. In certain embodiments, the substituent at the 1-position comprises tiglate. In certain embodiments, the substituent at the 1-position comprises angelate. In certain embodiments, the substituent at the 1-position comprises valerate. In certain embodiments, the substituent at the 1-position comprises acrylate, methacrylate, or cinnamate. In certain embodiments, the substituent at the 1-position comprises 2-imidazoleacrylate. In certain embodiments, the substituent at the 1-position comprises urocanate.
In certain embodiments, the MOGL-containing therapeutic compositions described above are characterized in that the substituent at the 1-position comprises an acyl- or ether-linked aromatic moiety substituted with an amine or an aminoalkyl group. In certain embodiments, the provided compositions are characterized in that the substituent at the 1-position comprises an acyl-linked aromatic moiety substituted with an amine. In certain embodiments, the acyl-linked aromatic moiety comprises a phenyl ring. In certain embodiments, the acyl-linked aromatic moiety comprises a substituted benzoyl group. In certain embodiments, the acyl-linked aromatic moiety comprises an optionally substituted aminobenzoyl group. In certain embodiments, the acyl-linked aromatic moiety comprises anthranilic acid. In certain embodiments, substituent at the 1-position comprises an ether-linked aromatic moiety substituted with an aminoalkyl group. In certain embodiments, the ether-linked aromatic moiety comprises a phenyl ring. In certain embodiments, a substituent at the 1-position comprises a phenolic ether where the phenyl ring of the phenol is substituted with an aminoalkyl group. In certain embodiments, the ether-linked aromatic moiety comprises a phenol substituted with an optionally substituted 2-aminoethyl group. In certain embodiments, the ether-linked aromatic moiety comprises tyramine. In certain embodiments, the ether-linked aromatic moiety comprises octopamine. In certain embodiments the the substituent at the 1-position comprises O-linked serotonin. In certain embodiments the the substituent at the 1-position comprises O-linked N-acetylserotonin (normelatonin). In certain embodiments the the substituent at the 1-position comprises O-linked dopamine. In certain embodiments the the substituent at the 1-position comprises 3-O-linked dopamine. In certain embodiments the the substituent at the 1-position comprises 4-O-linked dopamine. In certain embodiments the the substituent at the 1-position comprises 3-O-linked norepinepherine. In certain embodiments the the substituent at the 1-position comprises 4-O-linked norepinepherine. In certain embodiments the the substituent at the 1-position comprises 3-O-linked epinepherine. In certain embodiments the the substituent at the 1-position comprises 4-O-linked epinepherine.
In some embodiments, G1 in any of Formulae I, II, III, IV, V, VI, or VII above is selected from:
In some embodiments, G1 in any of Formulae I, II, III, IV, V, VI, or VII above is selected from:
In some embodiments, G1 in any of Formulae I, II, III, IV, V, VI, or VII above is selected from:
In some embodiments, G1 in any of Formulae I, II, III, IV, V, VI, or VII above is selected from:
In certain embodiments, therapeutic compositions of the present invention comprise a MOGL having a neurotransmitter (or a derivative or precursor of a neurotransmitter) linked to the 1-position of the glucose. In certain embodiments, such compositions comprise one or more compounds selected from the group:
where each of G2, G3, G6, and X is as defined above and in the genera and subgenera herein, and —NT comprises a neurotransmiltter, or a derivative or precursor of a neurotransmitter linked to the glucose through any suitable atom.
In certain embodiments, the neurotransmitter is linked to the glucose through a nitrogen or oxygen atom comprising part of the neurotransmitter structure. In some embodiments, the neurotransmitter is linked to the glucose through an atom from which a hydrogen is removed, with the resulting radical forming the point of attachment. In certain embodiments, the neurotransmitter is N-linked. In certain embodiments a neurotransmiltter is linked via a phenolic oxygen. In certain embodiments a neurotransmitter is linked via an acyl linkage.
In certain embodiments, the moiety —NT comprises a monoamine neurotransmitter or a derivative or precursor thereof. In certain embodiments, —NT comprises a catecholamine neurotransmitter or a derivative or precursor thereof. In certain embodiments —NT is selected from the group consisting of: dopamine, norepinepherine, epinepherine, histamine, and serotonin. In certain embodiments, —NT is selected from the group consisting of dopamine, norepinepherine, and epinepherine. In certain embodiments, —NT is selected from tryptamine, phenethylamine, N-methylphenethylamine, phenethanolamine, m-tyramine, p-tyramine, 3-methoxytyramine, N-methyltyramine, 3-indothyronamine, m-octopamine, p-octopamine, and synepherine.
In certain embodiments, the moiety —NT is selected from the group consisting of:
In certain embodiments, the moiety —NT is selected from the group consisting of
In certain embodiments, the moiety —NT is selected from the group consisting of
In certain embodiments, the moiety —NT is selected from the group consisting of
In certain embodiments, therapeutic compositions of the present invention comprise a MOGL having a nucleobase (or a derivative or precursor of a nucleobase) linked to the 1-position of the glucose. In certain embodiments, such compositions comprise one or more compounds selected from the group:
In certain embodiments, the nucleobase is linked to the glucose through a nitrogen or oxygen atom comprising part of the nucleobase structure. In some embodiments, the nucleobase is linked to the glucose through an atom from which a hydrogen is removed, with the resulting radical forming the point of attachment. In certain embodiments, the nucleobase is N-linked.
In certain embodiments, the moiety —NB is selected from the group consisting of:
In certain embodiments, the moiety —NB is selected from the group consisting of:
In certain embodiments, the moiety —NB is selected from the group consisting of:
In certain embodiments, the moiety —NB is selected from the group consisting of:
In certain embodiments, therapeutic compositions of the present invention comprise a MOGL having an alpha beta unsaturated acyl group linked to the 1-position of the glucose. In certain embodiments, such compositions comprise one or more compounds selected from the group:
In certain embodiments, the moiety -MCR comprises a C3-8 alpha beta unsaturated acyl group. In certain embodiments, the moiety -MCR comprises a C4-8 alpha beta unsaturated acyl group. In certain embodiments, the moiety -MCR comprises an acyl group corresponding to an ester of acrylic acid, methylacrylic acid, crotonic acid, methyl crotonic acid, valeric acid, 3-methylcrotonic acid or tiglic acid. In certain embodiments, the substituent at the 1-position comprises crotonate. In certain embodiments, the moiety -MCR comprises crotonate. In certain embodiments, the moiety -MCR comprises tiglate. In certain embodiments, the moiety MCR comprises angelate. In certain embodiments, the moiety -MCR comprises valerate. In certain embodiments, the moiety -MCR comprises acrylate, methacrylate, or cinnamate. In certain embodiments, the moiety MCR comprises 2-imidazoleacrylate. In certain embodiments, the moiety -MCR comprises urocanate.
In certain embodiments, the MOGL-containing therapeutic compositions described above are characterized in that the substituent at the 2-position comprises an optionally substituted aromatic or heteroaromatic acyl group. In certain embodiments, the substituent at the 2-position comprises an optionally substituted benzoate. In certain embodiments the optionally substituted benzoate is selected from the group consisting of: benzoate, anthranilate, and p-hydroxybenzoate. In certain embodiments, the substituent at the 2-position comprises an optionally substituted heteroaromatic acyl group. In certain embodiments, the substituent at the 2-position comprises a heteroaromatic acyl group with a 6-membered heteroaromatic moiety. In certain embodiments, the substituent at the 2-position comprises a pyridine or pyrimidine carboxylate ester. In certain embodiments, the 2-substituent comprises nicotinate. In certain embodiments, the 2-substituent comprises picolinate. In certain embodiments, the 2-substituent comprises isonicotinate. In certain embodiments, the substituent at the 2-position comprises a heteroaromatic acyl group with a 5-membered heteroaromatic moiety. In certain embodiments, the substituent at the 2-position comprises the ester of a pyrrole or imidazole carboxylic acid. In certain embodiments, the substituent at the 2-position is pyrrole-2-carboxylate.
In certain embodiments, the MOGL-containing therapeutic compositions described above are characterized in that the substituent at the 2-position comprises an optionally substituted aliphatic acyl group. In certain embodiments, the acyl group at the 2-position comprises an optionally substituted aliphatic group. In certain embodiments, the acyl group at the 2-position comprises an optionally substituted C1-40 aliphatic group, an optionally substituted C2-24 aliphatic group, an optionally substituted C12-24 aliphatic group, an optionally substituted C2-18 aliphatic group, an optionally substituted C2-12 aliphatic group, an optionally substituted C2-8 aliphatic group, or an optionally substituted C1-6 aliphatic group. In certain embodiments, the acyl group at the 2-position comprises a hydroxylated C1-40 aliphatic group. In certain embodiments, the acyl group at the 2-position comprises an epoxidized substituted C1-40 or C2-40 aliphatic group. In certain embodiments, such optionally substituted aliphatic groups are saturated. In certain embodiments, optionally substituted aliphatic groups have one or more sites of unsaturation. In certain embodiments, such unsaturated aliphatic groups have unsaturation adjacent to the carbonyl of the acyl linkage (e.g. they are alpha-beta unsaturated esters). In certain embodiments, an acyl substituent at the 2-position comprises a C2-8 aliphatic group with alpha beta unsaturation. In certain embodiments, the substituent at the 2-position comprises crotonate. In certain embodiments, the substituent at the 2-position comprises tiglate. In certain embodiments, the substituent at the 2-position comprises angelate. In certain embodiments, the substituent at the 2-position comprises acrylate, methacrylate, 3-methylcrotonate, isocrotonate, or optionally substituted cinnamate.
In some embodiments, the present invention provides a compound of Formulae XI-a, XI-b, XI-c, XI-d, XI-e, XI-f, or XI-g:
In some embodiments, Rn1 is hydrogen and Rn2 is optionally substituted C1-12 aliphatic. In some embodiments, Rn1 is hydrogen and Rn2 is optionally substituted C1-6 aliphatic. In some embodiments, Rn1 is methyl and Rn2 is optionally substituted C1-6 aliphatic. In some embodiments, Rn1 is hydrogen and Rn2 is optionally substituted aryl. In some embodiments, Rn1 is hydrogen and Rn2 is optionally substituted aryl. In some embodiments, Rn1 is methyl and R2 is optionally substituted aryl. In some embodiments, Rn1 is hydrogen and Rn2 is optionally substituted C3-12 heterocyclic. In some embodiments, Rn1 is hydrogen and Rn2 is optionally substituted C3-12 heterocyclic. In some embodiments, Rn1 is methyl and Rn2 is optionally substituted C3-6 heterocyclic. In some embodiments, Rn1 is hydrogen and Rn2 is optionally substituted acyl. In some embodiments, Rn1 is hydrogen and Rn2 is optionally substituted acyl.
In some embodiments, Rn1 is methyl and Rn2 is optionally substituted acyl. In some embodiments, —NRn1Rn2 comprises a monoamine neurotransmitter or a derivative or precursor thereof. In certain embodiments, —NRn1Rn2 comprises a catecholamine neurotransmitter or a derivative or precursor thereof. In certain embodiments —NRn1Rn2 is selected from the group consisting of: dopamine, norepinepherine, epinepherine, histamine, and serotonin. In certain embodiments, —NRn1Rn2 is selected from the group consisting of dopamine, norepinepherine, and epinepherine. In certain embodiments, —NRn1Rn2 is selected from tryptamine, phenethylamine, N-methylphenethylamine, phenethanolamine, m-tyramine, p-tyramine, 3-methoxytyramine, N-methyltyramine, 3-indothyronamine, m-octopamine, p-octopamine, and synepherine.
In some embodiments, G1 in any of the formulae above is selected from:
In some embodiments, G1 in any of the formulae above is selected from:
In some embodiments, G2 in any of the formulae above is selected from:
wherein n is 1-35 (e.g., 1-24, 1-18, 1-12, 1-8, or 1-6) and m is an integer dependent upon n to provide a stable saturated, unsaturated, or polyunsaturated aliphatic group.
In some embodiments, G2 in any of the formulae above is selected from:
wherein n is 1-35 (e.g., 1-24, 1-18, 1-12, 1-8, or 1-6) and m is an integer dependent upon n to provide a stable saturated, unsaturated, or polyunsaturated aliphatic group.
In some embodiments, G2 in any of the formulae above is selected from:
In certain embodiments, the MOGL-containing therapeutic compositions described above are characterized in that the substituent at the 3-position of the glucose comprises a phosphate—this may be a simple phosphate (e.g. —OPO3H2) or may comprise a di-, tri- or higher phosphate (e.g. —O—(P(O3H)n—H, where n is an integer greater than 1), or a phosphate derivative such as a salt or an ester. In certain embodiments, the 3-substituent is phosphate. In certain embodiments, the 3-substituent is diphosphate. In certain embodiments, the 3-substituent is triphosphate. In certain embodiments, the composition is provided in a form wherein the phosphate moiety at the 3-position is protonated. In certain embodiments, the compositions is provided in a form wherein the phosphate moiety at the 3-position comprises a salt (e.g. where one or more of —H groups on the phosphate are replaced by a metal cation or organic or inorganic ‘onium’ group).
In certain embodiments, the MOGL-containing therapeutic compositions described above are characterized in that the substituent at the 6-position comprises an optionally substituted moiety selected from the group consisting of: an acyl-linked amino acid, an aromatic acyl group and an aliphatic acyl group. In certain embodiments, the substituent at the 6-position comprises an acyl linked amino acid. In certain embodiments, the amino acid is an alpha amino acid. In certain embodiments, the amino acid comprises a proteinogenic amino acid. In certain embodiments, the amino acid comprises one of the 20 encoded proteogenic amino acids. In certain embodiments, the amino acid is phenylalanine. In certain embodiments, the substituent at the 6-position comprises a peptide linked to the glucose via an ester bond.
In certain embodiments, the substituent at the 6-position comprises an aromatic acyl group. In certain embodiments, the substituent at the 6-position comprises an optionally substituted benzoate. In certain embodiments, the optionally substituted benzoate is selected from the group consisting of: benzoate, anthranilate, and p-hydroxybenzoate. In certain embodiments, the substituent at the 6-position comprises anthranilate.
In certain embodiments, the substituent at the 6-position comprises a heteroaromatic acyl group. In certain embodiments, the substituent at the 6-position comprises an optionally substituted heteroaromatic acyl group with a 6-membered heteroaromatic moiety. In certain embodiments, the substituent at the 6-position comprises a pyridine or pyrimidine carboxylate ester. In certain embodiments, the 6-substituent comprises nicotinate. In certain embodiments, the 6-substituent comprises picolinate. In certain embodiments, the 6-substituent comprises isonicotinate. In certain embodiments, the substituent at the 6-position comprises a heteroaromatic acyl group with a 5-membered heteroaromatic moiety. In certain embodiments, the substituent at the 6-position comprises the ester of a pyrrole or imidazole carboxylic acid. In certain embodiments, the substituent at the 6-position is pyrrole-2-carboxylate.
In certain embodiments, the MOGL-containing therapeutic compositions described above are characterized in that the substituent at the 6-position comprises an optionally substituted aliphatic acyl group. In certain embodiments, the acyl group at the 6-position comprises an optionally substituted aliphatic group. In certain embodiments, the acyl group at the 6-position comprises an optionally substituted C1-30 aliphatic group, an optionally substituted C2-24 aliphatic group, an optionally substituted C12-24 aliphatic group, an optionally substituted C2-18 aliphatic group, an optionally substituted C2-12 aliphatic group, an optionally substituted C2-8 aliphatic group, or an optionally substituted C1-6 aliphatic group. In certain embodiments, the acyl group at the 6-position comprises phenylacetate. In certain embodiments, optionally substituted aliphatic groups are saturated. In certain embodiments, acyl groups at the 6-position have one or more sites of unsaturation. In certain embodiments, the 6-substituent comprises an unsaturated aliphatic group having unsaturation adjacent to the carbonyl of the acyl linkage (e.g. they are alpha-beta unsaturated esters). In certain embodiments, an acyl substituent at the 6-position comprises a C2-8 aliphatic group with alpha beta unsaturation. In certain embodiments, the substituent at the 6-position comprises crotonate. In certain embodiments, the substituent at the 6-position comprises tiglate. In certain embodiments, the substituent at the 6-position comprises angelate. In certain embodiments, the substituent at the 6-position comprises acrylate, methacrylate, or cinnamate. In certain embodiments, the substituent at the 6-position comprises 2-imidazoleacrylate. In certain embodiments, the substituent at the 6-position comprises urocanate.
In some embodiments, G6 in any of the formulae above is selected from:
In some embodiments, G6 in any of the formulae above is selected from:
wherein n is 1-35 (e.g., 1-24, 1-18, 1-12, 1-8, or 1-6) and m is an integer dependent upon n to provide a stable saturated, unsaturated, or polyunsaturated aliphatic group.
In some embodiments, G6 in any of the formulae above is selected from:
In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula I:
wherein:
In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula IV:
In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula V:
In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula VI:
In certain embodiments, the present invention encompasses compositions of matter comprising a therapeutically effective amount of one or more compounds of Formula VII:
In another aspect, the present invention encompasses methods of improving the health of an animal or of treating or ameliorating a health disorder in an animal by administering to the animal an effective amount of any one or more of the therapeutic compositions described above. In certain embodiments, the method comprises administering such a composition to a mammal. In certain embodiments, the method comprises administering such a composition to a human.
In another aspect, the present invention comprises methods of making therapeutic compositions comprising formulating an effective amount of one or more purified or synthetically-produced MOGLs (or a pharmaceutically-acceptable salt, prodrug or derivative thereof) into a pharmaceutical composition selected from the group consisting of injectible liquid, tablet, capsule, pill, solution or suspension for oral administration, solid for suspension or dissolution into a drinkable or injectible liquid, dermal patch, eye drop, cream, ointment, gel, powder, spray, and inhalable.
In another aspect, the present invention provides pharmaceutical compositions containing MOGLs. In certain embodiments, the invention encompasses a pharmaceutical composition or a single unit dosage form of any of the compounds described above. In certain embodiments, pharmaceutical compositions and single unit dosage forms of the invention comprise a prophylactically or therapeutically effective amount of one or more of the MOGLs describe above, or their pro-drugs, and typically one or more pharmaceutically acceptable carriers or excipients. In a specific embodiment and in this context, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government (or equivalent in other countries) or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Examples of suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by E. W. Martin.
Typical pharmaceutical compositions and dosage forms comprise one or more excipients.
Suitable excipients are well-known to those skilled in the art of pharmacy, and non-limiting examples of suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art including, but not limited to, the way in which the dosage form will be administered to a patient and the specific active ingredients in the dosage form. The composition or single unit dosage form, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
Lactose-free compositions of the invention can comprise excipients that are well known in the art and are listed, for example, in the U.S. Pharmocopia (USP) SP (XXI)/NF (XVI). In general, lactose-free compositions comprise an active ingredient, a binder/filler, and a lubricant in pharmaceutically compatible and pharmaceutically acceptable amounts. Preferred lactose-free dosage forms comprise an active ingredient, microcrystalline cellulose, pre-gelatinized starch, and magnesium stearate.
This invention further encompasses anhydrous pharmaceutical compositions and dosage forms comprising active ingredients (e.g any of the MOGLs described above and herein), since water can facilitate the degradation of some compounds. For example, the addition of water (e.g., 5%) is widely accepted in the pharmaceutical arts as a means of simulating long-term storage in order to determine characteristics such as shelf-life or the stability of formulations over time. See, e.g., Jens T. Carstensen, Drug Stability: Principles & Practice, 2d. Ed., Marcel Dekker, NY, N.Y., 1995, pp. 379-80. In effect, water and heat accelerate the decomposition of some compounds. Thus, the effect of water on a formulation can be of great significance since moisture and/or humidity are commonly encountered during manufacture, handling, packaging, storage, shipment, and use of formulations.
Anhydrous pharmaceutical compositions and dosage forms of the invention can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms that comprise lactose and at least one active ingredient that comprises a primary or secondary amine are preferably anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected.
An anhydrous pharmaceutical composition should be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions are preferably packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.
The invention further encompasses pharmaceutical compositions and dosage forms that comprise any one or more MOGLs and one or more compounds that reduce the rate by which an active ingredient will decompose. Such compounds, herein referred to as “stabilizers,” include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.
The pharmaceutical compositions and single unit dosage forms can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such compositions and dosage forms will contain a prophylactically or therapeutically effective amount of a prophylactic or therapeutic agent preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration. In certain embodiments, the pharmaceutical compositions or single unit dosage forms are sterile and in suitable form for administration to a subject, preferably an animal subject, more preferably a mammalian subject, and most preferably a human subject.
A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include, but are not limited to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), intranasal, transdermal (topical), transmucosal, intra-tumoral, intra-synovial and rectal administration. In a specific embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous, subcutaneous, intramuscular, oral, intranasal or topical administration to human beings. In certain embodiments, a pharmaceutical composition is formulated in accordance with routine procedures for subcutaneous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocane to ease pain at the site of the injection. Examples of dosage forms include, but are not limited to: tablets; caplets; capsules, such as soft elastic gelatin capsules; cachets; troches; lozenges; dispersions; suppositories; ointments; cataplasms (poultices); pastes; powders; dressings; creams; plasters; solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosage forms suitable for oral or mucosal administration to a patient, including suspensions (e.g., aqueous or non-aqueous liquid suspensions, oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions, and elixirs; liquid dosage forms suitable for parenteral administration to a patient; and sterile solids (e.g., crystalline or amorphous solids) that can be reconstituted to provide liquid dosage forms suitable for parenteral administration to a patient.
The composition, shape, and type of dosage forms of the invention will typically vary depending on their use. For example, a dosage form used in the acute treatment of inflammation or a related disorder may contain larger amounts of one or more of the active ingredients it comprises than a dosage form used in the chronic treatment of the same disease. Also, the therapeutically effective dosage form may vary among different types of cancer. Similarly, a parenteral dosage form may contain smaller amounts of one or more of the active ingredients it comprises than an oral dosage form used to treat the same disease or disorder. These and other ways in which specific dosage forms encompassed by this invention will vary from one another will be readily apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton Pa. (1990).
Generally, the ingredients of compositions of the invention are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. Typical dosage forms of the invention comprise a compound of the invention, or a pharmaceutically acceptable salt thereof lie within the range of from about 1 mg to about 1000 mg per day, given as a single once-a-day dose in the morning but preferably as divided doses throughout the day taken with food.
Pharmaceutical compositions of the invention that are suitable for oral administration can be presented as discrete dosage forms, such as, but are not limited to, tablets (e.g., chewable tablets), caplets, capsules, and liquids (e.g., flavored syrups). Such dosage forms contain predetermined amounts of active ingredients, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton Pa. (1990).
Typical oral dosage forms of the invention are prepared by combining the active ingredient(s) in an intimate admixture with at least one excipient according to conventional pharmaceutical compounding techniques. Excipients can take a wide variety of forms depending on the form of preparation desired for administration. For example, excipients suitable for use in oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents. Examples of excipients suitable for use in solid oral dosage forms (e.g., powders, tablets, capsules, and caplets) include, but are not limited to, starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents.
Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit forms, in which case solid excipients are employed. If desired, tablets can be coated by standard aqueous or nonaqueous techniques. Such dosage forms can be prepared by any of the methods of pharmacy. In general, pharmaceutical compositions and dosage forms are prepared by uniformly and intimately admixing the active ingredients with liquid carriers, finely divided solid carriers, or both, and then shaping the product into the desired presentation if necessary.
For example, a tablet can be prepared by compression or molding. Compressed tablets can be prepared by compressing in a suitable machine the active ingredients in a free-flowing form such as powder or granules, optionally mixed with an excipient. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
Examples of excipients that can be used in oral dosage forms of the invention include, but are not limited to, binders, fillers, disintegrants, and lubricants. Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, (e.g., Nos. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.
Examples of fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g., granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof. The binder or filler in pharmaceutical compositions of the invention is typically present in from about 50 to about 99 weight percent of the pharmaceutical composition or dosage form.
Suitable forms of microcrystalline cellulose include, but are not limited to, the materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICEL RC-581, AVICEL-PH-105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, Pa.), and mixtures thereof. A specific binder is a mixture of microcrystalline cellulose and sodium carboxymethyl cellulose sold as AVICEL RC-581. Suitable anhydrous or low moisture excipients or additives include AVICEL-PH-103.TM. and Starch 1500 LM.
Disintegrants are used in the compositions of the invention to provide tablets that disintegrate when exposed to an aqueous environment. Tablets that contain too much disintegrant may disintegrate in storage, while those that contain too little may not disintegrate at a desired rate or under the desired conditions. Thus, a sufficient amount of disintegrant that is neither too much nor too little to detrimentally alter the release of the active ingredients should be used to form solid oral dosage forms of the invention. The amount of disintegrant used varies based upon the type of formulation, and is readily discernible to those of ordinary skill in the art. Typical pharmaceutical compositions comprise from about 0.5 to about 15 weight percent of disintegrant, specifically from about 1 to about 5 weight percent of disintegrant.
Disintegrants that can be used in pharmaceutical compositions and dosage forms of the invention include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums, and mixtures thereof.
Lubricants that can be used in pharmaceutical compositions and dosage forms of the invention include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethyl laureate, agar, and mixtures thereof. Additional lubricants include, for example, a syloid silica gel (AEROSIL 200, manufactured by W.R. Grace Co. of Baltimore, Md.), a coagulated aerosol of synthetic silica (marketed by Degussa Co. of Plano, Tex.), CAB-O-SIL (a pyrogenic silicon dioxide product sold by Cabot Co. of Boston, Mass.), and mixtures thereof. If used at all, lubricants are typically used in an amount of less than about 1 weight percent of the pharmaceutical compositions or dosage forms into which they are incorporated.
Delayed Release Dosage Forms Active ingredients of the invention can be administered by controlled release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; and 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,566, each of which is incorporated herein by reference. Such dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled-release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the active ingredients of the invention. The invention thus encompasses single unit dosage forms suitable for oral administration such as, but not limited to, tablets, capsules, gelcaps, and caplets that are adapted for controlled-release.
All controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include extended activity of the drug, reduced dosage frequency, and increased patient compliance. In addition, controlled-release formulations can be used to affect the time of onset of action or other characteristics, such as blood levels of the drug, and can thus affect the occurrence of side (e.g., adverse) effects.
Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, temperature, enzymes, water, or other physiological conditions or compounds.
Parenteral dosage forms can be administered to patients by various routes including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial. Because their administration typically bypasses patients' natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions.
Suitable vehicles that can be used to provide parenteral dosage forms of the invention are well known to those skilled in the art. Examples include, but are not limited to: Water for Injection USP; aqueous vehicles such as, but not limited to, Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, and Lactated Ringer's Injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and polypropylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.
Compounds that increase the solubility of one or more of the active ingredients disclosed herein can also be incorporated into the parenteral dosage forms of the invention.
Transdermal, topical, and mucosal dosage forms of the invention include, but are not limited to, ophthalmic solutions, sprays, aerosols, creams, lotions, ointments, gels, solutions, emulsions, suspensions, or other forms known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences, 16th and 18th eds., Mack Publishing, Easton Pa. (1980 & 1990); and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger, Philadelphia (1985). Dosage forms suitable for treating mucosal tissues within the oral cavity can be formulated as mouthwashes or as oral gels. Further, transdermal dosage forms include “reservoir type” or “matrix type” patches, which can be applied to the skin and worn for a specific period of time to permit the penetration of a desired amount of active ingredients.
Suitable excipients (e.g., carriers and diluents) and other materials that can be used to provide transdermal, topical, and mucosal dosage forms encompassed by this invention are well known to those skilled in the pharmaceutical arts, and depend on the particular tissue to which a given pharmaceutical composition or dosage form will be applied. With that fact in mind, typical excipients include, but are not limited to, water, acetone, ethanol, ethylene glycol, propylene glycol, butane-1,3-diol, isopropyl myristate, isopropyl palmitate, mineral oil, and mixtures thereof to form lotions, tinctures, creams, emulsions, gels or ointments, which are non-toxic and pharmaceutically acceptable. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms if desired. Examples of such additional ingredients are well known in the art. See, e.g., Remington's Pharmaceutical Sciences, 16th and 18th eds., Mack Publishing, Easton Pa. (1980 & 1990).
Depending on the specific tissue to be treated, additional components may be used prior to, in conjunction with, or subsequent to treatment with active ingredients of the invention. For example, penetration enhancers can be used to assist in delivering the active ingredients to the tissue. Suitable penetration enhancers include, but are not limited to: acetone; various alcohols such as ethanol, oleyl, and tetrahydrofuryl; alkyl sulfoxides such as dimethyl sulfoxide; dimethyl acetamide; dimethyl formamide; polyethylene glycol; pyrrolidones such as polyvinylpyrrolidone; Kollidon grades (Povidone, Polyvidone); urea; and various water-soluble or insoluble sugar esters such as Tween 80 (polysorbate 80) and Span 60 (sorbitan monostearate).
The pH of a pharmaceutical composition or dosage form, or of the tissue to which the pharmaceutical composition or dosage form is applied, may also be adjusted to improve delivery of one or more active ingredients. Similarly, the polarity of a solvent carrier, its ionic strength, or tonicity can be adjusted to improve delivery. Compounds such as stearates can also be added to pharmaceutical compositions or dosage forms to advantageously alter the hydrophilicity or lipophilicity of one or more active ingredients so as to improve delivery. In this regard, stearates can serve as a lipid vehicle for the formulation, as an emulsifying agent or surfactant, and as a delivery-enhancing or penetration-enhancing agent. Different salts of the active ingredients can be used to further adjust the properties of the resulting composition.
In another aspect, the present invention encompasses novel compositions of matter including compositions of novel molecules. While some of the MOGLs are naturally occurring molecules that have been detected in the bodies of nematodes and in some cases have been found in low concentrations in the media in which nematodes are cultured, pure samples of these molecules and in particular bulk samples of the pure MOGLs free from other biological materials are not found in nature. Additionally, many of the MOGLs described above have not been detected in nature, even with the aid of highly sensitive and selective analytical techniques such as HPLC-coupled high resolution mass spectroscopy. As such, many of the compounds described above constitute novel compositions of matter.
In certain embodiments, the present invention provides a pure sample of any of the MOGLs described above and in the genera and subgenera herein. In certain embodiments, the present invention provides samples comprising bulk quantities of such molecules in substantially pure form. In certain embodiments, the present invention provides novel compositions comprising mixtures of between two and ten different MOGLs.
In some embodiments, a provided compound is an isolated compound. In some embodiments, a provided compound is a pure compound (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% free of other components).
In some embodiments, a compound or composition described herein is provided outside of a C. elegans worm body. In some embodiments, a compound or composition described herein is provided free of C. elegans tissue or other biological materials typically contained within or excreted by C. elegans.
In another aspect, the present invention encompasses methods of improving the health of an animal or of treating or ameliorating a health disorder in an animal by administering to the animal an effective amount of any one or more of the therapeutic compositions described above. In certain embodiments, the method comprises administering such a composition to a mammal. In certain embodiments, the method comprises administering such a composition to a human.
Without being bound by theory or thereby limiting the scope of the present invention, it is believed that MOGLs containing a neurotransmitter-like moiety, for example those derived from serotonin, N-acetyl serotonin, adrenaline, dopamine, tyramine, histidine, or octopamine as well as MOGLs derived from synthetic ligands of neurotransmiltter receptors, e.g. selective serotonin re-uptake inhibitors (SSRIs), have utility as therapeutics to cure or ameliorate neurological disease. Formation of the ester bonds in these neurotransmiltter-derived MOGLs (NeuroMOGs) via esterases (e.g. homologs of the carboxylesterase cest-1.2, such as mammalian cocaine esterase, CES2) is known to be reversible in living systems. Similarly, formation of glycosidic bonds such as those linking glucose to neurotransmitters in the NeuroMOGs is known to be reversible. Moreover, glycosides, including phosphorylated glycosides, are known to be readily transported through the vascular system. Therefore, NeuroMOGs produced in the gut, or NeuroMOG-based therapeutics taken up through the gut, skin, or other modes of administration offer an effective means to alter neurotransmitter-dependent physiological responses by taking advantage of endogenous transport and release mechanisms.
Treatment with NeuroMOGs can be used to improve the mental or emotional state of a patient or to treat anxiety disorders and depression (e.g. by regulating the levels of serotonin or SSRIs), tic disorders (by regulating adrenaline levels), learning disorders and cognitive decline (e.g. in Parkinson patients by elevating dopamine levels), behavioral disorders, and digestive disorders. Selection of the specific moieties attached to the glucose allows for targeting of the NeuroMOGs to specific tissues (e.g. as a result of different lipophilicities) and further enables control of the time scale at which active species (e.g, a neurotransmitters, SSRIs, or neurotransmitter glucoside) are released.
Therefore in certain embodiments, the present invention provides methods of improving the mental or emotional state of an animal (including humans) by administering a therapeutically effective amount of a MOGL comprising a neurotransmitter, or a neurotransmitter-like moiety covalenty linked to the 1-position of the present invention provides methods of treating, ameliorating or curing a neurological or emotional disorder of an animal (including humans) by administering a therapeutically effective amount of a MOGL comprising a neurotransmitter, or a neurotransmitter-like moiety covalenty linked to the 1-position of the MOGL. In certain embodiments, the neurological or emotional disorder comprises anxiety, depression, obsessive or compulsive disorders or behaviors, tics, bipolar disorder, schizophrenia, learning disorders, cognitive decline, behavioral disorders, learning disability, hyperactivity and the like.
In certain embodiments, such methods comprise administering an effective dose of a MOGL having a neurotransmitter (or a derivative or precursor of a neurotransmitter) linked to the 1-position of the glucose. In certain embodiments, such compositions comprise one or more compounds selected from the group:
Kinases play a central role for many types of human diseases. For example, (Cicenas J. Zalyte E, Bairoch A, Gaudet P. Kinases and Cancer. Cancers (Basel). 2018; 10(3):63. Published 2018 Mar. 1. doi:10.3390/cancers10030063) report that mutated kinases that are constitutively active are drivers of many types of cancers, e.g. the V600E mutation of BRAF colorectal cancer, melanoma, thyroid cancer, and non-small cell lung cancer. Other examples include driver mutations in KIT, EGFR, and FTL3. In addition to mutations, epigenetic changes can result in cancer-driving changes of kinase expression levels. As a result, kinase inhibitors and modulators have been a major focus of cancer research over the past 40 years, which has yielded important cancer drugs in current clinical use, e.g. imatinib (Gleevec), which can extend survival of chronic myelogenous leukemia patients often by a decade or more. Most kinases bind ATP or other nucleotides, and many synthetic kinase inhibitors act as ATP-competitive mechanism or otherwise interact with the nucleotide-binding domain, whereby additional interactions with nearby hydrophobic pockets often play an important role. See for example, Roskoski, Pharmacol. Res., 100:1-23 (2015).
Without being bound by theory, or thereby limiting the scope of the present invention, it is believed the MOGLs described above that comprise a nucleobase or other aromatic moiety, e.g. indole, 5-hydroxyindole, anthranilic acid, or nicotinic acid at the 1-position can play a role in regulating kinase activity and therefore have utility for the treatment of cancer and other kinase dependent disorders or diseases including, for example, hypertension, Parkinson's disease, and autoimmune diseases.
Members of this family of nucleotide-related MOGLs (NuMOGs), representative members of which were recently discovered in the model organism C. elegans, structurally mimic ATP and other nucleotides that kinases are known to bind to, and feature additional hydrophilic and hydrophobic moieties. The combination of polar (phosphate sugar) and less polar (acyl moieties) moieties in the structures of the NuMOGs can be used to tailor affinity and specificity to different kinases, which can be used to target disease-relevant kinases selectively. A subset of NuMOGs featuring one or two acyl groups may also serve as a precursor or pro-drugs of NuMOGs with fewer acyl groups, based on the finding that enzymes of the carboxylesterase family (e.g. CES2 in humans, a homolog of cest-1.2 in C. elegans) are able to hydrolyze ester bonds. The ability to tailor lipophilicity via additional acyl moieties facilitates design of NuMOGs or pro-drugs of NuMOGs that have desirable properties, such as high bioavalability in the gut or high tissue penetration. As inhibitors and modulators of kinase activity, NUMOGs can be used to treat cancer, but also offer new opportunities for the treatment of other diseases in which kinases are known to play an important role, including hypertension, Parkinson's disease, and autoimmune diseases. See for example, Roskoski, Pharmacol. Res., 100:1-23 (2015).
Therefore, in certain embodiments, the present invention provides methods of amelieorating or curing a kinase-dependent disease or disorder. In certain embodiments, such methods comprise administering to a patient a pharmaceutically effective dose of one or more MOGLs. In certain embodiments, the MOGL(s) administered are characterized in that they comprise a nucleobase or other aromatic moiety at the 1-position. In certain embodiments, such MOGLs are selected from the group consisting of:
Upregulation of nucleoside metabolism is a hallmark of cancer, and correspondingly chemotherapeutics that target nucleoside biosynthesis and oligonucleotide production are important components of cancer treatments. Similar to cancerous cells, virally infected cells also increase nucleotide synthesis, for example by inhibiting the tumor suppressor p53, and enhanced nucleotide production is necessary for viral replication. Correspondingly, nucleotide metabolism is an important target of established treatments of cancer and viral diseases. See for example, Ariav et. al., Science Advances, 7(21):1-8 (May 19, 2021). In certain embodiments, the present invention relates to therapies for the treatment of disorders that result in or arise from changes to nucleotide synthesis including, but not limited to cancer and viral diseases.
In certain embodiments, such methods comprise treating an animal with a therapeutically effective amount of a MOGLs comprising a nucleoside or nucleoside derivative (e.g., adenine glucoside, 4-N-methylcytosine glucoside, guanosine, methylguanosine, or methyladenine).
Without being bound by theory or thereby limiting the scope of this invention, it is believed members of this family of nucleotide-related MOGLs (NuMOGs), structurally mimic canonical ribonucleotides and can interfere with production of ribonucleotides by inhibiting enzymes required for their biosynthesis. In addition, NuMOGs, due to their structural similarity with ribonucleotides, can interfere with assembly of oligonucleotides, e.g. RNA and DNA and thereby interfere with cell division (e.g. of tumor cells) or viral replication. These properties indicate that NuMOGs can be useful as anti-cancer drugs and antivirals. A subset of NuMOGs featuring one or two acyl groups may also serve as a precursor or pro-drugs of NuMOGs with fewer acyl groups, based on the finding that enzymes of the carboxylesterase family (e.g. CES2 in humans, a homolog of cest-1.2 in C. elegans) are able to hydrolyze ester bonds. The ability to tailor lipophilicity via additional acyl moieties facilitates design of NuMOGs or pro-drugs of NuMOGs that have desirable properties, such as high bioavailability in the gut or high tissue penetration.
Therefore, in certain embodiments, the present invention provides methods of amelieorating or curing a nucleotide synthesis-related disease or disorder. In certain embodiments, such methods comprise administering to a patient a pharmaceutically effective dose of one or more MOGLs. In certain embodiments, the MOGL(s) administered are characterized in that they comprise a a nucleobase or other aromatic moiety at the 1-position. In certain embodiments, such MOGLs are selected from the group consisting of:
Modular glucosides (MOGLs) derived from glucosides of methylcrotonate-related moieties (MeMOGs), which are naturally produced in a TOR- (Target Of Rapamycin-) dependent manner in some organisms (e.g. the model organism C. elegans) offer new opportunities for the treatment of important human disease. The TOR signaling network, see for example, Loewith and Hall, Genetics, 189(4):1177-1201 (2011), is a central regulator of nutrient-dependent signaling and growth, and the amino acid leucine and its downstream metabolite 3-methylcrotonate are known to play an important role in regulating TOR function. Our finding that MeMOG production is dependent on TOR indicates that MeMOGs offer new perspectives for modulating TOR. Modulating TOR activity, e.g. via the FDA-approved drug rapamycin, has been employed successfully in three major therapeutic areas: immunosuppression/organ transplantation, cancer, and coronary artery disease. Similarly, MeMOGs can be used (i) to suppress or otherwise modulate immune responses (e.g. in the context of organ rejection or autoimmune disorder), (ii) to suppress proliferation of tumor cells (in analogy to the action of rapamycin, which blocks cancer growth directly and further prevents the growth of new blood vessels (angiogenesis) that supply oxygen and nutrients to tumors), and (iii) prevent restenosis after angioplasty (again in analogy to rapamycin).
Therefore, in certain embodiments, the present invention provides methods of amelieorating or curing a disease or disorder responsive to regulation of TOR function. In certain embodiments, such methods comprise administering to a patient a pharmaceutically effective dose of one or more MOGLs. In certain embodiments, the MOGL(s) administered are characterized in that they comprise an alpha-beta unsaturated acyl moiety. In certain embodiments, such MOGLs comprise a crotonate or methyl crotonate moiety. In certain embodiments, such MOGLs are characterized in that a substituent at the 1-position independently comprises a C3-8 acyl group with alpha beta unsaturation. In certain embodiments, the substituent at the 1-position comprises crotonate. In certain embodiments, the substituent at the 1-position comprises methylcrotonate. In certain embodiments, the substituent at the 1-position comprises tiglate. In certain embodiments, the substituent at the 1-position comprises angelate. In certain embodiments, the subsituent at the 1-position comprises acrylate, methacrylate, 3-methylcrotonate, or isocrotonate.
In certain embodiments, the present invention provides methods of amelieorating or curing a disease or disorder responsive to regulation of TOR function comprising administering to a patient a therapeutically effective dose of one or more compounds selected from the group:
In certain embodiments, the method comprises treatment with an effective amount of a MeMOG based on alpha or beta-glycosides of 3-methylcrotonate, isobutyric acid, or isovaleric acid, optionally bearing a phosphate or phosphate derivative in position 3 of the sugar, as well as acyl groups selected from any variable substituent as defined above for G2 or G6 (i.e., at the oxygens in positions 2 and 6 of the glucose). Selection of the specific moieties attached to the glucose allows for targeting of the MeMOGs to specific tissues (e.g. as a result of different lipophilicities) and further enables control of the time scale at which active species (e.g, a monoacylated 3-methylcrotonyl glucoside) are released.
Without being bound by theory or thereby limiting the scope of the present invention, it is believed that MOGLs containing a neurotransmitter-like moiety (e.g., NeuroMOGs) have utility as modulators of the proteasome. Function of the proteasome (i.e., protein degradation) requires assembly of seven well-folded subunits to form a ring complex, and conformational changes in one or more of the subunits can be expected to significantly enhance or reduce activity of proteolysis. Resulting modulation of proteasome activity can provide important advantages for the treatment of human disease. Inhibition of proteasome function is one important strategy for the treatment of cancer. See for example, Irvine et. al., J Cell Commun. Signal, 5(2): 101-110 (2011); Rastogi and Mishra, Cell Div., 7:26, 1-10 (2012); Adams, Cancer Cell, 5(5): 417-421 (2004). Specific structural MOGLs could also increase proteasome function, which would offer new treatment opportunities for many aging-related diseases and neurodegenerative disorders that are derived from protein misfolding, including Alzheimer's and Parkinson's and Huntington's disease. See for example, Hodgson et. al. Translational Neurodegeneration 6:6, 1-13 (2017).
In certain embodiments, the present invention provides methods of treating a disease or disorder responsive to modulation of the proteasome, comprising administering to a patient in need thereof a therapeutically effective amount of a compound (e.g., MOGL) described herein. In some embodiments, a MOGL is a proteasome inhibitor. In some embodiments, a MOGL is a proteasome activator. In some embodiments, a compound is a MOGL having a neurotransmitter (or a derivative or precursor of a neurotransmitter) linked to the 1-position of the glucose. In certain embodiments, a compound is selected from:
The following numbered embodiments, while non-limiting, are exemplary of certain aspects of the disclosure:
1. A glucose derivative substituted at the 1-, 2-, 3-, and 6-positions, wherein:
2. The glucose derivative of embodiment 1, characterized in that the glucose derivative is not substituted at the 4-position.
3. A glucose derivative substituted at the 1-, 2-, and 3-positions, wherein:
4. The glucose derivative of embodiment 3, characterized in that the glucose derivative is not substituted at the 4- or 6-position.
5. A glucose derivative substituted at the 1-, 3-, and 6-positions, wherein:
6. The glucose derivative of embodiment 5, characterized in that the glucose derivative is not substituted at the 2- or 4-position.
7. A glucose derivative substituted at the 1- and 3-positions, wherein:
8. The glucose derivative of embodiment 7, characterized in that the glucose derivative is not substituted at the 2-, 4-, or 6-position.
9. A glucose derivative substituted at the 1-, 2-, and 6-positions, wherein:
10. The glucose derivative of embodiment 9, characterized in that the glucose derivative is not substituted at the 3- or 4-position.
11. A glucose derivative substituted at the 1- and 2-, positions, wherein:
12. The glucose derivative of embodiment 11, characterized in that the glucose derivative is not substituted at the 3-, 4-, or 6-position.
13. A glucose derivative substituted at the 1- and 6-positions, wherein:
14. The glucose derivative of embodiment 13, characterized in that the glucose derivative is not substituted at the 2-, 3-, or 4-position.
15. The glucose derivative of any one of the preceding embodiments, wherein the N-linked heterocycle is a nucleobase (e.g., adenine, cytosine, guanine, thymine, or uracil).
16. The glucose derivative of any one of the preceding embodiments, wherein the substituent at the 1-position comprises an acyl- or ether-linked aromatic moiety substituted with an amine or an aminoalkyl group.
17. The glucose derivative of any one of the preceding embodiments, wherein the substituent at the 1-position comprises a phenolic ether where the phenyl ring of the phenol is substituted with an aminoalkyl group.
18. The glucose derivative of any one of the preceding embodiments, wherein the substituent at the 2-position comprises an optionally substituted benzoate.
19. The glucose derivative of any one of the preceding embodiments, wherein the substituent at the 2-position comprises the ester of a pyrrole or imidazole carboxylic acid.
20. The glucose derivative of any one of the preceding embodiments, wherein the substituent at the 2-position comprises an optionally substituted C1-6 aliphatic group.
21. The glucose derivative of any one of the preceding embodiments, wherein the substituent at the 2-position comprises acrylate, methacrylate, 3-methylcrotonate, isocrotonate, or optionally substituted cinnamate.
22. The glucose derivative of any one of the preceding embodiments, wherein the substituent at the 3-position of the glucose comprises a phosphate (e.g. —OPO3H2).
23. The glucose derivative of any one of the preceding embodiments, wherein the substituent at the 6-position comprises an acyl linked amino acid (e.g., an alpha amino acid).
24. The glucose derivative of any one of the preceding embodiments, wherein the substituent at the 6-position comprises an optionally substituted aliphatic acyl group.
25. The glucose derivative of any one of the preceding embodiments, wherein an N-linked heterocyclic group is heteroaryl.
26. The glucose derivative of any one of the preceding embodiments, wherein each aromatic group is independently aryl.
27. A compound that is any one of the glucose derivatives of embodiments 1-26.
28. A compound of Formula I:
29. A compound of Formula I:
30. A compound of Formula III:
31. A compound of Formula IV:
32. A compound of Formula V:
33. A compound of Formula VI:
34. A compound of Formula VII:
35. A compound having the formulae:
36. The compound of embodiment 35, wherein the moiety —NT comprises a monoamine neurotransmitter or a derivative or precursor thereof.
37. The compound of embodiment 35, wherein —NT comprises a catecholamine neurotransmitter or a derivative or precursor thereof.
38. The compound of embodiment 35, wherein —NT is selected from the group consisting of: dopamine, norepinepherine, epinepherine, histamine, and serotonin.
39. The compound of embodiment 35, wherein —NT is selected from tryptamine, phenethylamine, N-methylphenethylamine, phenethanolamine, m-tyramine, p-tyramine, 3-methoxytyramine, N-methyltyramine, 3-indothyronamine, m-octopamine, p-octopamine, and synepherine.
40. The compound of embodiment 35, wherein —NT is selected from:
41. The compound of embodiment 35, wherein —NT is selected from:
42. The compound of embodiment 35, wherein —NT is selected from:
43. The compound of embodiment 35, wherein —NT is selected from:
44. The compound having the formulae:
45. The compound of embodiment 44, wherein —NB is selected from:
46. The compound of embodiment 44, wherein —NB is selected from:
47. The compound of embodiment 44, wherein —NB is selected from:
48. The compound of embodiment 44, wherein —NB is selected from:
49. A compound having the formulae:
50. The compound of embodiment 49, wherein the moiety -MCR comprises a C3-8 alpha beta unsaturated acyl group.
51. The compound of embodiment 49, wherein the moiety -MCR comprises an acyl group corresponding to an ester of acrylic acid, methylacrylic acid, crotonic acid, methyl crotonic acid, valeric acid, 3-methylcrotonic acid or tiglic acid.
52. The compound of embodiment 49, wherein the moiety -MCR comprises acrylate, methacrylate, or cinnamate.
53. A compound of any one of the preceding embodiments, wherein G1 is selected from:
54. A compound of any one of the preceding embodiments, wherein G1 is selected from:
55. A compound of any one of the preceding embodiments, wherein G1 is selected from:
56. A compound of any one of the preceding embodiments, wherein G1 or —NT is selected from:
57. A compound of Formulae XI-a, XI-b, XI-c, XI-d, XI-e, XI-f, or XI-g:
58. The compound of any one of the preceding embodiments, wherein Rn1 is hydrogen and Rn2 is optionally substituted C1-12 aliphatic.
59. The compound of any one of the preceding embodiments, wherein Rn1 is hydrogen and Rn2 is optionally substituted C1-6 aliphatic.
60. The compound of any one of the preceding embodiments, wherein Rn1 is methyl and Rn2 is optionally substituted C1-6 aliphatic.
61. The compound of any one of the preceding embodiments, wherein Rn1 is hydrogen and Rn2 is optionally substituted aryl.
62. The compound of any one of the preceding embodiments, Rn1 is methyl and Rn2 is optionally substituted aryl.
63. The compound of any one of the preceding embodiments, wherein Rn1 is hydrogen and R2 is optionally substituted C3-12 heterocyclic.
64. The compound of any one of the preceding embodiments, wherein Rn1 is hydrogen and R2 is optionally substituted C3-12 heterocyclic.
65. The compound of any one of the preceding embodiments, wherein Rn1 is methyl and Rn2 is optionally substituted C3-6 heterocyclic.
66. The compound of any one of the preceding embodiments, wherein Rn1 is hydrogen and Rn2 is optionally substituted acyl.
67. The compound of any one of the preceding embodiments, wherein Rn1 is hydrogen and Rn2 is optionally substituted acyl.
68. The compound of any one of the preceding embodiments, wherein Rn1 is methyl and Rn2 is optionally substituted acyl.
69. The compound of any one of the preceding embodiments, wherein —NRn1Rn2 comprises a monoamine neurotransmitter or a derivative or precursor thereof
70. The compound of any one of the preceding embodiments, wherein —NRn1Rn2 comprises a catecholamine neurotransmitter or a derivative or precursor thereof.
71. The compound of any one of the preceding embodiments, wherein —NRn1Rn2 is selected from the group consisting of: dopamine, norepinepherine, epinepherine, histamine, and serotonin.
72. The compound of any one of the preceding embodiments, wherein —NRn1Rn2 is selected from tryptamine, phenethylamine, N-methylphenethylamine, phenethanolamine, m-tyramine, p-tyramine, 3-methoxytyramine, N-methyltyramine, 3-indothyronamine, m-octopamine, p-octopamine, and synepherine.
73. The compound of any one of the preceding embodiments, wherein G1 is selected from:
74. A compound of any one of the preceding embodiments, wherein G1 or —NT is selected from:
75. The compound of any one of the preceding embodiments, wherein G2 is selected from:
wherein n is 1-35 (e.g., 1-24, 1-18, 1-12, 1-8, or 1-6) and m is an integer dependent upon n to provide a stable saturated, unsaturated, or polyunsaturated aliphatic group.
76. The compound of any one of the preceding embodiments, wherein G2 is selected from:
wherein n is 1-35 (e.g., 1-24, 1-18, 1-12, 1-8, or 1-6) and m is an integer dependent upon n to provide a stable saturated, unsaturated, or polyunsaturated aliphatic group.
77. The compound of any one of the preceding embodiments, wherein G2 is selected from:
78. The compound of any one of the preceding embodiments, wherein each X is hydrogen.
79. The compound of any one of the preceding embodiments, wherein one X is hydrogen and the other X is a phosphate or diphosphate.
80. The compound of any one of the preceding embodiments, wherein one X is hydrogen and the other X is M+.
81. The compound of any one of the preceding embodiments, wherein one X is hydrogen and the other X is Z+.
82. The compound of any one of the preceding embodiments, wherein G6 is selected from:
83. The compound of any one of the preceding embodiments, wherein G6 is selected from:
84. The compound of any one of the preceding embodiments, wherein G6 is selected from:
85. The compound of any one of the preceding embodiments, wherein G2 and G6 are hydrogen.
86. A compound of Formula A-1 or A-2:
87. The compound of any one of the preceding embodiments, wherein X is hydrogen.
88. The compound of any one of the preceding embodiments, wherein G1 is —OR10.
89. The compound of any one of the preceding embodiments, wherein R10 is optionally substituted aryl or optionally substituted heteroaryl.
90. The compound of any one of the preceding embodiments, wherein R10 is substituted with a group containing a nitrogen atom.
91. The compound of any one of the preceding embodiments, wherein R10 is substituted with an amino (—NH2) group.
92. The compound of any one of the preceding embodiments, wherein R10 is optionally indole (e.g., indole substituted with —(CH2)0-4N(R∘)C(O)R∘).
93. The compound of any one of the preceding embodiments, wherein R10 is aryl or heteroaryl substituted with —(CH2)0-4N(R∘)2, —(CH2)0-4N(R∘)C(O)R∘, or —(CH2)0-4C(O)N(R∘)2.
94. The compound of any one of the previous embodiments, wherein G1 is an N-linked nucleobase.
95. The compound of any one of the previous embodiments, wherein G1 is selected from the group consisting of:
96. The compound of any one of the previous embodiments, wherein G1 is:
97. The compound of any one of the previous embodiments, wherein G1 is selected from the group consisting of:
98. The compound of any one of the previous embodiments, wherein G1 is other than unsubstituted adenine.
99. The compound of any one of the previous embodiments, wherein G1 is —OR10.
100. The compound of any one of the previous embodiments, wherein G1 is —OR10 and R10 is optionally substituted aryl or optionally substituted heteroaryl.
101. The compound of any one of the previous embodiments, wherein G1 is —OR10 and R10 is optionally substituted phenyl, optionally substituted 5- to 6-membered monocyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 8- to 10-membered bicyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
102. The compound of any one of the previous embodiments, wherein R10 is substituted with a group containing a nitrogen atom.
103. The compound of any one of the previous embodiments, wherein R10 is substituted with an amino (—NH2) group.
104. The compound of any one of the previous embodiments, wherein R10 is substituted with —(CH2)0-4N(R∘)2, —(CH2)0-4N(R∘)C(O)R∘, —(CH2)0-4C(O)N(R∘)2.
105. The compound of any one of the previous embodiments, wherein G1 is —OC(O)R11.
106. The compound of any one of the previous embodiments, wherein G1 is —OC(O)R11 and R11 is optionally substituted aryl or optionally substituted heteroaryl.
107. The compound of any one of the previous embodiments, wherein G1 is —OC(O)R11 and R11 is optionally substituted phenyl, optionally substituted 5- to 6-membered monocyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 8- to 10-membered bicyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
108. The compound of any one of the previous embodiments, wherein R11 is substituted with a group containing a nitrogen atom.
109. The compound of any one of the previous embodiments, wherein R11 is substituted with an amino (—NH2) group.
110. The compound of any one of the previous embodiments, wherein R11 is substituted with —(CH2)0-4N(R∘)2, —(CH2)0-4N(R∘)C(O)R∘, —(CH2)0-4C(O)N(R∘)2.
111. The compound of any one of the preceding embodiments, wherein R10 and R11 do not comprise a nitro group.
112. The compound of any one of the preceding embodiments, wherein G1 does not comprise a pyrrole or indole.
113. The compound of any one of the preceding embodiments, wherein G2 and G6 are not acetyl or benzoyl.
114. The compound of any one of the preceding embodiments, wherein the compound 5 is other than one or more more of the following compounds:
115. The compound of any one of the preceding embodiments, wherein the compound 5 is other than one of more of the following compounds:
116. The compound of any one of the preceding embodiments, wherein the compound is other than one or more of the following compounds:
117. A compound of Table S4a, or a pharmaceutically acceptable salt thereof.
118, A compound of Table S4b, or a pharmaceutically acceptable salt thereof.
119. A compound of Table S5, or a pharmaceutically acceptable salt thereof.
120. The compound of any one of the preceding embodiments, wherein the compound is a compound depicted in
122. The compound of any one of the preceding embodiments, wherein the compound is a pure compound.
123. The compound of any one of the preceding embodiments, wherein the compound is provided outside of a C. elegans worm body.
124. The compound of any one of the preceding embodiments, wherein the compound is provided free of C. elegans tissue or other biological materials typically contained within or excreted by C. elegans.
125. A compound of any one of the preceding embodiments for use in medicine.
126. A therapeutic composition comprising a therapeutically effective amount of a compound of any one of the preceding embodiments.
127. A therapeutic composition for treating a disease or disorder, wherein the composition comprises one or more MOGLs of Formula I:
or a pharmaceutically acceptable salt thereof wherein:
128. The therapeutic composition of embodiment 127, where the disease or disorder is a neurological disease,
129. The therapeutic composition of embodiment 128, wherein the —NT comprises a monoamine neurotransmitter or a derivative or precursor thereof.
130. The therapeutic composition of embodiment 128, wherein the —NT is selected from the group consiting of: catecholamine neurotransmitters or derivatives or precursors thereof, dopamine, norepinepherine, epinepherine, histamine, serotonin, tryptamine, phenethylamine, N-methylphenethylamine, phenethanolamine, m-tyramine, p-tyramine, 3-methoxytyramine, N-methyltyramine, 3-indothyronamine, m-octopamine, p-octopamine, and synepherine.
131. The therapeutic composition of embodiment 127, where the disease or disorder is cancer, a kinase dependent disorder or disease such as hypertension, Parkinson's disease, and autoimmune disease, or a disorder that resulst in or arises from changes to nucleotide synthesis including, but not limited to cancer and viral diseases,
132. The therapeutic composition of embodiment 131, wherein —NB comprises a nucleobase linked to the glucose through a nitrogen or oxygen atom comprising part of the nucleobase structure.
133. The therapeutic composition of embodiment 131, wherein —NB is selected from the group consisting of:
134. The therapeutic composition of embodiment 131, wherein —NB is selected from the group consisting of:
135. The therapeutic composition of embodiment 131, wherein —NB is selected from the group consisting of:
136. The therapeutic composition of embodiment 131, wherein —NB is selected from the group consisting of:
137. The therapeutic composition of embodiment 127, where the disease or disorder is responsive to regulation of TOR function, and wherein the composition comprises one or more MOGLs selected from the group consisting of:
138. The therapeutic composition of embodiment 137, wherein -MCR comprises a C3-8 alpha beta unsaturated acyl group, or wherein the moiety -MCR comprises a C4-8 alpha beta unsaturated acyl group, or wherein the moiety -MCR comprises an acyl group corresponding to an ester of acrylic acid, methylacrylic acid, crotonic acid, methyl crotonic acid, valeric acid, 3-methylcrotonic acid, or tiglic acid.
139. The therapeutic composition of embodiment 137, wherein -MCR is selected from the group consisting of: crotonate, tiglate, valerate, acrylate, methacrylate, cinnamate, 2-imidazoleacrylate and urocanate.
140. A therapeutic composition for treatment of a disease or disorder responsive to regulation of proteasome function, wherein the composition comprises one or more MOGLs of Formula A-1:
141. The compound or composition of any one of the preceding embodiments, wherein G2 is an optionally substituted aliphatic acyl, optionally substituted aromatic acyl, optionally substituted heteroaromatic acyl, or optionally substituted heteroaliphatic acyl group.
142. The compound or composition of any one of the preceding embodiments, wherein G6 is an optionally substituted aliphatic acyl, optionally substituted aromatic acyl, optionally substituted heteroaromatic acyl, or optionally substituted heteroaliphatic acyl group.
143. The compound or composition of any one of the preceding embodiments, wherein each N-linked heterocycle is independently heteroaryl.
144. The compound or composition of any one of the preceding embodiments, wherein each aromatic is independently aryl (e.g., phenyl).
145. The compound or composition of any one of the preceding embodiments, wherein each heteroaliphatic is an independently an aliphatic group having 1-24 (e.g., 1-12, 1-8, or 1-6) carbons where 1-6 (e.g., 1-4, 1-3, or 1-2) carbons are independently replaced by a heteroatom selected from oxygen, sulfur, nitrogen, and phosphorus.
146. The compound or composition of any one of the preceding embodiments, wherein a heteroaryl ring is 5- to 6-membered monocyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 8- to 10-membered bicyclic heteroaryl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
147. The compound or composition of any one of the preceding embodiments, wherein a heterocylic ring is 3- to 7-membered saturated or partially unsaturated monocyclic heterocyclyl having 1-3 heteroatoms independently selected from nitrogen, oxygen, and sulfur, or 7- to 10-membered saturated or partially unsaturated bicyclic heterocyclyl having 1-4 heteroatoms independently selected from nitrogen, oxygen, and sulfur.
148. A pharmaceutical composition comprising a compound or composition of any one of the preceding embodiments and a pharmaceutically acceptable carrier or excipient.
149. A method of making a therapeutic composition comprising formulating an effective amount a compound or composition of any one of the preceding embodiments (or a pharmaceutically-acceptable salt, prodrug or derivative thereof) into a pharmaceutical composition selected from the group consisting of: injectible liquid, tablet, capsule, pill, solution or suspension for oral administration, solid for suspension or dissolution into a drinkable or injectible liquid, dermal patch, eye drop, cream, ointment, gel, powder, spray, and inhalable.
150. A method of making a therapeutic composition comprising formulating an effective amount of one or more purified or synthetically-produced MOGLs (or a pharmaceutically-acceptable salt, prodrug or derivative thereof) into a pharmaceutical composition selected from the group consisting of: injectible liquid, tablet, capsule, pill, solution or suspension for oral administration, solid for suspension or dissolution into a drinkable or injectible liquid, dermal patch, eye drop, cream, ointment, gel, powder, spray, and inhalable.
151. A method comprising administering to a mammal a therapeutically effective dose of one or more compounds of the preceding embodiments.
152. A method of improving the mental or emotional state of a patient comprising administering to a patient in need thereof a therapeutically effective amount of a compound of any one of the preceding embodiments.
153. A method of treating anxiety in a patient comprising administering to a patient in need thereof a therapeutically effective amount of a compound of any one of the preceding embodiments.
154. A method of treating depression in a patient comprising administering to a patient in need thereof a therapeutically effective amount of a compound of any one of the preceding embodiments.
155. A method of treating a neurological disorder in a patient comprising administering to a patient in need thereof a therapeutically effective amount of a compound of any one of the preceding embodiments.
156. The method of embodiment 155, wherein the neurological disorder is anxiety, depression, obsessive or compulsive disorders or behaviors, tics, bipolar disorder, schizophrenia, learning disorders, cognitive decline, behavioral disorders, learning disability, or hyperactivity.
157. A method of treating a kinase-dependent disease or disorder in a patient comprising administering to a patient in need thereof a therapeutically effective amount of a compound of any one of the preceding embodiments.
158. A method of treating diseases or disorders that result in or arise from changes to nucleotide synthesis comprising administering to a patient in need thereof a therapeutically effective amount of a compound of any one of the preceding embodiments.
159. The method of embodiment 158, wherein the disease or disorder is a cancer or a viral infection.
160. A method of treating a disease or disorder responsive to modulation of the proteasome, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of any one of the preceding embodiments.
161. The method of embodiment 160, wherein the disease or disorder is cancer or neurodegenerative disease.
162. The method of embodiment 161, wherein the disease or disorder is Alzheimer's, Parkinson's, or Huntington's disease.
163. The method of any one of embodiments 160-162, wherein the compound is sngl #1, sngl #2, or a pharmaceutically acceptable salt thereof.
164. A method of treating or ameliorating a disease, disorder, or condition associated with a cellular or environmental stress response, comprising administering to a patient in need thereof a therapeutically effective amount of a compound of any one of the preceding embodiments.
165. The method of embodiment 164, wherein the stress response is oxidative stress response.
166. The method of embodiment 164 or 165, where the condition is shortened life span.
167. The method of embodiment 164 or 165, wherein the disease is cancer or a neurodegenerative disease.
168. The method of any one of embodiments 165-167, wherein the compound comprises an indole moiety at the 1-position (e.g., G1).
169. A method treating a disease or disorder, comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising one or more MOGLs of Formula I:
or a pharmaceutically acceptable salt thereof
170. The method of embodiment 169, where the disease or disorder is a neurological disease, and wherein the composition comprises one or more MOGLs selected from the group consisting of:
171. The method of embodiment 170, wherein the —NT comprises a monoamine neurotransmitter or a derivative or precursor thereof.
172. The method of embodiment 170, wherein the —NT is selected from the group consiting of catecholamine neurotransmitters or derivatives or precursors thereof, dopamine, norepinepherine, epinepherine, histamine, serotonin, tryptamine, phenethylamine, N-methylphenethylamine, phenethanolamine, m-tyramine, p-tyramine, 3-methoxytyramine, N-methyltyramine, 3-indothyronamine, m-octopamine, p-octopamine, and synepherine.
173. The method of embodiment 169, where the disease or disorder is cancer, a kinase dependent disorder or disease such as hypertension, Parkinson's disease, and autoimmune disease, or a disorder that resulst in or arises from changes to nucleotide synthesis including, but not limited to cancer and viral diseases,
174. The method of embodiment 173, wherein —NB comprises a nucleobase linked to the glucose through a nitrogen or oxygen atom comprising part of the nucleobase structure.
175. The method of embodiment 173, wherein —NB is selected from the group consisting of:
176. The method of embodiment 173, wherein —NB is selected from the group consisting of:
177. The method of embodiment 173, wherein —NB is selected from the group consisting of:
178. The method of embodiment 173, wherein —NB is selected from the group consisting of:
179. The method of claim 169, where the disease or disorder is responsive to regulation of TOR function, and wherein the composition comprises one or more MOGLs selected from the group consisting of:
180. The method of claim 179, wherein -MCR comprises a C3-8 alpha beta unsaturated acyl group, or wherein the moiety -MCR comprises a C4-8 aloha beta unsaturated acyl group, or wherein the moiety -MCR comprises an acyl group corresponding to an ester of acrylic acid, methylacrylic acid, crotonic acid, methyl crotonic acid, valeric acid, 3-methylcrotonic acid, or tiglic acid.
181. The method of claim 179, wherein -MCR is selected from the group consisting of crotonate, tiglate, valerate, acrylate, methacrylate, cinnamate, 2-imidazoleacrylate and urocanate.
182. A method for treating a disease or disorder responsive to regulation of proteasome function, comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising one or more MOGLs of Formula A-1:
183. A method comprising administering to a mammal a composition comprising a therapeutically effective amount of one or more MOGLs of Formula I:
or a pharmaceutically acceptable salt thereof
184. The method of claim 183, where the disease or disorder is cancer or another other kinase dependent disorder or disease such as hypertension, Parkinson's disease, and autoimmune disease, or a disorder that results in or arises from changes to nucleotide synthesis including cancer and viral diseases,
185. The method of claim 183, where the disease or disorder is a neurological disease, and wherein the one or more MOGLs selected from the group consisting of:
186. The method of claim 183, where the disease or disorder is one responsive to regulation of TOR function, and wherein the composition comprises one or more MOGLs selected from the group consisting of:
187. A method comprising administering to a mammal a composition comprising a therapeutically effective amount of one or more MOGLs of Formula A-1 or A-2:
188. The method of any one of the preceding claims, wherein the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient or carrier.
The following Examples are useful to confirm aspects of the disclosure described above and to exemplify certain embodiments of the disclosure.
Cel-CEST-1.2 contributes to biosynthesis of >150 MOGLs. Following the initial discovery of certain MOGLs,7,14,16 we noted that their production is greatly increased under starvation conditions. Surveying published transcriptomic datasets for starvation-induced cest-homologs, we noted that Cel-cest-1.2 expression is rapidly induced 4-5-fold by starvation (
To investigate the biosynthetic role of Cel-cest-1.2, we obtained a mutant lacking the first 1500 bp of the coding sequence, including the serine at the putative active site (
Next, we categorized the large number of Cel-cest-1.2-dependent metabolites based on their MS/MS fragmentation patterns, which enabled putative assignment to families of MOGLs based on several different scaffolds, e.g. N- or O-glucosylated indole and anthranilic acid (
These results suggested that Cel-CEST-1.2 may be required for site-selective acylation of the parent glucoside scaffolds. To determine whether Cel-CEST-1.2 is responsible for 2- or 6-O-acylation, we selected the 2-O-acylated variants of three mono-acylated MOGLs for total synthesis via established methods (
Cbr-CEST-2 is the functional ortholog of Cel-CEST-1.2. Cel-CEST-1.2 appears to be well conserved across the genus Caenorhabditis and possibly other nematode genera, e.g. Pristionchus (
Using CRISPR/Cas9, we generated two Cbr-cest-2 null mutant strains and compared their endo- and exo-metabolomes with C. briggsae wildtype via HPLC-HRMS-based comparative metabolomics, as above. We found that Cbr-cest-2 mutants are defective in the production of >150 different MOGLs, including 97 MOGLs also produced by C. elegans, all of which are Cel-cest-1.2-dependent (
Lifestage- and starvation-dependent roles of Cel-CEST-1.2. Biosynthesis of small molecules in C. elegans is often strongly dependent on developmental stage and nutritional state.25-27 Previous transcriptomic analysis showed that Cel-cest-1.2 expression peaks at the third larval stage (L3) and is induced by starvation (
Biosynthesis of most Cel-cest-1.2-dependent MOGLs was strongly induced by starvation. Pyrrolic acid-containing MOGLs were most strongly upregulated (e.g. iglu #58 (40)), whereas MOGLs incorporating nicotinic acid were not increased or even slightly downregulated (e.g. iglu #601 (42))
C. elegans is an important model for how starvation and dietary restriction affect lifespan in animals,28-31 and small molecules have been shown to play a major role in the underlying mechanisms.32 Because MOGL biosynthesis is strongly upregulated during starvation, we tested whether Cel-CEST-1.2 is required for starvation survival (
These results demonstrate that Cel-CEST-1.2 and Cbr-CEST-2 are required for 2-O-acylation in the biosynthetic pathways of >150 different MOGLs. The product ranges in the two nematode species largely overlap, and differences may be due primarily to differences in available substrate pools. Despite the very large number of Cel-CEST-1.2/Cbr-CEST-2-dependent metabolites, their biosynthetic roles appear to be specific to 2-O-acylation, since every significant metabolic feature strongly downregulated or abolished in Cel-cest-1.2 or Cbr-cest-2 mutants, as detected in our comparative metabolomic analysis, could be assigned to a 2-O-acylated glucoside. Members of the α/β hydrolase family are known to exhibit broad substrate promiscuity,36 for example, the human Cel-CEST-1.2 homolog, carboxylesterase 2 (CES2) is capable of cleaving a diverse range of xenobiotics.37
In conjunction with the previous finding that Cel-cest-4 is specifically required for 6-O-attachment of anthranilate in indole glucosides (e.g. iglu #4 (11) in
Whereas none of the abundant diacylated MOGLs are strictly cest-4-dependent,7 production of a large number of diacylated MOGLs is fully abolished in Cel-cest-1.2 mutants, suggesting that Cel-CEST-1.2 is primarily responsible for 2-O-acylation, whereas there must be additional homologs mediating 6-O-acylation, in addition to Cel-CEST-4, which compared to Cel-CEST-1.2, appears to have a much narrower substrate scope. Attempts to recapitulate the biosynthetic activities of CESTs in vitro have been unsuccessful so far, likely due to the presence of the C-terminal transmembrane domain which may cause improper folding under in vitro conditions.7,9,38
Our results further demonstrate that MOGL biosynthesis is highly regulated during development and depends on nutritional conditions. Different compound profiles at different life stages likely result in part from regulation of cest-expression, but may also reflect changes in substrate pools. For example, starvation is generally associated with increased protein turnover, which may result in an increase in amino acid degradation-derived building blocks, e.g. pyrrolic acid from proline or isovaleric and tiglic acid from leucine and isoleucine, respectively.39,40 Further, the relatives abundance of MOGLs may also depend on bacterial metabolism.22 For example, most bacteria occurring naturally with C. elegans produce much smaller amounts of indole than E. coli OP50.41 Correspondingly, we observed that C. elegans fed Providencia alcalifaciens JUb39, a bacterial species found with C. elegans in the wild, produce less indole-derived MOGLs compared to OP50-fed worms, whereas production of tyramine-derived MOGLs is increased, consistent with increased tyramine production in C. elegans fed Jub39 bacteria (
Notably, MOGLs are mostly retained in the worm body and not excreted, suggesting that they serve specific intra-organismal function(s), paralleling the role of ascarosides in inter-organismal signaling. Their highly context-specific production further supports the hypothesis that MOGLs may serve diverse biological functions. Our finding that Cel-cest-1.2 plays an important role for starvation survival and is conserved across other species provides a starting point for elucidating the role of MOGLs in C. elegans and other nematodes.
Nematode and bacterial strains. Unless indicated otherwise, worms were maintained on Nematode Growth Medium (NGM) 6 cm diameter Petri dish plates with E. coli OP50 (www.wormbook.org/methods).1 Nematode strains used in this study are listed below:
C. elegans wildtype
C. briggsae wildtype
Metabolite nomenclature. All newly detected metabolites for which a structure could be proposed were named using SMIDs. SMIDs (Small Molecule IDentifiers) have been introduced as a search-compatible naming system for metabolites newly identified from C. elegans and other nematodes. The SMID database (www.smid-db.org) is an electronic resource maintained in collaboration with WormBase (www.wormbase.org). A complete list of SMIDs can be found at www.smid-db.org/browse.
Amino acid sequence alignment. Alignments of Cel-CEST-1.1 with Cel-CEST-1.2 and Cbr-CEST-2 were done using T-Coffee Multiple Sequence alignment.3 Protein sequences are from WormBase. Amino acids were colored based on chemical properties: AVFPMILW=red (small+hydrophobic), DE=blue (acidic), RHK=magenta (basic), STYHCNGQ=green (hydroxyl+sulfhydryl+amine+glycine).
C. briggsae phylogenetic tree. The protein sequence of Cel-CEST-1.1 was submitted to an NCBI BLASTp search (restricted to species C. briggsae, conditional compositional BLOSUM62, gap open cost: 11, gap extension cost: 1, word size: 6).4 The top 36 BLAST hits by E-value and only the best scoring transcript variant was kept for each protein sequence hit. These 42 hits along with the 8 C. elegans esterase strains were then imported into MEGAX and aligned using MUSCLE5 (settings: gap open penalty: −2.9, gap extend 0, hydrophobicity multiplier 1.2, max. iterations 8, clustering method for all iterations: UPGMB, minimal diagonal length: 24). The evolutionary history was inferred using the Neighbor-Joining method.6 The optimal tree is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (200 replicates) are shown next to the branches.7 The evolutionary distances were computed using the JTT matrix-based method8 and are in the units of the number of amino acid substitutions per site. This analysis involved 44 amino acid sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 1248 positions in the final dataset. Evolutionary analyses were conducted in MEGA X.9
Caenorhabditis Cel-CEST-1.1 homologs tree. The protein sequence of Cel-CEST-1.1 was submitted to an NCBI BLASTp search (restricted to various Caenorhabditis species, conditional compositional BLOSUM62, gap open coast:11, gap extension cost: 1, word size: 6).4 Hits with Bit-score above ˜300 were kept for each species. These 17 sequences were then imported into MEGAX10 and aligned using MUSCLE5 (settings: gap open penalty: −2.9, gap extend 0, hydrophobicity multiplier 1.2, max. iterations 8, clustering method for all iterations: UPGMB, minimal diagonal length: 24). The evolutionary history was inferred using the Neighbor-Joining method.6 The bootstrap consensus tree inferred from 200 replicates is taken to represent the evolutionary history of the taxa analyzed.7 Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (200 replicates) are shown next to the branches.7 The evolutionary distances were computed using the JTT matrix-based method and are in the units of the number of amino acid substitutions per site.8 This analysis involved 17 amino acid sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 1803 positions in the final dataset. Evolutionary analyses were conducted in MEGA X.9
C. briggsae CRISPR mutagenesis for generation of Cbr-cest-2 null mutants. The Cbr-cest-2 mutants PS9060 and PS9061 were both created using the briggsae-adaptation of the STOP-IN cassette method as described previously.n11,12 Both strains were made via insertion of the STOP-IN cassette into the middle of the first exon using the guide CATTACTCATACAAGCTGGA.
Nematode cultures. Cultures were started by chunking C. elegans or C. briggsae onto 10 cm NGM plates (each seeded with 800 μL of OP50 E. coli grown to stationary phase in Lennox Broth) and incubated at 22° C. Once most food was consumed, each plate was washed with 25 mL of S-complete medium into a 125 mL Erlenmeyer flask, and 1 mL of OP50 E. coli was added (E. coli cultures were grown to stationary phase in Lennox Broth, pelleted and resuspended at 1 g wet mass per 1 mL M9 buffer), shaking at 220 RPM and 22° C. After 70 hr, cultures were centrifuged at 1000 g for 1 min. After discarding supernatant, 24 mL H2O was added along with 6 mL bleach, 900 μL 10 M NaOH, and the mixture was shaken for 3 min to prepare eggs. Eggs were centrifuged at 1000 g, the supernatant was removed, and the egg pellet was washed with 25 mL M9 buffer twice and then suspended in a final volume of 5 mL M9 buffer in a 50 mL centrifuge tube. Eggs were counted and placed on a rocker and allowed to hatch as L1 larvae for 24 hr at 22° C. 70,000 L1 larvae were seeded in 25 mL cultures of S-complete with 1 mL of OP50 and incubated at 220 RPM and 22° C. in a 125 mL Erlenmeyer flask. After 72 hr, worms were spun down at 1000 g for 5 min, and media was separated from worm body pellet. Separated media and worm pellet were flash frozen over liquid nitrogen and then lyophilized. Two to four biological replicates were grown for each strain. Mutants were grown with parallel wildtype controls, and biological replicates were started on different days.
Nematode cultures with Providencia Jub39.13 Approximately 10,000 mixed stage C. elegans wildtype (N2) animals were reared on either E. coli OP50 or Providencia alcalifaciens JUb39 at a density of 2,000 animals per 10 cm NGM plate. Animals were collected in 15 mL conical tubes by serially washing the plates with M9 buffer. Animals were washed three times with 10 mL M9 before transfer to 1.5 mL microfuge tubes, then snap frozen in liquid nitrogen. Samples were lyophilized for 18-24 hr using a VirTis BenchTop 4K Freeze Dryer. After the addition of two stainless steel grinding balls and 1 mL of 80% methanol, samples were sonicated for 5 min (2 sec on/off pulse cycle at 90 A) using a Qsonica Q700 Ultrasonic Processor with a water bath cup hom adaptor (Model 431C2). Following sonication, microfuge tubes were centrifuged at 10,000 g for 5 min in an Eppendorf 5417R centrifuge. 800 μL of the resulting supernatant was transferred to a clean 4 mL glass vial, and 800 μL of fresh methanol added to the sample. The sample was sonicated and centrifuged as described, and the resulting supernatant was transferred to the same receiver vial and concentrated to dryness in an SC250EXP Speedvac Concentrator coupled to an RVT5105 Refrigerated Vapor Trap (Thermo Scientific). The resulting powder was suspended in 120 μL of 100% methanol, followed by vigorous vortex and brief sonication. This solution was transferred to a clean microfuge tube and subjected to centrifugation at 20,000 g for 10 min in an Eppendorf 5417R centrifuge to remove precipitate. The resulting supernatant was transferred to an HPLC vial and analyzed by HPLC-MS.
Metabolite extraction. Lyophilized pellet or media samples were crushed and homogenized by shaking with 2.5 mm steel balls at 1300 RPM for 3 min in 30 s pulses while chilled with liquid nitrogen (SPEX sample prep miniG 1600). Powdered media and pellet samples were extracted with 10 mL methanol in 50 mL centrifuge tubes, rocking overnight at 22° C. Extractions were pelleted at 5000 g for 10 min at 4° C., and supernatants were transferred to 20 mL glass scintillation vials. Samples were then dried in a SpeedVac (Thermo Fisher Scientific) vacuum concentrator. Dried materials were resuspended in 1 mL methanol and vortexed for 1 min. Samples were pelleted at 10,000 g for 5 min at 22° C., and supernatants were transferred to 2 mL HPLC vials and dried in a SpeedVac vacuum concentrator. Samples were resuspended in 100 μL of methanol, transferred into 1.7 mL Eppendorf tubes, and centrifuged at 18,000 g for 20 min at 4° C. Clarified extracts were transferred to HPLC vials and stored at −20° C. until analysis.
Preparation of endo-metabolome samples from staged starved and fed cultures. 40,000 synchronized L1 larvae were added to 125 mL Erlenmeyer flasks containing 30 mL of S-complete medium. Worms were fed with 4 mL of concentrated OP50 and incubated at 20° C. with shaking at 160 RPM for: 12 hr (L1), 24 hr (L2), 32 hr (L3), 40 hr (L4) and 58 hr (gravid adults). For preparation of starved samples, each of the stages was starved for 24 hr after reaching their desired developmental stage in S-complete without OP50. After incubation for the desired time, liquid cultures were centrifuged (1000 g, 22° C., 1 min) and supernatants were collected. Supernatant was separated from intact OP50 by centrifuging (3000 g, 22° C., 5 min), and the resulting supernatants (exo-metabolome) were lyophilized. Lyophilized samples were homogenized with a dounce homogenizer in 10 mL methanol and extracted on a stirring plate (22° C., 12 hr). The resulting suspension was centrifuged (4000 g, 22° C., 5 min) to remove any precipitate before carefully transferred to HPLC vials. Three biological replicates were started on different days.
Mass spectrometric analysis. High resolution LC-MS analysis was performed on a Thermo Fisher Scientific Vanquish Horizon UHPLC System coupled with a Thermo Q Exactive hybrid quadrupole-orbitrap high-resolution mass spectrometer equipped with a HESI ion source. 1 μL of extract was injected and separated using a water-acetonitrile gradient on a Thermo Scientific Hypersil GOLD C18 column (150 mm×2.1 mm 1.9 um particle size 175 Å pore size, Thermo Scientific) and maintained at 40° C. Solvents were all purchased from Fisher Scientific as HPLC grade. Solvent A: 0.1% formic acid in water; solvent B: 0.1% formic acid in acetonitrile. A/B gradient started at 1% B for 3 min, then from 1% to 100% B over 20 min, 100% for 5 min, then down to 1% B for 3 min. Mass spectrometer parameters: 3.5 kV spray voltage, 380° C. capillary temperature, 300° C. probe heater temperature, 60 sheath flow rate, 20 auxiliary flow rate, 2.0 spare gas; S-lens RF level 50.0, resolution 240,000, m/z range 150-1000, AGC target 3e6. Instrument was calibrated with positive and negative ion calibration solutions (Thermo Fisher) Pierce LTQ Velos ESI pos/neg calibration solutions. Peak areas were determined using Xcalibur 2.3 QualBrowser version 2.3.26 (Thermo Scientific) using a 5 ppm window around the m/z of interest. HPLC-MS peak areas were normalized to the measured abundance of ascr #3 (www.smid-db.org/detail/ascr %233) in each sample for all graphs in this manuscript, except for
Feature detection and characterization. LC-MS RAW files from each sample were converted to mzXML (centroid mode) using MSConvert (ProteoWizard), followed by analysis using the XCMS14 analysis feature in Metaboseek (metaboseek.com). Peak detection was carried out with the centWave algorithm15 values set as: 4 ppm, 320 peakwidth, 3 snthresh, 3100 prefilter, FALSE fitgauss, 1 integrate, TRUE firstBaselineCheck, 0 noise, wMean mzCenterFun, −0.005 mzdiff XCMS feature grouping values were set as: 0.2 minfrac, 2 bw, 0.002 mzwid, 500 max, 1 minsamp, FALSE usegroup. Metaboseek peak filling values set as: 5 ppm_m, 5 rtw, TRUE rtrange. Resulting tables were then processed with the Metaboseek Data Explorer. Molecular features were filtered for each particular null mutant against all other mutants. Filter values were set as: 10 to max minFoldOverCtrl, 15000 to max meanInt, 120 to 1500 rt, 0.95 to max Peak Quality as calculated by Metaboseek. Features were then manually curated by removing isotopic and adducted redundancies. Remaining masses were put on the inclusion list for MS/MS (ddMS2) characterization. Positive and negative mode data were processed separately. In both cases we checked if a feature had a corresponding peak in the opposite ionization mode, since fragmentation spectra in different modes often provide complementary structural information. To acquire MS/MS spectra, we ran a top-10 data dependent MS2 method on a Thermo QExactive-HF mass spectrometer with MS1 resolution 60,000, AGC target 1×10{circumflex over ( )}6, maximum IT (injection time) 50 ms, MS/MS resolution 45,000, AGC target 5×10{circumflex over ( )}5, maximum IT 80 ms, isolation window 1.0 m/z, stepped NCE (normalized collision energy) 25, 50, dynamic exclusion 3 s.
Starvation survival assay. 20-30 gravid adults were placed on 6 cm NGM plates seeded with 75 μL OP50 bacteria grown overnight in LB media (ad libitum, AL plates) and allowed to lay eggs for 2 hr. 15-20 single embryos were isolated onto fresh 3.5 cm AL plates and grown for 60 hr, before starting egg laying. Single worms were transferred to 3.5 cm NGM plates without peptone and without bacteria (starvation plates) for 2 hr to get rid of remaining OP50 bacteria. They were then transferred to fresh starvation plates and monitored for the timepoint of first egg laying. From 70 hr on, worms were monitored for death caused by internal hatching events (bagging/exploding phenotype) and for rarely occurring death events not caused by internal hatching. Worms that crawled off the agar were censored from the analysis. The assay was repeated three times.
Developmental assay. Developmental timing in wildtype (N2) and Cel-cest-1.2 mutant worms grown up under high density (HD) conditions was measured as previously described by determining the time point of first egg laying.16 Briefly, around 40 gravid young adults were allowed to lay eggs for 1 hr on NGM plates seeded with OP50 E. coli bacteria. 25 Single eggs were then transferred to a fresh plate. After 59 hr animals were scored for the timepoint of first egg laying using a Leica S6E stereo microscope.
13C6-Leu isotope tracing experiment. Approximately 60,000 synchronized N2 (wildtype C. elegans) and Cel-daf-22 mutant L1 larvae were seeded in 125 mL Erlenmeyer flasks containing 20 mL S-Complete medium. Worms were fed with 3 mg/mL freeze-dried OP50 powder (InVivoBiosystems, formerly NemaMetrix Inc., cat. #OP-50-31772) and supplemented with either L-Leucine (Sigma-Aldrich cat. #L8000) or 13C6-L-Leucine (Cambridge Isotope Laboratories cat. #CLM-2262-H—PK) at a final concentration of 2 mM. Worms were incubated at 20° C. with shaking at 180 RPM for approx. 70 hr, at which time the population was a mixture of young and gravid adults, determined by microscopic inspection. Liquid cultures were centrifuged (500 g, 22° C., 1 min), and the resulting supernatant was snap frozen. Worm pellet was washed three times with M9 before snap freezing in liquid nitrogen. Frozen samples were lyophilized and extracted as above (Metabolite extraction).
It will appreciated that certain compounds of Tables S4a and S4b observed in C. elegans and C. briggsae have been chemically synthesized in order to confirm structural assignments. Such syntheses are described in the ensuing examples. The skilled person will recognize that individual compounds not explicity described synthetically below can be made using methods similar to those described, substituting appropriate starting materials or intermediates to arrive at the desired compound.
General synthetic procedures. Unless noted otherwise, all chemicals and reagents were purchased from Sigma-Aldrich. All oxygen and moisture-sensitive reactions were carried out under argon atmosphere in flame-dried glassware. Solutions and solvents sensitive to moisture and oxygen were transferred via standard syringe and cannula techniques. All commercial reagents were purchased as reagent grade and, unless otherwise stated, were purchased from Sigma-Aldrich and used without any further purification. Boc-2-Abz-OH was purchased from Chem-impex. Acetic acid (AcOH), acetonitrile (ACN), dichloromethane (DCM), ethyl acetate (EtOAc), formic acid, hexanes and methanol (MeOH) used for chromatography and as a reagent or solvent were purchased from Fisher Scientific. Thin-layer chromatography (TLC) was performed using J. T. Baker Silica Gel IB2F plates. Flash chromatography was performed using Teledyne Isco CombiFlash systems and Teledyne Isco RediSep Rf silica and C18 columns. All deuterated solvents were purchased from Cambridge Isotopes. Nuclear Magnetic Resonance (NMR) spectra were recorded on Bruker INOVA 500 (500 MHz) and Varian INOVA 600 (600 MHz) spectrometers at Cornell University's NMR facility and Bruker AVANCE III HD 800 MHz (800 MHz) or Bruker AVANCE III HD 600 MHz (600 MHz) at SUNY ESF's NMR facility. 1H NMR chemical shifts are reported in ppm (δ) relative to residual solvent peaks (7.26 ppm for chloroform-d, 3.31 ppm for methanol-d4, 2.50 for DMSO-d6). NMR-spectroscopic data are reported as follows: chemical shift, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br=broad), coupling constants (Hz), and integration and often tabulated including 2D NMR data. 13C NMR chemical shifts are reported in ppm (δ) relative to residual solvent peaks (77.16 ppm for chloroform-d, 49.00 ppm for methanol-d4, 39.52 for DMSO-d6). All NMR data processing was done using MNOVA 14.2.1 (mestrelab.com).
HPLC-HRMS, high performance liquid chromatography-high resolution mass spectrometry; MOGL, modular glucoside; MS/MS, tandem mass spectrometry; LRO, lysosome related organelle; UGT, uridine diphosphoglucuronosyltransferase; UDP, uridine 5′-diphosphate; CEST, carboxylesterase; ESI-, electrospray ionization negative mode; ESI+, electrospray ionization positive mode; mCPBA, 3-chloroperoxybenzoic acid.
iglu #1 (4) was synthesized as described previously.17
iglu #3 (10) was synthesized as described previously.2
Iglu #301 (31) was synthesized as described previously.2
To 2.5 mL of DMF was added iglu #1 (4, 144.6 mg, 0.518 mmol, 1.0 equiv.) and imidazole (155.0 mg, 2.28 mmol, 4.4 equiv.). The stirred mixture was cooled to 0° C. before adding 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (215 μL, 0.673 mmol, 1.3 equiv.). The reaction mixture was stirred at room temperature for 30 min, diluted with DCM, and then quenched with water. The organics were washed with sat. aq. NaHCO3, dried with Na2SO4, and concentrated in vacuo. Flash column chromatography on silica using a gradient of 0-20% MeOH in DCM afforded S1 (250.5 mg, 93%) as an orange oil. 1H NMR (500 MHz, chloroform-d): δ (ppm) 7.60 (d, J=7.8 Hz, 1H), 7.50 (d, J=8.2 Hz, 1H), 7.24 (d, J=3.5 Hz, 1H), 7.19 (ddd, J=1.2, 7.6, 8.6 Hz, 1H), 7.12 (ddd, J=0.9, 7.5, 8.0 Hz, 1H), 5.36 (d, J=8.9 Hz, 1H), 4.13 (dd, J=2.0, 12.7 Hz, 1H), 4.04 (t, J=7.7 Hz, 1H), 4.02 (t, J=8.2 Hz, 1H), 3.97 (dd, J=1.6, 12.7 Hz, 1H), 3.80 (t, J=9.0 Hz, 1H), 3.47 (dt, J=1.6, 9.0 Hz, 1H), 1.17-1.03 (m, 28H).
To a stirred solution of benzoic acid (14.4 mg, 0.118 mmol, 1.0 equiv.) in DCM, EDC·HCl (45.2 mg, 0.236 mmol, 2.0 equiv.) was added. The mixture was stirred at room temperature for 40 min, and S1 (73.8 mg, 0.142 mmol, 1.2 equiv.) and DMAP (36.0 mg, 0.295 mmol, 2.5 equiv.) were added. The reaction mixture was stirred at room temperature for 2.5 hours. The reaction mixture was concentrated in vacuo followed by flash column chromatography on silica using a gradient of 5-80% EtOAc in hexanes affording 16 (72.3 mg, 98%). 1H NMR (500 MHz, chloroform-d): δ (ppm) 7.77 (dd, J=1.2, 8.5 Hz, 2H), 7.52 (d, J=7.8 Hz, 1H), 7.49-7.44 (m, 2H), 7.32-7.28 (m, 3H), 7.18 (ddd, J=1.0, 7.7, 8.3 Hz, 1H), 7.07 (ddd, J=0.8, 7.6, 8.3 Hz, 1H), 6.46 (d, J=3.4 Hz, 1H), 5.72 (d, J=9.2 Hz, 1H), 5.66 (t, J=9.0 Hz, 1H), 4.17 (dd, J=2.1, 12.6 Hz, 1H), 4.14 (t, J=9.0 Hz, 1H), 4.08 (t, J=9.0 Hz, 1H), 4.02 (dd, J=1.2, 12.6 Hz, 1H), 3.55 (dt, J=1.6, 9.2 Hz, 1H), 1.19-1.03 (m, 28H).
To a solution of 16 (32.5 mg, 0.052 mmol, 1.0 equiv.) in 0.8 mL DCM was added dibenzyl N,N-diisopropylphosphoramidite (105 μL, 0.312 mmol, 6.0 equiv.) and 1H-tetrazole (0.45 M in ACN, 693 μL, 0.312 mmol, 6.0 equiv.). The reaction mixture was stirred at room temperature for 30 min. Then the solution was cooled to −78° C. under argon before adding 3-chloroperoxybenzoic acid (mCPBA, ˜77%, 81.6 mg, 0.364 mmol, 7.0 equiv.). The solution was stirred at room temperature for 2 hr. The mixture was diluted with DCM and washed with sat. aq. NaHCO3, dried with Na2SO4, and concentrated in vacuo. Flash column chromatography on silica using a gradient of 0-90% EtOAc in hexanes afforded 19 (32.2 mg, 70%) as a roughly 1:1 mixture with excess dibenzyl diisopropylphosphoramidite. 1H NMR (500 MHz, chloroform-d): δ (ppm) 7.77 (dd, J=1.3, 8.3 Hz, 2H), 7.49 (d, J=7.7 Hz, 1H), 7.47-7.40 (m, 2H), 7.27 (m, 2H), 7.38-7.28 (m, 9H, with impurity), 7.21-7.14 (m, 3H, with impurity), 7.23 (d, J=3.4 Hz, 1H), 7.14 (m, 1H), 7.03 (ddd, J=0.8, 7.4, 8.3 Hz, 1H), 6.92 (m, 2H), 6.44 (d, J=3.4 Hz, 1H), 5.80 (t, J=9.1 Hz, 1H), 5.66 (d, J=9.1 Hz, 1H), 4.92 (d, J=9.1 Hz, 1H), 4.86 (dd, J=6.8, 11.7 Hz, 1H), 4.78-4.69 (m, 2H), 4.51 (dd, J=9.7, 11.8 Hz, 1H), 4.32 (t, J=9.3 Hz, 1H), 4.19 (dd, J=1.9, 12.7 Hz, 1H), 4.03 (dd, J=1.2, 12.7 Hz, 1H), 1.18 (d, J=7.1 Hz, 3H), 1.17 (d, J=7.8 Hz, 3H), 1.10-0.99 (m, 19H), 0.94 (d, J=6.9 Hz, 3H).
To a solution of 19 (32.2 mg, 0.0364 mmol, 1.0 equiv.) in 1 mL THF was added acetic acid (6 μL, 0.109 mmol, 3.0 equiv.), and the mixture was cooled to −10° C. Tetrabutylammonium fluoride (1M in THF, 109 μL, 0.109 mmol, 3.0 eq) was added, and the solution was stirred for 10 min. Subsequently, acetic acid (15 μL, 0.262 mmol, 7.2 equiv.) was added, and the reaction mixture was concentrated in vacuo. Flash column chromatography on silica using a gradient of 0-40% MeOH in DCM afforded 22 (23.1 mg, 99%). Product contained 45% of impurity dibenzyl diisopropylphosphoramidite. 1H NMR (500 MHz, methanol-d4): δ (ppm) 7.70 (dd, J=1.2, 8.3 Hz, 2H), 7.60 (d, J=8.4 Hz, 1H), 7.43 (m, 1H), 7.41 (br, 1H), 7.24 (m, 2H), 7.10 (m, 1H), 6.97 (m, 1H), 6.94 (m, 2H), 6.40 (d, J=3.3 Hz, 1H), 6.03 (d, J=9.2 Hz, 1H), 6.44 (t, J=9.2 Hz, 1H), 5.07-4.92 (m, 5H), 4.00-3.95 (m, 2H), 3.88-3.90 (m, 2H).
To a mixture of 1:1 MeOH/EtOAc (v/v, 2 mL) and 22 (23.1 mg, 0.0359 mmol, 1.0 equiv.) was added Pd/C (10% w/w) (20 mg). The reaction mixture was purged with argon for 2 min, then H2 gas was bubbled through for 45 min at room temperature, and the reaction vessel was again purged with argon for 2 min. The reaction mixture was filtered through Celite and concentrated in vacuo. The crude mixture was purified by reversed-phase flash chromatography with a C18 column using a gradient of 0-60% ACN in H2O (with 0.1% formic acid), which afforded iglu #121 (25, 2.4 mg, 14%) as clear oil. See Table S1 for NMR spectroscopic data of iglu #121 (25).
To a stirred solution of pyrrole-2-carboxylic acid (13.4 mg, 0.121 mmol, 1.0 equiv.) in DCM, EDC·HCl (46.0 mg, 0.240 mmol, 2.0 equiv.) was added. The mixture was stirred at room temperature for 30 min, and S1 (75.2 mg, 0.144 mmol, 1.2 equiv.) and DMAP (36.7 mg, 0.30 mmol, 2.5 equiv.) were added. The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated in vacuo followed by flash column chromatography on silica using a gradient of 0-80% EtOAc in hexanes, affording 17 (38.7 mg, 44%). 1H NMR (500 MHz, chloroform-d): δ (ppm) 8.92 (s, 1H), 7.52 (d, J=7.8 Hz, 1H), 7.47 (d, J=8.3 Hz, 1H), 7.26 (d, J=3.4 Hz, 1H), 7.19 (ddd, J=1.0, 7.2, 9.3 Hz, 1H), 7.08 (ddd, J=0.8, 7.0, 8.6 Hz, 1H), 6.76 (m, 1H), 6.72 (m, 1H), 6.45 (d, J=3.4 Hz, 1H), 6.11 (m, 1H), 5.65 (d, J=9.2 Hz, 1H), 5.49 (t, J=9.2 Hz, 1H), 4.15 (dd, J=2.0, 12.7 Hz, 1H), 4.09 (dd, J=3.4, 8.7 Hz, 1H), 4.03-3.98 (m, 2H), 3.52 (dt, J=1.6, 9.2 Hz, 1H), 1.17-1.01 (m, 28H).
To a solution of 17 (38.7 mg, 0.063 mmol, 1.0 equiv.) in 1 mL DCM was added dibenzyl N,N-diisopropylphosphoramidite (64 μL, 0.189 mmol, 3.0 equiv.) and 1H-tetrazole (0.45 M in ACN, 420 μL, 0.189 mmol, 3.0 equiv.). The reaction mixture was stirred at room temperature for 30 min. Then the solution was cooled to −78° C. under argon before added 3-chloroperoxybenzoic acid (mCPBA, ˜77%, 44.0 mg, 0.196 mmol, 3.1 equiv.). The solution was stirred to up room temperature over a 2-hr period. The mixture was diluted with DCM and washed with sat. aq. NaHCO3, dried with Na2SO4, and concentrated in vacuo. Flash column chromatography on silica using a gradient of 0-80% EtOAc in hexanes afforded 20 (47.8 mg, 87%). 1H NMR (500 MHz, chloroform-d): δ (ppm) 9.68 (s, 1H), 7.53 (dt, J=1.0, 7.7 Hz, 1H), 7.45 (d, J=8.3 Hz, 1H), 7.37-7.32 (m, 3H), 7.27 (d, J=3.4 Hz, 1H), 7.24-7.20 (m, 4H), 7.13 (ddd, J=1.1, 6.9, 7.9 Hz, 1H), 7.02 (ddd, J=0.9, 6.9, 7.9 Hz, 1H), 6.92 (dd, J=1.4, 7.7 Hz, 1H), 6.78 (m, 1H), 6.48 (d, J=3.4 Hz, 1H), 6.11 (m, 1H), 5.63 (t, J=9.0 Hz, 1H), 5.58 (d, J=8.9 Hz, 1H), 5.02 (m, 1H), 4.97 (dd, J=4.8, 12.0 Hz, 1H), 4.95-4.88 (m, 2H), 4.68 (dd, J=7.2, 11.7 Hz, 1H), 4.53 (dd, J=8.4, 11.7 Hz, 1H), 4.29 (t, J=9.4 Hz, 1H), 4.17 (dd, J=1.9, 12.7 Hz, 1H), 4.01 (dd, J=1.2, 12.7 Hz, 1H), 3.50 (m, 1H), 1.24 (d, J=6.7 Hz, 3H), 1.18 (d, J=6.9 Hz, 3H), 1.11-0.93 (m, 22H).
To a solution of 20 (47.8 mg, 0.0547 mmol, 1.0 equiv.) in 1 mL THF was added acetic acid (9.4 μL, 0.164 mmol, 3.0 equiv.) and cooled to −10° C. The solution was added tetrabutylammonium fluoride (1M in THF, 164 μL, 0.164 mmol, 3.0 equiv.) and stirred for 1.5 hr. The reaction mixture was added acetic acid (10 μL, 0.175 mmol, 3.2 equiv.) and concentrated in vacuo. Flash column chromatography on silica using a gradient of 0-40% MeOH in DCM afforded 23 (28.4 mg, 82%). 1H NMR (500 MHz, methanol-d4): δ (ppm) 7.59 (d, J=8.4 Hz, 1H), 7.44 (d, J=7.8 Hz, 1H), 7.40 (d, J=3.4 Hz, 1H), 7.31-7.19 (m, 7H), 7.12 (ddd, J=0.9, 7.1, 8.1 Hz, 1H), 6.98 (m, 2H), 6.85 (m, 1H), 6.73 (dd, J=1.5, 3.9 Hz, 1H), 6.41 (d, J=3.4 Hz, 1H), 6.06 (dd, J=2.5, 3.7 Hz, 1H), 5.94 (d, J=9.2 Hz, 1H), 5.77 (t, J=9.3 Hz, 1H), 5.06 (dd, J=7.3, 11.8 Hz, 1H), 4.97 (dd, J=8.3, 11.8 Hz, 1H), 4.89 (q, J=8.9 Hz, 1H), 4.76 (dd, J=7.3, 11.8 Hz, 1H), 4.63 (dd, J=8.3, 11.8 Hz, 1H), 3.98-3.91 (m, 2H), 3.82 (dd, J=5.4, 12.0 Hz, 1H), 3.78 (ddd, J=1.8, 5.3, 9.7 Hz, 1H).
To a 1:1 mixture of MeOH/EtOAc (v/v, 2 mL) 23 (28.4 mg, 0.0449 mmol, 1.0 equiv.) and Pd/C (10% w/w) (23 mg) were added. The reaction mixture was purged with argon for 2 min, subjected to H2 for 1 hr, at room temperature, and again purged with argon for 2 min. The reaction mixture was filtered through Celite and concentrated in vacuo. The residue was purified by reversed-phase flash chromatography with a C18 column using a gradient of 0-60% ACN in H2O (with 0.1% formic acid), which afforded iglu #101 (26, 9.2 mg, 45%) as a clear oil. See Table S3 for NMR spectroscopic data of iglu #101 (26).
To a stirred solution of Boc-2-Abz-OH (24.0 mg, 0.101 mmol, 1.0 equiv.) in DCM, EDC·HCl (38.7 mg, 0.202 mmol, 2.0 equiv.) was added. The mixture was stirred at room temperature for 30 min, and S1 (63.0 mg, 0.121 mmol, 1.2 equiv.) and DMAP (30.8 mg, 0.252 mmol, 2.5 equiv.) were added. The reaction mixture was stirred at room temperature for 4 hr. The reaction mixture was concentrated in vacuo followed by flash column chromatography on silica using a gradient of 5-90% EtOAc in hexanes, which afforded 18 (21.6 mg, 29%). 1H NMR (500 MHz, chloroform-d): δ (ppm) 9.76 (s, 1H), 8.33 (dd, J=0.8, 8.6 Hz, 1H), 7.69 (dd, J=1.6, 8.2 Hz, 1H), 7.47 (d, J=8.3 Hz, 1H), 7.41 (ddd, J=1.3, 1.6, 7.9 Hz, 1H), 7.25 (d, 1H), 7.18 (ddd, J=1.0, 1.0, 7.7 Hz, 1H), 7.07 (ddd, J=0.8, 0.8, 7.5 Hz, 1H), 6.84 (ddd, J=1.0, 1.0, 7.7 Hz, 1H), 6.47 (d, J=3.4 Hz, 1H), 5.71-5.64 (m, 2H), 4.16 (dd, J=1.9, 12.6 Hz, 1H), 4.14-4.09 (m, 2H), 4.03 (dd, J=1.1, 12.6 Hz, 1H), 3.55 (m, 1H), 1.49 (s, 9H), 1.18-1.02 (m, 28H).
To a solution of 18 (21.6 mg, 0.0292 mmol, 1.0 equiv.) in 0.8 mL DCM was added dibenzyl N,N-diisopropylphosphoramidite (29 μL, 0.0875 mmol, 3.0 equiv.) and 1H-tetrazole (0.45 M in ACN, 194 μL, 0.0875 mmol, 3.0 equiv.). The reaction mixture was stirred at room temperature for 45 min. Then the solution was cooled to −78° C. under argon before adding 3-chloroperoxybenzoic acid (mCPBA, ˜77%, 20 mg, 0.364 mmol, 3.0 equiv.). The solution was stirred at room temperature for 2-hr. The mixture was diluted with DCM and washed with sat. aq. NaHCO3, dried with Na2SO4, and concentrated in vacuo. Flash column chromatography on silica using a gradient of 5-90% EtOAc in hexanes afforded 21 (25.5 mg, 87%). 1H NMR (500 MHz, chloroform-d): δ (ppm) 9.48 (s, 1H), 8.25 (d, J=8.4 Hz, 1H), 7.85 (dd, J=1.1, 8.0 Hz, 1H), 7.50 (d, J=8.0 Hz, 1H), 7.48 (d, J=8.5 Hz, 1H), 7.39 (m, 1H), 7.36-7.33 (m, 2H), 7.25-7.09 (m, 8H), 7.05 (ddd, J=0.7, 7.6, 7.8 Hz, 1H), 6.95 (m, 2H), 6.88 (ddd, J=0.7, 7.6, 8.4 Hz, 1H), 6.43 (d, J=3.3 Hz, 1H), 5.75 (d, J=9.1 Hz, 1H), 5.61 (d, J=9.0 Hz, 1H), 4.90-4.82 (m, 2H), 4.77 (dd, J=7.2, 11.6 Hz, 1H), 4.69 (dd, J=8.3, 11.6 Hz, 1H), 4.46 (t, J=11.2 Hz, 1H), 4.31 (t, J=9.2 Hz, 1H), 4.20 (dd, J=1.3, 12.7 Hz, 1H), 4.05 (dd, J=1.0, 12.7 Hz, 1H), 3.53 (d, J=9.3 Hz, 1H), 1.41 (s, 9H), 1.32-1.26 (m, 7H), 1.19 (d, J=6.8 Hz, 3H), 1.17 (d, J=6.8 Hz, 3H), 1.04-1.00 (m, 6H), 0.95 (d, J=6.8 Hz, 3H), 0.89 (d, J=6.8 Hz, 3H), 0.88 (d, J=7.1 Hz, 3H). 13C NMR (125 MHz, chloroform-d): 171.3, 167.3, 152.7, 142.6, 136.4, 135.5, 135.4, 135.0, 134.97, 134.92, 131.4, 129.3, 128.3, 128.2, 128.1, 127.41, 127.38, 125.2, 122.6, 121.4, 121.2, 120.8, 118.5, 113.9, 109.8, 104.4, 80.4, 79.9, 69.38, 69.34, 68.13, 68.09, 68.06, 59.2, 31.7, 28.4, 22.8, 21.2, 18.3, 17.43, 17.42, 17.39, 17.2, 17.0, 14.3, 14.2, 13.5, 13.3, 12.9, 12.6.
To a solution of 21 (25.5 mg, 0.0255 mmol, 1.0 equiv.) in 1 mL THF was added acetic acid (4.4 μL, 0.0765 mmol, 3.0 equiv.), and the mixture was cooled to −10° C. To the solution was added tetrabutylammonium fluoride (1M in THF, 77 μL, 0.0765 mmol, 3.0 eq) and the resulting mixture stirred for 1.4 hr. Subsequently, acetic acid (10 μL, 0.175 mmol, 6.8 equiv.) was added and the mixture concentrated in vacuo. Flash column chromatography on silica using a gradient of 5-20% MeOH in DCM afforded 24 (17.1 mg, 88%), containing about 20% of dibenzyl diiylphosphoramidite as an impurity. 1H NMR (600 MHz, chloroform-d): δ (ppm) 9.64 (s, 1H), 8.33 (dd, J=1.1, 8.6 Hz, 1H), 7.71 (dd, J=1.6, 8.1 Hz, 1H), 7.54 (dt, J=0.9, 7.9 Hz, 1H), 7.43-7.39 (m, 2H), 7.36-7.34 (m, 2H), 7.30 (d, J=2.2 Hz, 1H), 7.30-7.27 (m, 2H), 7.24 (m, 1H), 7.21 (m, 1H), 7.14 (m, 2H), 7.10 (ddd, J=0.9, 7.5, 8.0 Hz, 1H), 7.03 (m, 2H), 6.80 (dt, J=1.1, 7.6 Hz, 1H), 6.51 (d, J=3.4 Hz, 1H), 5.80 (t, J=9.3 Hz, 1H), 5.68 (d, J=9.3 Hz, 1H), 4.96 (dd, J=8.2, 11.7 Hz, 1H), 4.83 (dd, J=7.9, 11.7 Hz, 1H), 4.80 (d, J=8.7 Hz, 1H), 4.68 (dt, J=7.2, 9.0 Hz, 1H), 4.02-3.94 (m, 2H), 3.88 (dd, J=5.1, 12.1 Hz, 1H), 3.72 (ddd, J=3.3, 5.1, 9.6 Hz, 1H), 1.44 (s, 9H). 13C NMR (125 MHz, chloroform-d): 175.4, 170.0, 152.5, 142.4, 136.3, 135.24, 135.19, 135.0, 134.93, 134.87, 130.7, 129.2, 128.69, 128.68, 128.62, 128.58, 127.8, 124.7, 122.5, 121.3, 121.1, 120.8, 118.6, 112.9, 109.8, 104.4, 83.1, 82.3, 82.2, 80.7, 78.6, 70.85, 70.81, 70.16, 70.11, 70.08, 70.04, 69.79, 69.78, 61.9, 50.6, 28.3.
To a solution of 24 (17.1 mg, 0.0226 mmol, 1.0 equiv.) in 1.5 mL DCM was added TFA (0.1 mL, 1.31 mmol, 58 equiv.). The reaction mixture was stirred at room temperature for 20 min and then concentrated in vacuo. Flash column chromatography on silica using a gradient of 0-20% MeOH in DCM afforded 27 (14.7 mg, 99%), containing 27% of dibenzyl diisopropylphosphoramidite as impurity. 1H NMR (600 MHz, DMSO-d4): δ (ppm) 7.72 (d, J=8.4 Hz, 1H), 7.61 (dd, J=1.4, 8.2 Hz, 1H), 7.48 (d, J=3.4 Hz, 1H), 7.45 (d, J=7.8 Hz, 1H), 7.39-7.19 (m, 14H, with impurity), 7.13 (dt, J=1.7, 7.1 Hz, 1H), 7.00 (t, J=7.6 Hz, 1H), 6.97 (m, 2H), 6.61 (d, J=8.4 Hz, 1H), 6.43 (d, J=3.3 Hz, 1H), 6.37 (dt, J=1.0, 7.5 Hz, 1H), 6.20 (d, J=9.2 Hz, 1H), 5.90 (br, 1H), 5.76 (d, J=9.2 Hz, 1H), 4.94 (m, 2H), 4.70 (dd, J=7.1, 12.0 Hz, 1H), 4.56 (dd, J=8.0, 12.0 Hz, 1H), 3.85-3.73 (m, 3H), 3.60 (dd, J=5.6, 12.3 Hz, 1H).
To a 1:1 mixture of MeOH/EtOAc (v/v, 2 mL) 27 (14.7 mg, 0.0223 mmol, 1.0 equiv.) and Pd/C (10% w/w) (14 mg) were added. The reaction mixture was purged with argon for 2 min, subjected to H2 for 1 hr at room temperature, and again purged with argon for 2 min. The reaction mixture was filtered through Celite and concentrated in vacuo. The residue was purified by reversed-phase flash chromatography with a C18 column using a gradient of 0-60% ACN in H2O (with 0.1% formic acid), which afforded iglu #401 (28, 1.6 mg, 15%) as a clear oil. See Table S2 for NMR spectroscopic data of iglu #401 (28).
All oxygen and moisture-sensitive reactions were carried out under argon atmosphere in flame-dried glassware. Solutions and solvents sensitive to moisture and oxygen were transferred via standard syringe and cannula techniques. Trimethylsilyl trifluoromethanesulfonate (TMSOTf) was transferred to a Schlenk flask prior to use and stored at −20° C. Methanolic ammonia (7N) was purchased from Acros Organics. All commercial reagents were purchased as reagent grade and, unless otherwise stated, were purchased from Sigma-Aldrich and used without any further purification. Acetic acid (AcOH), acetonitrile (ACN), dichloromethane (DCM), ethylacetate (EtOAc), N,N-dimethylformamide (DMF), tetrahydrofuran (THF), dimethylsulfoxide (DMSO), formic acid, hexanes, and methanol (MeOH) used for chromatography and as a reagent or solvent were purchased from ThermoFisher Scientific. Acetyl chloride (1-13C, 99%) was purchased from Cambridge Isotope Laboratories, N-acetylserotonin (NAS) was obtained from Biosynth International, Boc-2-aminobenzoic acid (Boc-2-Abz-OH) was from Chem-Impex International, and trifluoroacetic acid (TFA) was from Tokyo Chemical Industry, fluoxetine hydrochloride was from Spectrum Chemical. Dichloromethane (DCM), and N,N-dimethylformamide (DMF) were dried with 3 Å molecular sieves prior to use. Thin-layer chromatography (TLC) was performed using J. T. Baker Silica Gel IB2F plates. Flash chromatography was performed using Teledyne IscoCombiFlash systems and Teledyne Isco RediSep Rf silica and C18 reverse phase columns. All deuterated solvents were purchased from Cambridge Isotopes. Abbreviations used: triethylamine (TEA), 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), trichloroacetonitrile (CCl3CN), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), trifluoromethanesulfonate (TMSOTf), N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl), 4-dimethylaminopyridine (DMAP), 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane(TIPDSiCl2), 3-chloroperoxybenzoic acid (m-CPBA). Nuclear Magnetic Resonance (NMR) spectra were recorded on Bruker INOVA 500 (500 MHz) and Varian INOVA 600 (600 MHz) spectrometers at Cornell University's NMR facility and Bruker AVANCE III HD 800 MHz (800 MHz) or Bruker AVANCE III HD 600 MHz (600 MHz) at SUNY ESF's NMR facility. 1H NMR chemical shifts arereported in ppm (δ) relative to residual solvent peaks (7.26 ppm for chloroform-d, 3.31 ppm for methanol-d4, 2.05 ppm for acetone-d6). NMR-spectroscopic data are reported as follows: chemical shift, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet, br=broad), coupling constants (Hz), and integration and often tabulated including 2D NMR data. 13CNMR chemical shifts are reported in ppm (δ) relative to residual solvent peaks (77.16 ppm for chloroform-d, 49.00 ppm for methanol-d4, 29.9 ppm for acetone-d6). All NMR data processing was done using MestreLab MNOVA version 14.2.1-27684 (mestrelab.com).
Several methods for chromatographic separation were utilized due to varying polarity of metabolites of interest. High resolution LC-MS analysis was performed on a Thermo Fisher Scientific Vanquish Horizon UHPLC System coupled with a Thermo Q Exactive HF hybrid quadropole-orbitrap high resolution mass spectrometer quipped with a HESI ion source. 1 μL of synthetic and natural endo- and exo-metabolome extracts (C. elegans N2, C. briggsae AF-16, C. elegans him-5, and C. elegans fem-3 (gf)) were injected and separated according to the methods provided below:
Method A—water-acetonitrile gradient on a Hypersil GOLD C18 column (150 mm×2.1 mm 1.9 um particle size 175 Å pore size, Thermo Scientific) and maintained at 40° C. Solvent A: 0.1% formic acid in water; solvent B: 0.1% formic acid in acetonitrile. A/B gradient started at 1% B for 3 min, then from 1% to 99% B over 17 min, 99% B for 5 min, then rapidly down to 1% B over 0.5 min and held for 2.5 min to equilibrate the column.
Method B—water-acetonitrile gradient on a Hypersil GOLD C18 column (150 mm×2.1 mm 1.9 um particle size 175 Å pore size, Thermo Scientific) and maintained at 40° C. Solvent A: 0.1% formic acid in water; solvent B: 0.1% formic acid in acetonitrile. A/B gradient started at 1% B for 3 min, then from 1% to 35% B over 37 min, then from 35% to 100% B over 15 min, held at 100% B for 2 min, then rapidly down to 1% B over 0.5 min, and held for 2.5 min to equilibrate the column.
Method C—water-acetonitrile gradient on a Zorbax HILIC Plus column (150 mm×2.1 mm 1.8 um particle size 95 Å pore size, Agilent) and maintained at 40° C. Solvent A: 0.1% formic acid in water; solvent B: 0.1% formic acid in acetonitrile. A/B gradient started at 95% B for 4 min, then from 95% to 55% B over 15 min, then rapidly down to 5% B and held for 3 min, then back to 95% B and equilibrated for 3 min.
Method D—water-acetonitrile gradient on a XBridge Amide column (150 mm×2.1 mm 3.5 um particle size 130 Å pore size, Waters) and maintained at 40° C. Solvent A: 90% acetonitrile and 10% water prepared with 0.4% (v/v) of 25% ammonia in water solution combined with 0.1% (v/v) formic acid, solvent B: 30% acetonitrile and 70% water prepared with 0.4% (v/v) of 25% ammonia in water solution combined with 0.1% (v/v) formic acid. A/B gradient started at 1% B for 3 min, then from 1% to 60% B over 17 min, then from 60% to 100% B over 6 min and held for 1.5 min, then back to 1% B over 0.5 min and equilibrated for 2 min.
Method E—water-acetonitrile gradient on a XBridge Amide column (150 mm×2.1 mm 3.5 um particle size 130 Å pore size, Waters) and maintained at 40° C. Solvent A: 90% acetonitrile and 10% water prepared with 0.4% (v/v) of 25% ammonia in water solution combined with 0.1% (v/v) formic acid, solvent B: 30% acetonitrile and 70% water prepared with 0.4% (v/v) of 25% ammonia in water solution combined with 0.1% (v/v) formic acid. A/B gradient started at 1% B for 3 min, then from 1% to 35% B over 37 min, then from 35% to 100% B over 15 min and held for 2 min, then back to 1% B over 0.5 min and equilibrated for 2.5 min.
Mass spectrometer parameters: 3.5 kV spray voltage, 380° C. capillary temperature, 300° C. probe heater temperature, 60 sheath flow rate, 20 auxiliary flow 15 rate, 1 spare gas; S-lens RF level 50.0, resolution 240,000, m/z range 100-1200 m/z, AGC target 3e6. Instrument was calibrated with positive and negative ion calibration solutions (Thermo-Fisher) Pierce LTQ Velos ESI pos/neg calibration solutions. Peak areas were determined using Xcalibur 2.3 QualBrowser version 2.3.26 (Thermo Scientific) using a 5-10 ppm window around the m/z of interest.
To a solution of serotonin hydrochloride (128.1 mg, 0.602 mmol, 1.0 equiv.) in DMF (6 mL) was added succinic anhydride (78.3 mg, 0.783 mmol, 1.3 equiv.) and pyridine (0.6 mL). The mixture was stirred at room temperature for 24 hours and concentrated in vacuo. Flash column chromatography on silica using a gradient of 0-50% MeOH in DCM afforded 14 (165.4 mg, 99%) as clear oil. 1H NMR (500 MHz, methanol-d4): δ (ppm) 7.16 (d, J=8.6 Hz, 1H), 6.98 (s, 1H), 6.96 (d, J=2.3 Hz, 1H), 6.69 (dd, J=2.3, 8.6 Hz, 1H), 3.42 (t, J=7.2 Hz, 2H), 2.83 (t, J=7.2 Hz, 2H), 2.57 (t, J=7.0 Hz, 2H), 2.43 (t, J=7.0 Hz, 2H). 13C NMR (125 MHz, methanol-d4): δ (ppm) 176.3, 174.3, 151.0, 132.9, 129.3, 124.3, 112.7, 112.4, 112.3, 103.5, 41.3, 31.5. 30.2, 26.1. HRMS (ESI) m/z calcd for C14H16N2O4 [M−H]− 275.1037, found 275.1043.
To a suspension of serotonin hydrochloride (132 mg, 0.621 mmol, 1.0 equiv.) in DCM (5 mL) was added TEA (433 μL, 3.10 mmol, 5.0 equiv.). The stirred mixture was cooled to 0° C. before 1-13C-acetyl chloride (93 μL, 1.30 mmol, 2.1 equiv.) was added. The mixture was slowly warmed to room temperature and stirred for 24 hours. The reaction mixture was then diluted with DCM, the organics were washed with water, dried with Na2SO4, and concentrated in vacuo. Crude intermediates were dissolved in MeOH (10 mL), and K2CO3 (85.8 mg, 0.621 mmol, 1.0 equiv.) was added. The reaction was stirred at room temperature for 2 hours and concentrated to 2 mL in vacuo. The residue was diluted with water and extracted with EtOAc twice. The organics were separated, washed with brine, and dried with Na2SO4. Flash column chromatography on silica using a gradient of 0-50% MeOH in DCM afforded 28 (98.0 mg, 72%) as light-yellow oil. 1H NMR (600 MHz, methanol-d4): δ (ppm) 7.15 (dd, J=0.6, 8.6 Hz, 1H), 6.99 (s, 1H), 6.93 (dd, J=0.6, 2.4 Hz, 1H), 6.66 (dd, J=2.4, 8.6 Hz, 1H), 3.42 (ddd, J=3.7, 7.3, 8.2 Hz, 2H), 2.85 (dt, J=0.6, 7.3 Hz, 2H), 1.91 (d, J=6.1 Hz, 3H). 13C NMR (125 MHz, methanol-d4): δ (ppm) 175.9 (12C), 173.4 (13C), 151.1, 133.1, 129.5, 124.2, 112.6, 112.4, 103.5, 41.4, 26.2, 22.6 (d, J=50.3 Hz). HRMS (ESI) m/z calcd for C1113CH14N2O2 [M+H]+ 220.1161, found 220.1160.
To a solution of 2,3,4,6-tetra-O-benzyl-D-glucopyranose (412 mg, 0.761 mmol, 1.0 equiv.) in DCM (2 mL) was added trichloroacetonitrile (152 μL, 1.52 mmol, 2.0 equiv.) and DBU (21 μL, 0.152 mmol, 0.2 equiv.) under argon. The mixture was stirred at room temperature for 1.5 hours and concentrated in vacuo. Flash column chromatography on silica using a gradient of 25% ethyl acetate in hexanes afforded intermediate 44 (502.4 mg, 97%) as clear oil. A well-stirred solution of 44 (502.4 mg, 0.745 mmol, 2.0 equiv.) and N-acetylserotonin (806 mg, 0.368 mmol, 1.0 equiv.) in DCM (4 mL) and DMF (0.8 mL) was cooled to 0° C., followed by addition of TMSOTf (66 μL, 0.368 mmol, 1.0 equiv.), and the solution was allowed to warm to room temperature within 30 minutes. After stirring at 45° C. for 18 hours, the mixture was concentrated in vacuo. Flash column chromatography on silica using a gradient of 0-15% MeOH in DCM afforded 45 (59.7 mg, 22%) as clear oil. 1H NMR (500 MHz, chloroform-d): δ (ppm) 7.41-7.26 (m, 20H), 7.17-7.14 (m, 2H), 7.04-7.01 (m, 2H), 5.50 (d, J=3.4 Hz, 1H), 5.44 (m, 1H), 5.08 (d, J=10.8 Hz, 1H), 4.90 (d, J=11.0 Hz, 1H), 4.88 (d, J=10.9 Hz, 1H), 4.81 (d, J=12.0 Hz, 1H), 4.72 (d, J=12.0 Hz, 1H), 4.57 (d, J=11.9 Hz, 1H), 4.50 (d, J=10.8 Hz, 1H), 4.41 (d, J=12.0 Hz, 1H), 4.25 (t, J=9.2 Hz, 1H), 4.03 (m, 1H), 3.78-3.71 (m, 3H), 3.62 (dd, J=1.9, 10.8 Hz, 1H), 3.53 (dt, J=6.2, 6.6 Hz, 2H), 2.87 (t, J=6.6 Hz, 2H), 1.90 (s, 3H). 13C NMR (125 MHz, chloroform-d): δ (ppm) 170.1, 151.2, 139.0, 138.4, 138.2, 138.0, 132.7, 128.62, 128.58, 128.54, 128.48, 128.19, 128.13, 128.05, 128.02, 127.87, 127.82, 127.78, 123.1, 114.3, 113.2, 111.9, 105.8, 96.8, 82.2, 80.0, 77.8, 76.0, 75.3, 75.5, 10.8, 68.7, 39.6, 25.4, 23.5.
To a solution of 45 (59.2 mg, 0.080 mmol, 1.0 equiv.) in a mixture of MeOH and EtOAc (3 mL, v/v=1:1) was added Pd/C (10% w/w, 38 mg). The stirred reaction mixture was purged with argon for 5 minutes, flushed with hydrogen and then subjected to a hydrogen atmosphere for 2 hours at room temperature, and again purged with argon for 5 minutes. The mixture was filtered through Celite and concentrated in vacuo, affording 36 as clear oil (29.8 mg, 98%). HRMS (ESI) m/z calcd for C18H24N2O7 [M+Na]+ 403.1476, found 403.1486.
To a solution of N-acetylserotonin (210.2 mg, 0.963 mmol, 1.0 equiv.) in TFA (4 mL) was added triethylsilane (185 μL, 1.15 mmol, 1.2 equiv.). The mixture was stirred at 45° C. for 4 hours and concentrated in vacuo. Flash column chromatography on silica using a gradient of 0-40% MeOH in DCM afforded 46 (209.0 mg, 99%). 1H NMR (500 MHz, methanol-d4): δ (ppm) 7.17 (d, J=8.6 Hz, 1H), 6.79 (d, J=2.2 Hz, 1H), 6.74 (dd, J=2.2, 8.6 Hz, 1H), 3.95-3.88 (m, 1H), 3.62-3.55 (m, 1H), 3.49-3.42 (m, 2H), 3.29-3.20 (m, 2H), 2.02-1.94 (m, 1H), 1.88 (s, 3H), 1.72-1.63 (m, 1H). 13C NMR (125 MHz, methanol-d4): δ (ppm) 173.5 (br), 160.2, 141.3, 128.5, 120.1, 116.6, 112.7, 52.5, 40.6, 38.0, 34.5, 22.5. HRMS (ESI) m/z calcd for C12H16N2O [M+H]+ 221.1284, found 221.1272.
To a solution of 46 (209 mg, 0.953 mmol, 1.0 equiv.) in TFA (1.5 mL) was added α-D-glucose (867 mg, 4.82 mmol, 5.0 equiv.). The mixture was refluxed for 2 hours and concentrated in vacuo. The crude intermediate was redissolved in pyridine (15 mL) and acetic anhydride (8 mL, 86.7 mmol, 90 equiv.) was added. The resulting mixture was stirred at room temperature for 1 hour and then diluted with water and extracted with DCM:MeOH (v/v=95:5) for three times. The combined organics were washed with sat. aq. NaHCO3 and brine and dried with Na2SO4. Flash column chromatography on silica using a gradient of 0-30% isopropanol in toluene afforded 47 (mixture of diastereomers, 19.5 mg, 3.8%) as yellow oil. 1H NMR (600 MHz, chloroform-d): δ (ppm) 6.86-6.78 (m, 2H), 6.52 (d, J=8.4 Hz, 0.5H), 6.50 (d, J=8.5 Hz, 0.5H), 5.67 (m, 0.5H), 5.57 (m, 0.5H), 5.33 (dt, J=6.7, 9.4 Hz, 1H), 5.23 (dt, J=8.2, 9.2 Hz, 1H), 5.07 (td, J=3.3, 9.7 Hz, 1H), 4.91 (d, J=10.0 Hz, 1H), 4.25 (ddd, J=5.0, 10.9, 12.4 Hz, 1H), 4.04 (ddd, J=2.4, 12.3, 17.5 Hz, 1H), 3.77-3.71 (m, 2H), 3.34-3.28 (m, 3H), 3.21 (m, 1H), 2.35 (s, 3H), 2.04 (d, J=1.7 Hz, 3H), 2.03 (d, J=1.7 Hz, 3H), 2.01-1.98 (6H), 1.94 (d, J=11.8 Hz, 3H), 1.76-1.62 (m, 2H). HRMS (ESI) m/z calcd for C28H36N2O12 [M+H]+ 593.2341, found 593.2299.
To a solution of 47 (19.5 mg, 0.0324 mmol, 1.0 equiv.) in 1,4-dioxane (1 mL) was added DDQ (8.8 mg, 0.039 mmol, 1.2 equiv.), and the mixture was stirred at room temperature. After 1.5 hours, the reaction mixture was cooled to 0° C. ice bath, diluted with sat. aq. NaHCO3, and extracted with EtOAc for three times. Combined organics were washed with brine, dried with Na2SO4, and then concentrated in vacuo. Flash column chromatography on silica using 100% DCM afforded 48 (15.2 mg, 79%). 1H NMR (600 MHz, chloroform-d): δ (ppm) 7.31 (d, J=8.9 Hz, 1H), 7.23 (d, J=2.1 Hz, 1H), 7.15 (s, 1H), 6.98 (dd, J=2.1, 8.9 Hz, 1H), 5.91 (m, 1H), 5.53 (d, J=9.0 Hz, 1H), 5.46 (t, J=9.5 Hz, 1H), 5.35 (t, J=9.4 Hz, 1H), 5.25 (t, J=9.8 Hz, 1H), 4.32 (dd, J=5.0, 12.6 Hz, 1H), 4.16 (dd, J=2.1, 12.6 Hz, 1H), 4.11 (q, J=7.2 Hz, 1H), 4.01 (ddd, J=2.2, 5.0, 10.2 Hz, 1H), 3.67 (m, 1H), 3.42 (m, 1H), 2.93 (m, 1H), 2.81 (m, 1H), 2.31 (s, 3H), 2.084 (s, 3H), 2.078 (s, 3H), 2.02 (s, 3H), 1.94 (s, 3H), 1.55 (s, 3H). 13C NMR (125 MHz, chloroform-d): δ (ppm) 170.7, 170.6, 170.4, 170.1, 169.6, 169.2, 144.9, 134.6, 128.8, 123.6, 117.1, 115.7, 111.8, 109.8, 83.0, 75.0, 72.8, 71.5, 68.3, 62.0, 51.0, 39.1, 23.3, 21.3, 20.9, 20.73, 20.70, 20.2. HRMS (ESI) m/z calcd for C28H34N2O12 [M+H]+ 591.2184, found 591.2151.
To a solution of 48 (15.2 mg, 0.0257 mmol, 1.0 equiv.) in MeOH (1.5 mL) was added 8% NaOH (0.3 mL). The mixture was stirred at room temperature for 25 min. and concentrated in vacuo. Flash column chromatography on silica using a gradient of 0-90% MeOH in DCM afforded 37 as clear oil (5.7 mg, 58%). HRMS (ESI) m/z calcd for C18H24N2O7 [M+Na]+ 403.1476, found 403.1471.
To a 20 mL glass vial containing 38 (1.52 g, 8.35 mmol, 3 equiv.), N-acetylserotonin (607 mg, 2.78 mmol, 1.0 equiv.) and Ca(OH)2 (618 mg, 8.35 mmol, 3 equiv.) was added water (3 mL). The reaction mixture was stirred vigorously for 35 minutes. The crude mixture was purified by reversed-phase flash chromatography with a C18 column using a gradient of 0-40% MeOH in H2O, which afforded sngl #1 (29, 779.0 mg, 74%) as a white solid. HRMS (ESI) m/z calcd for C18H24N2 NaO7+ [M+Na]+ 403.1476, found 403.1485.
To a mixture of DCM/DMF (3 mL, v/v=1:2) was added Boc-2-aminobenzoic acid (15.4 mg, 0.065 mmol, 1.2 equiv.) and EDC·HCl (31.2 mg, 0.163 mmol, 3.0 equiv.). The mixture was stirred at room temperature for 30 minutes, and DMAP (26.5 mg, 0.217 mmol, 4.0 equiv.) and sngl #1 (29, 20.6 mg, 0.0542 mmol, 1.0 equiv.) were added. After 5 days, the reaction mixture was concentrated in vacuo. Flash column chromatography on silica using a gradient of 0-30% MeOH in DCM afforded intermediate 49 (4.1 mg, 13%).
Intermediate 49 was redissolved in DCM (1 mL), followed by slow addition of TFA (0.1 mL). The reaction mixture was stirred at room temperature for 1.5 hours and concentrated in vacuo. Preparative HPLC provided a pure sample of sngl #3 (31, 0.3 mg, 1.1%). HRMS (ESI) m/z calcd for C25H29N3O8 [M+H]+ 500.2027, found 500.2005.
To a solution of sngl #1 (29, 194 mg, 0.511 mmol, 1.0 equiv.) in DMF was added imidazole (152 mg, 1.84 mmol, 4.4 equiv.) was cooled to 0° C. before TIPDSiCl2 (228 μL, 0.713 mmol, 1.4 equiv.) was added. The reaction mixture was allowed to warm to room temperature over 1.5 hours and stirred for another 30 minutes. The mixture was then diluted with DCM and quenched with water. The organics were washed with sat. aq. NaHCO3, dried with Na2SO4, and concentrated in vacuo. Flash column chromatography on silica using a gradient of 0-10% MeOH in DCM afforded 50 as a white solid (227.6 mg, 72%). 1H NMR (500 MHz, chloroform-d): δ (ppm) 8.16 (s, 1H), 7.25-7.20 (m, 2H), 7.04-6.95 (m, 2H), 4.89 (d, J=7.3 Hz, 1H), 4.13 (d, J=11.9 Hz, 1H), 4.01 (d, J=12.5 Hz, 1H), 3.93 (t, J=8.9 Hz, 1H), 3.75-3.64 (m. 2H), 3.52 (m, 2H), 3.36 (m, 1H), 3.88 (m, 2H), 1.94 (s, 3H), 1.10-0.99 (m, 28H). HRMS (ESI) m/z calcd for C30H50N2O8Si2, [M+H]+ 623.3178, found 623.3157.
To a solution of 50 (227 mg, 0.365 mmol, 1.0 equiv.) in DCM was added DMAP (147 mg, 1.20 mmol, 3.3 equiv.) and DMF (50 μL). The mixture was cooled to 0° C. before added benzyl chloroformate (233 μL, 1.64 mmol, 4.5 equiv.). The reaction mixture was allowed to warm to room temperature within 30 minutes and stirred for another 1.3 hours. The mixture was diluted with DCM and then quenched with water. The aqueous layer was separated and extracted with DCM for three times. The combined organics were washed with sat. aq. NaHCO3 and brine, dried with Na2SO4, and concentrated in vacuo. Flash column chromatography of the residue on silica using a gradient of 0-20% isopropanol in toluene afforded 51 as a white solid (196.1 mg, 66%). 1H NMR (600 MHz, chloroform-d): δ (ppm) 8.22 (s, 1H), 7.40-7.37 (m, 2H), 7.36-7.31 (m, 3H), 7.19-7.16 (m, 2H), 6.99 (s, 1H), 6.82 (dd, J=2.2, 8.7 Hz, 1H), 5.64 (m, 1H), 5.26 (d, J=12.1 Hz, 1H), 5.21 (d, J=12.1 Hz, 1H), 4.97 (d, J=8.0 Hz, 1H), 4.93 (dd, J=8.7, 9.3 Hz, 1H), 4.12 (dd, J=1.9, 12.7 Hz, 1H), 4.05 (dd, J=1.2, 12.7 Hz, 1H), 3.98 (t, J=1.2, 9.3 Hz, 1H), 3.81 (t, J=1.2, 9.1 Hz, 1H), 3.51-3.47 (m, 2H), 3.34 (dt, J=1.2, 9.4 Hz, 1H), 2.83 (t, J=6.6 Hz, 2H), 1.88 (s, 3H), 1.14-1.01 (m, 28H). 13C NMR (125 MHz, chloroform-d): δ (ppm) 170.5, 155.1, 151.7, 135.2, 133.1, 128.74, 128.68, 128.48, 128.35, 127.8, 126.4, 114.5, 113.0, 111.8, 106.6, 101.5, 77.9, 76.7, 75.2, 70.2, 69.7, 60.9, 39.8, 25.2, 23.3, 17.57, 17.47, 17.43, 17.37, 17.33, 17.31, 17.25, 13.7, 13.3, 12.7, 12.6. HRMS (ESI) m/z calcd for C38H56N2O10Si2 [M+H]+ 757.3546, found 757.3517.
To a solution of 51 (145.8 mg, 0.188 mmol, 1.0 equiv.) in DCM was added dibenzyl N,N-diisopropylphosphoramidite (221 μL, 0.659 mmol, 3.5 equiv.) and 1H-tetrazole (0.45 M in ACN, 1.5 mL, 0.659 mmol, 3.5 equiv.). The reaction mixture was stirred at room temperature for 1 hour. The solution was cooled to −78° C. under argon, and m-CPBA (≤77%, 143.0 mg, 0.638 mmol, 3.4 equiv.) in DCM (1.5 mL) was added slowly to the reaction mixture. The solution was stirred at −78° C. for 0.5 hour, and slowly warmed to room temperature and reacted for another 1 hour, then was diluted with DCM and washed with 10% Na2SO4 twice, sat. aq. NaHCO3, and brine, dried with Na2SO4, and concentrated in vacuo. Flash column chromatography on silica using a gradient of 5-100% EtOAc in hexanes afforded 52 as a white solid (141.3 mg, 74%). 1H NMR (500 MHz, chloroform-d): δ (ppm) 8.31 (s, 1H), 7.36-7.27 (m, 15H), 7.22-7.18 (m, 2H), 7.04 (d, J=2.2 Hz, 1H), 6.78 (dd, J=2.2, 8.7 Hz, 1H), 5.53 (m, 1H), 5.23 (d, J=12.2 Hz, 1H), 5.13 (dd, J=8.0, 9.4 Hz, 1H), 5.08-4.91 (m, 6H), 4.61 (dt, J=8.6, 8.9 Hz, 1H), 4.20-4.14 (m, 2H), 4.09 (d, J=12.6 Hz, 1H), 3.59-3.47 (m, 2H), 3.33 (dt, J=1.7, 9.4 Hz, 1H), 2.87 (t, J=6.6 Hz, 2H), 1.92 (s, 3H), 1.16-0.99 (m, 28H). 13C NMR (125 MHz, chloroform-d): 170.2, 154.6, 151.7, 136.14, 136.08, 135.90, 135.85, 135.3, 133.2, 128.60, 128.57, 128.53, 128.49, 128.36, 128.12, 128.06, 127.8, 123.3, 114.6, 113.1, 111.8, 106.8, 101.6, 80.3 (d, J=6.5 Hz), 76.6 (d, J=4.6 Hz), 70.0, 69.6 (t, J=5.9 Hz), 68.7 (d, J=5.2 Hz), 60.9, 39.8, 25.3, 23.5, 17.54, 17.50, 17.46, 17.41, 17.36, 17.34, 17.28, 17.11, 13.35, 13.26, 12.97, 12.95. HRMS (ESI) m/z calcd for C52H69N2O13PSi2 [M+H]+ 1017.4149, found 1017.4105.
To a solution of 52 (141.3 mg, 0.139 mmol, 1.0 equiv.) in THF (6 mL) was added acetic acid (24 μL, 0.417 mmol, 3.0 equiv.). The solution was cooled to −10° C. before tetrabutylammonium fluoride solution (1M in THF, 417 μL, 00.417 mmol, 3.0 equiv.) was added. The reaction mixture was stirred for 1.5 hours in cold and concentrated in vacuo. Flash column chromatography on silica using a gradient of 0-15% MeOH in DCM afforded 53 as a white solid (92.3 mg, 86%). 1H NMR (500 MHz, chloroform-d): δ (ppm) 8.32 (s, 1H), 7.36-7.21 (m, 15H), 7.14 (d, J=8.7 Hz, 1H), 6.96 (d, J=2.1 Hz, 1Hd), 6.71 (dd, J=2.1, 8.7 Hz, 1H), 5.82 (m, 1H), 5.12 (d, J=12.2 Hz, 1H), 5.10-4.94 (m, 7H), 4.49 (dt, J=7.2, 8.9 Hz, 1H), 3.99 (dd, J=2.8, 12.2 Hz, 1H), 3.84-3.74 (m, 2H), 3.55-3.46 (m, 2H), 3.40 (m, 1H), 2.91-2.77 (m, 2H), 1.89 (s, 3H). 13C NMR (125 MHz, chloroform-d): δ (ppm) 171.1, 154.5, 151.2, 135.0, 133.1, 128.81, 128.75, 128.72, 128.71, 128.69, 128.66, 123.4, 114.2, 113.1, 111.8, 106.9, 100.6, 81.7 (d, J=5.6 Hz), 76.17, 76.0 (d, J=6.2 Hz), 70.4, 70.25 (d, J=6.0 Hz), 70.17, 70.13 (d, J=6.0 Hz), 62.3, 40.3, 25.4, 23.4. HRMS (ESI) m/z calcd for C40H43N2O12P [M−H]− 773.2481, found 773.2488.
To a solution of 53 (26.9 mg, 0.0347 mmol, 1.0 equiv.) in a mixture of MeOH and EtOAc (2 mL, v/v=1:1) was added Pd/C (10% w/w, 18 mg). The reaction mixture was purged with argon for 5 minutes, flushed with hydrogen, and then subjected to hydrogen atmosphere for 1.5 hours at room temperature, and subsequently again purged with argon for 5 minutes. The mixture was filtered through Celite and concentrated in vacuo. The crude mixture was purified by reversed-phase flash chromatography with a C18 column using a gradient of 0-10% ACN in H2O with 0.1% formic acid, which afforded sngl #2 as a clear oil (30, 9.3 mg, 58%). HRMS (ESI) m/z calcd for C18H25N2O10P [M−H]− 459.1174, found 459.1185.
((2R,3R,4S,5R,6S)-6-((3-(2-acetamidoethyl)-1H-indol-5-yl)oxy)-5-(((benzyloxy)carbonyl)oxy)-4-((bis(benzyloxy)phosphoryl)oxy)-3-hydroxytetrahydro-2H-pyran-2-yl)methyl 2-((tert-butoxycarbonyl)amino)benzoate (54)
To a mixture of dry DCM/DMF (2 mL, v/v=100:1) was added Boc-2-aminobenzoic acid (70.7 mg, 0.298 mmol, 2.5 equiv.) and EDC·HCl (68.4 mg, 0.444 mmol, 3.0 equiv.). The mixture was stirred at room temperature for 25 minutes, and DMAP (58.2 mg, 0.476 mmol, 4.0 equiv.) and 53 (92.3 mg, 0.119 mmol, 1.0 equiv.) were added. After 25 hours, the reaction mixture was concentrated in vacuo. Flash column chromatography on silica using a gradient of 0-12% MeOH in DCM afforded 54 as a white solid (72.0 mg, 61%). 1H NMR (600 MHz, chloroform-d): δ (ppm) 8.42 (d, J=8.4 Hz, 1H), 7.97 (dd, J=1.1, 8.0 Hz, 1H), 7.49 (dd, J=1.1, 7.8 Hz, 1H), 7.33-7.15 (m, 16H), 7.09 (d, J=8.7 Hz, 1H), 7.00 (d, J=1.1 Hz, 1H), 6.91 (t, J=7.8 Hz, 1H), 6.82 (dd, J=2.0, 8.7 Hz, 1H), 5.61 (m, 1H), 5.15-4.95 (m, 8H), 4.71 (dd, J=2.0, 12.0 Hz, 1H), 4.56 (dd, J=6.1, 12.0 Hz, 1H), 4.49 (m, 1H), 3.84 (t, J=9.4 Hz, 1H), 3.77 (m, 1H), 3.49-3.44 (m, 2H), 2.75 (t, J=6.9 Hz, 2H), 1.90 (s, 3H), 1.50 (s, 9H). HRMS (ESI) m/z calcd for C52H56N3O15P [M+H]+ 994.3522, found 994.3489.
To a solution of 54 (72.0 mg, 72.5 μmol, 1.0 equiv.) in DCM (2 mL) was added TFA (200 μL). The yellow mixture was stirred at room temperature for 1 hour and turned purple. The reaction mixture was then concentrated in vacuo. Flash column chromatography on silica using a gradient of 0-10% MeOH in DCM afforded 55 (54.9 mg, 85%). 1H NMR (600 MHz, acetone-d6): δ (ppm) 7.89 (dd, J=1.5, 8.1 Hz, 1H), 7.40-7.24 (m, 17H), 7.22 (d, J=8.5 Hz, 1H), 7.15 (s, 1H), 6.84 (dd, J=2.3, 8.7 Hz, 1H), 6.80 (dd, J=0.6, 8.3 Hz, 1H), 6.56 (ddd, J=1.1, 7.1, 8.3 Hz, 1H), 5.32 (d, J=8.1 Hz, 1H), 5.24 (d, J=12.2 Hz, 1H), 5.17-5.03 (m, 6H), 4.84 (m, 1H), 4.75 (dd, J=1.2, 12.2 Hz, 1H), 4.53 (dd, J=5.6, 11.8 Hz, 1H), 4.10 (m, 1H), 4.03 (t, J=9.1 Hz, 1H), 3.50-3.39 (m, 2H), 2.83 (t, J=7.2 Hz, 2H), 1.87 (s, 3H). 13C NMR (125 MHz, acetone-d6): δ (ppm) 168.4, 155.4, 152.4, 152.0, 137.1, 136.5, 132.0, 129.34, 129.32, 129.26, 129.18, 129.13, 129.07, 129.04, 128.84, 128.81, 128.68, 124.44, 117.3, 116.1, 113.8, 113.5, 112.5, 110.4, 106.8, 101.1, 81.6 (d, J=5.8 Hz), 77.1 (d, J=4.6 Hz), 74.6, 70.54, 70.46, 70.3 (d, J=5.5 Hz), 70.2 (d, J=5.5 Hz), 63.7, 40.4, 26.3, 23.0. HRMS (ESI) m/z calcd for C47H56N3015P [M+H]+ 894.2998, found 894.2957.
To a solution of 55 (54.9 mg, 61.4 μmol, 1.0 equiv.) in a mixture of MeOH and EtOAc (2.5 mL, v/v=2:3) was added Pd/C (10% w/w) (32 mg). The reaction mixture was purged with argon for 5 minutes, flushed with hydrogen, and then subjected to hydrogen atmosphere for 3 hours at room temperature, and again purged with argon for 5 minutes. The mixture was filtered through Celite and concentrated in vacuo, affording sngl #4 (32, 33.4 mg, 94%). HRMS (ESI) m/z calcd for C33H36N3013P, [M−H]− 578.1545, found 578.1554.
To BC-1 (503 mg, 0.62 mmol, 1.00 equiv.) in a high-pressure flask was added 15 mL of MeNH2 (40% in H2O) and 2 mL MeOH. The flask was sealed and heated to 100° C., at which the solution was stirred for 2 hr. The resulting solution was allowed to cool to room temp, at which a precipitate slowly formed, filtered, and washed with cold methanol/water, affording BC-2 (maglu #3, 266 mg, 82%) as a white solid. 1H NMR (600 MHz, DMSO-d6): δ 8.30 (s, 1H), 8.23 (br s, 1H), 7.70 (br s, 1H), 5.40 (d, J=9.4 Hz, 1H), 5.31 (d, J=5.8 Hz, 1H), 5.28 (d, J=4.6 Hz, 1H), 5.14 (d, J=5.4 Hz, 1H), 4.59 (t, J=5.9 Hz, 1H), 3.99 (td, J=9.1, 5.8 Hz, 1H), 3.70 (ddd, J=11.7, 5.7, 1.7 Hz, 1H), 3.43 (dt, J=11.9, 6.1 Hz, 1H), 3.41-3.34 (m, 2H), 3.24 (td, J=9.2, 5.6 Hz, 1H), 2.95 (br s, 3H). 13C NMR (126 MHz, DMSO-d6): δ 155.0, 152.6, 139.4, 82.8, 80.0, 77.3, 71.3, 69.8, 60.9, 29.7. HRMS (ESI) m/z: [M+H]+ calcd for C12H18N5O5 312.1302; found 312.1290.
To BC-1 (1.00 g, 2.06 mmol, 1.00 equiv.) in a high-pressure flask was added 5 mL of MeOH and methanolic ammonia (7N, 29 mL, 206 mmol, 100 equiv.). The flask was sealed and heated to 100° C., at which the resulting yellow solution was stirred for 8 hr. The solution was transferred to a round-bottom flask and concentrated to dryness in vacuo. The reaction crude was then re-dissolved in MeOH upon heating, silica gel (11 g) was added, and the mixture was concentrated to dryness in vacuo (for dry-loading). Flash column chromatography on silica using a gradient of 30-60% MeOH in DCM was performed, affording BC-3 (420 mg, 68%) as an off-white power. 1H NMR (600 MHz, DMSO-d6): δ 8.31 (s, 1H), 8.14 (s, 1H), 7.23 (s, 2H), 5.39 (d, J=9.4 Hz, 1H), 5.30 (d, J=5.8 Hz, 1H), 5.25 (d, J=4.7 Hz, 1H), 5.12 (d, J=5.6 Hz, 1H), 4.57 (t, J=5.9 Hz, 1H), 3.99 (td, J=9.1, 5.8 Hz, 1H), 3.70 (ddd, J=11.7, 5.7, 1.7 Hz, 1H), 3.43 (dt, J=11.9, 6.1 Hz, 1H), 3.41-3.34 (m, 2H), 3.24 (td, J=9.2, 5.6 Hz, 1H). 13C NMR (126 MHz, DMSO-d6): δ 156.0, 152.6, 149.8, 139.7, 118.7, 82.8, 80.0, 77.3, 71.2, 69.8, 60.9. HRMS (ESI) m/z: [M+H]+ calcd for C11H16N5O5 298.1146; found 298.1136.
A solution of BC-3 (15 mg, 0.050 mmol, 1.00 equiv.) and Mel (12 μL, 0.193 mmol, 3.85 equiv.) in DMF (0.5 mL) was stirred for 48 hr at 40° C. The resulting yellow solution was concentrated to dryness in vacuo. Flash column chromatography on C18 using 100% H2O (w/ 0.1% acetic acid) afforded maglu #1 (BC-4, 20 mg, 90%) as a white solid. maglu #1 was compared to the corresponding peak in C. elegans wildtype (N2) endo-metabolome samples by HILIC-HRMS (Method C) and MS2 (see Figure Sla and S5 for co-elution and MS2 data, respectively). 1H NMR (500 MHz, methanol-d4): 8.56 (s, 1H), 8.55 (s, 1H), 5.63 (d, J=9.3 Hz, 1H), 4.02 (t, J=9.0 Hz, 1H), 3.91 (s, 3H), 3.88 (d, J=12.1 Hz, 1H), 3.73 (dd, J=12.1, 5.3 Hz, 1H), 3.62-3.56 (m, 2H), 3.53 (t, J=9.1 Hz). 13C NMR (126 MHz, methanol-d4): δ 152.7, 149.1, 148.7, 144.3, 120.3, 85.2, 81.3, 78.5, 73.7, 70.9, 62.3, 38.3. HRMS (ESI) m/z: [M+H]+ calcd for C12H18N5O5 312.1302; found 312.1294.
To a solution of BC-3 (350 mg, 1.18 mmol, 1.00 equiv.) in DMF (7 mL) at 0° C. was added TIPDSiCl2 (560 μL, 1.75 mmol, 1.48 equiv.) and imidazole (362 mg, 5.32 mmol, 4.51 equiv.). The reaction mixture was stirred for 15 min at 0° C. and then diluted with DCM, followed by addition of H2O. Organics were extracted 2× with DCM, combined, and then basified using sat. aq. NaHCO3. The organic layer was collected and remaining organics were extracted 3× with a 2:1 mixture of DCM:EtOAc. Combined organics were dried using MgSO4, filtered, and concentrated in vacuo. The reaction crude was then dissolved in a DCM/MeOH mixture, silica gel (2 g) was added, and the mixture was concentrated to dryness (for dry-loading). Flash column chromatography on silica using a gradient of 2.5-30% MeOH in DCM was performed, affording BC-5 (475 mg, 75%) as a white solid. 1H NMR (500 MHz, methanol-d4): δ 8.29 (s, 1H), 8.21 (s, 1H), 5.58 (d, J=9.4 Hz, 1H), 4.18 (dd, J=12.7, 2.2 Hz, 1H), 4.04 (t, J=9.2 Hz, 1H), 3.95 (t, J=9.1 Hz, 1H), 3.91 (dd, J=12.7, 0.8 Hz, 1H), 3.65 (t, J=9.0 Hz, 1H), 3.55 (dt, J=9.4, 1.8 Hz, 1H), 1.27-0.98 (m, 28H). 13C NMR (126 MHz, methanol-d4): δ 157.4, 154.0, 151.2, 140.8, 120.0, 84.8, 80.8, 78.3, 73.7, 70.4, 62.1, 18.0, 17.8, 17.7, 17.6, 14.9, 14.5, 14.0, 13.8.
To a solution of BC-5 (260 mg, 0.48 mmol, 1.00 equiv.) in 1:1 DCM:DMF (16 mL) at 40° C. was added dibenzyl N,N-diisopropylphosphoramidite (0.58 mL, 1.73 mmol, 3.60 equiv.), and 1H-tetrazole (0.45 M in ACN, 3.20 mL, 1.44 mmol, 3.00 equiv.). The reaction mixture was stirred at 40° C. for 1 hr and then cooled to −78° C. after which mCPBA (77% max, 300 mg, 1.34 mmol, 2.79 equiv.) was added. The resulting mixture was stirred at −78° C. for 10 min. and was then quenched with the addition of sat. aq. NaHCO3 (3 mL) after which H2O (10 mL) and DCM (50 mL) were added. The organic layer was washed 1× with sat. aq. NaHCO3 (10 mL total) and collected and the aqueous layer was extracted 2× with DCM (20 mL each). Combined organics were dried with MgSO4, filtered, and concentrated in vacuo. Flash column chromatography on silica using a gradient of 2.5-25% MeOH in DCM was performed, affording separable BC-6 (301 mg, 78%) and BC-7 (40 mg, 10%) as white solids. 2′-O isomer (BC-6): 1H NMR (600 MHz, methanol-d4): δ 8.30 (s, 1H), 8.14 (s, 1H), 7.33-7.22 (m, 6H), 7.24-7.17 (m, 2H), 7.00 -6.95 (m, 2H), 5.87 (d, J=9.3 Hz, 1H), 4.90 (dd, J=11.8, 7.5 Hz, 1H), 4.95-4.88 (m, 1H) 4.81 (dd, J=11.7, 8.5 Hz, 1H), 4.53-4.42 (m, 2H), 4.18 (dd, J=12.9, 2.1 Hz, 1H), 4.03 (t, J=9.2 Hz, 1H), 3.94 (dd, J=13.0, 1.2 Hz, 1H), 3.92 (t, J=9.0 Hz, 1H), 3.62 (dt, J=9.4, 1.8 Hz, 1H), 1.31-0.95 (m, 28H). 2′-O isomer (BC-6): 13C NMR (126 MHz, methanol-d4): δ 157.4, 154.1, 151.1, 140.9, 137.0 (d, J=7.3 Hz), 136.5 (d, J=7.3 Hz), 129.6, 129.5, 129.0, 128.6, 120.0, 82.9, 81.0, 80.1, 76.7 (d, J=2.7 Hz), 70.9 (d, J=6.0 Hz), 70.6, 70.4 (d, J=5.9 Hz), 61.9, 18.0, 17.8, 17.7, 17.6, 14.8, 14.5, 14.1, 13.8. 3′-O isomer (BC-7): 1H NMR (600 MHz, methanol-d4): δ 8.31 (s, 1H), 8.22 (s, 1H), 7.38-7.30 (m, 10H), 5.65 (d, J=9.2 Hz, 1H), 5.14-5.07 (m, 2H), 5.07-5.00 (m, 2H), 4.55 (q, J=8.5 Hz, 1H), 4.43 (t, J=9.1 Hz, 1H), 4.18 (t, J=9.1 Hz, 1H), 4.15 (dd, J=12.8, 2.0 Hz, 1H), 3.94 (dd, J=12.8, 1.8 Hz, 1H), 3.59 (dt, J=9.4, 1.9 Hz, 1H), 1.14-0.86 (m, 28H). 3′-O isomer (BC-7): 13C NMR (126 MHz, methanol-d4): δ 157.4, 154.0, 151.1, 141.1, 137.4 (d, J=7.0 Hz), 137.1 (d, J=7.2 Hz), 129.7, 129.6, 129.3, 120.1, 86.00, 85.4 (d, J=6.6 Hz), 85.0, 80.3, 72.2, 71.1 (d, J=5.6 Hz), 70.8 (d, J=5.3 Hz), 69.8 (d, J=5.4 Hz), 62.0, 18.0, 17.9, 17.8, 17.7, 17.5, 14.4, 14.3, 14.1.
Note: Some variability between experiments regarding amount of 1H-tetrazole and phosphoramidite needed. It is important to monitor conversion of sugar starting material to prevent bis-phosphorylation. Developed TLC plate (12:1 DCM:MeOH) was visualized using p-anisaldehyde stain, where 2′-O phosphate BC-6 stained brown and 3′-O phosphate BC-7 blue.
To a 10:1 solution of BC-6 and BC-7 (154 mg, 0.192 mmol, 1.00 equiv.), respectively, in THF (4 mL) at 0° C. was added TBAF (1M in THF, 480 μL, 0.48 mmol, 2.50 equiv.). After 15 min., AcOH (60 uL) was added, and the resulting solution was concentrated in vacuo. Flash column chromatography on silica using a gradient of 10-40% MeOH in DCM was performed, affording BC-8 (73 mg, 0.131 mmol, 68%) and BC-9 (20 mg, 0.036 mmol, 19%) which were able to be mostly separated after subsequent purification. 2′-O isomer (BC-9): 1H NMR (500 MHz, methanol-d4): δ 8.38 (s, 1H), 8.15 (s, 1H), 7.34-7.15 (m, 8H), 6.98-6.92 (m, 8H), 5.86 (d, J=9.3 Hz, 1H), 4.97-4.88 (m, 2H), 4.83 (dd, J=12.0, 8.5 Hz, 1H), 4.48 (dd, J=11.8, 6.8 Hz, 1H), 4.43 (dd, J=11.8, 8.5 Hz, 1H), 3.90 (dd, J=12.3, 1.5, 1H), 3.83 (t, J=8.8 Hz, 1H), 3.76 (dd, J=12.3, 5.0 Hz, 1H), 3.68-3.62 (m, 2H). 2′-O isomer (BC-9): 13C NMR (126 MHz, methanol-d4): δ 157.4, 154.1, 150.9, 141.4, 137.0 (d, J=7.2 Hz), 136.6 (d, J=7.4 Hz) 129.5, 128.9, 128.5, 120.0, 82.8, 81.4, 79.7, 77.3 (d, J=2.6 Hz), 71.1, 70.9 (d, J=5.8 Hz), 70.3 (d, J=6.0 Hz), 62.2. 3′-O isomer (BC-8): 1H NMR (500 MHz, methanol-d4): δ 8.32 (s, 1H), 8.22 (s, 1H), 7.40-7.24 (m, 10H), 5.65 (d, J=9.3 Hz, 1H), 5.16 (d, J=7.5 Hz, 2H), 5.07-5.00 (m, 2H), 4.54 (q, J=8.8 Hz, 1H), 4.40 (t, J=9.2 Hz, 1H), 3.91 (dd, J=12.2, 2.1, 1H), 3.87 (t, J=9.0 Hz, 1H), 3.80 (dd, J=12.2, 5.1 Hz, 1H), 3.67 (ddd, J=9.8, 5.0, 2.1 Hz, 1H). 3′-O isomer (BC-8): 13C NMR (126 MHz, methanol-d4): δ 157.4, 153.9, 150.8, 141.6, 137.5 (d, J=4.2 Hz), 137.4 (d, J=4.2 Hz), 129.6, 129.5, 129.0, 120.2, 85.9, 85.2, 80.7, 71.9 (d, J=3.2 Hz), 70.8 (d, J=5.8 Hz), 69.7 (d, J=3.2 Hz), 62.1. Note: Developed TLC plate (5:1 DCM:MeOH) was visualized using p-anisaldehyde stain, where 2′-O phosphate BC-9 stained brown and 3′-O phosphate BC-8 blue.
A solution of BC-8 (12 mg, 0.021 mmol) and Mel (5.2 μL, 0.083 mmol, 3.97 equiv.) in DMF (1 mL) was stirred for 24 hr at 40° C. The resulting yellow solution was then dissolved in 1.5 mL MeOH/1.0 mL H2O. NaHCO3 (7.5 mg, 0.089 mmol, 4.25 equiv.) and Pd/C (12 mg) were added, the suspension was sparged with Ar for 5 min. then switched to H2, through which the suspension was rapidly stirred under for 2 hr. After sparging with Ar, AcOH (20 μL) was added and the reaction mixture was filtered through celite. The collected filtrate was concentrated to dryness in vacuo. Flash column chromatography on C18 using 100% H2O (w/ 0.1% formic acid) afforded maglu #2 (BC-10, 6.8 mg, 80% over two steps) as a white solid. maglu #2 was compared to the corresponding peak in C. elegans wildtype (N2) endo-metabolome samples by HILIC-HRMS (Method D) and MS2 (see Figure S2b and S7 for co-elution and MS2 data, respectively). HRMS (ESI) m/z: [M+H]+ calcd for C12H19N5OsP+392.0966; found 392.0953. Note: Addition of NaHCO3 was required as non-basified solutions led to only mono-debenzylation.reference AcOH was added to prevent any partial Dimroth rearrangement of samples during the concentration step as well during evaluation of sample purity w/crude NMR.
Phenylacetic acid (19 mg, 0.141 mmol, 2.82 equiv.) and TBTU (45 mg, 0.141 mmol, 2.82 equiv.) were added to a solution of BC-8 (28 mg, 0.050 mmol, 1.00 equiv.) in 0.7 mL dry pyridine. The resulting mixture was stirred for 3 hr at room temp. and then concentrated to dryness in vacuo. Flash column chromatography on silica using a gradient of 2.5-30% MeOH in DCM was performed, affording BC-11 (21 mg, 62%) as a colorless oil, with some fractions containing co-eluting HOBt. 1H NMR (600 MHz, methanol-d4): δ 8.21 (s, 1H), 8.15 (s, 1H), 7.38-7.26 (m, 1OH), 7.22-7.12 (m, 5H), 5.63 (d, J=9.3 Hz, 1H), 5.17-5.07 (m, 4H), 4.58-4.49 (m, 2H), 4.35 (t, J=9.2 Hz, 1H), 4.32 (dd, J=12.4, 5.9 Hz), 3.88 (ddd, J=10.0, 5.8, 2.2 Hz, 1H), 3.79 (t, J=9.5 Hz, 1H), 3.63 (dd, J=15.0 Hz, 1H), 3.59 (d, J=15.0 Hz, 1H). 13C NMR (126 MHz, methanol-d4): δ 173.2, 157.5, 154.0, 151.0, 150.1, 141.3, 137.5 (d, J=2.8 Hz, 1H), 137.4 (d, J=2.8 Hz, 1H), 135.6, 130.4, 129.6, 129.5, 129.4, 129.0, 128.0, 125.6, 120.3, 85.6 (d, J=6.7 Hz, 1H), 84.8, 77.9, 71.9 (d, J=3.8 Hz, 1H), 70.8 (d, J=5.8 Hz, 1H), 70.1 (d, J=3.8 Hz, 1H), 64.4, 41.8.
A solution of BC-11 (6.0 mg, 8.88 μmol, 1.00 equiv.) and Mel (2.2 μL, 35.6 μmol, 4.01 equiv.) in dry DMF (0.3 mL) was stirred for 24 hr at 40° C. The resulting yellow solution (a 2:1 mixture of mono and bis-benzylated products, respectively) was concentrated in vacuo and then dissolved in 0.5 mL MeOH and 75 μL H2O. To the solution was added NaHCO3 (2.5 mg, 30.0 μmol, 3.38 equiv.) in 22.5 μL H2O and Pd/C (17 mg, 10% w/w). The suspension was sparged with Ar for 5 min. then switched to H2, through which the suspension was rapidly stirred under for 1.5 hr. After sparging with Ar for 5 min., AcOH (30 μL) was added and the reaction mixture was then filtered through celite using MeOH/H2O. The collected filtrate was concentrated to dryness in vacuo. Purification by preparative HPLC (see Methods) afforded maglu #11 (BC-12, 1.4 mg, 30% over two steps) as a white solid. maglu #3 was found to be identical to the corresponding peak on C18 in C. elegans wildtype (N2),fem-3 (gf), him-5 endo-metabolome samples by HPLC-HRMS (Method A) and MS2 (see
Compound BC-14 was synthesized according to a previously reported procedure. A suspension containing BC-13 (2.1 g, 4.05 mmol, 1.00 equiv.), NaOH (1.62 g, 40.5 mmol, 10 equiv.), H2O (50 mL), and 1,4-dioxane (20 mL) was heated at 100° C. for 4 hr. The resulting dark red solution was neutralized by the addition of AcOH and then concentrated to dryness in vacuo. Flash column chromatography on C18 using a gradient of 0-50% ACN in H2O (w/ 0.1% AcOH) afforded BC-14 (560 mg, 41%) as a yellow solid. 1H NMR (600 MHz, DMSO-d6): δ 8.30 (s, 1H), 5.37 (d, J=4.1 Hz, 1H), 5.30 (d, J=9.3 Hz, 1H), 5.30-5.23 (m, 1H), 5.20-5.08 (m, 1H), 4.65-4.51 (m, 1H), 3.89-3.83 (m, 1H), 3.70 (d, J=11.4 Hz, 1H), 3.46-3.34 (m, 3H), 3.24 (t, J=9.1 Hz, 1H). Reference: MODERNA THERAPEUTICS INC—WO2017/66793, 2017, A1
A solution containing BC-14 (330 mg, 0.99 mmol, 1.00 equiv.) and MeNH2 (12 mL, 40% in H2O, 154 mmol, 156 equiv.) was heated at 100° C. in a sealed container for 15 hr. The resulting solution was acidified to pH ˜5 w/ AcOH, transferred to a round-bottom flask, and then concentrated to dryness in vacuo. Flash column chromatography on C18 using a gradient of 0%-30% ACN in H2O (w/ 0.1% AcOH) afforded BC-15 (mgglu #3, 210 mg, 65%) as an off-white solid. mgglu #3 was found to be identical to the corresponding peak using HILIC-MS (Method C) in C. elegans wildtype (N2) samples by HPLC-HRMS (see
HRMS (ESI) m/z: [M+Na]+ calcd for C12H17N5O6Na+ 350.1071; found 350.1053.
To BC-14 (60 mg, 0.181 mmol, 1.00 equiv.) in a high-pressure flask was added 40% NHMe2 in H2O (2.5 mL). The flask was sealed and heated to 100° C., at which the resulting solution was stirred for 14 hr. The solution was transferred to a round-bottom flask and concentrated to dryness in vacuo. Flash column chromatography on C18 using a gradient of 0-100% ACN (w/0.1% formic acid) in H2O (w/ 0.1% formic acid), followed by additional purification with flash column chromatography on silica using a gradient of 20-60% MeOH in DCM afforded BC-16 (48 mg, 77%) as a white solid. dmgglu #3 (BC-16) was compared to isomer peaks using HILIC-MS (Method C) in C. elegans and C. briggsae wildtype samples by HILIC-HRMS (see Figure S1c). 1H NMR (500 MHz, DMSO-d6): δ 10.68 (br s, 1H), 7.85 (s, 1H), 5.53-5.23 (m, 2H), 5.20 (d, J=9.2 Hz, 1H), 5.18 (br s, 1H), 4.61 (br s, 1H), 3.85 (t, J=9.1 Hz, 1H), 3.69 (d, J=12.0, 1H), 3.42 (dd, J=12.0, 6.1 Hz, 1H), 3.36-3.30 (m, 3H), 3.20 (t, J=9.2 Hz, 1H), 3.07 (s, 6H). 13C NMR (126 MHz, DMSO-d6): 157.5, 153.0, 151.1, 136.3, 115.6, 82.3, 79.9, 77.2, 71.3, 69.8, 60.9, 37.6. HRMS (ESI) m/z: [M+Na]+ calcd for C13H19N5O6Na+ 364.1227; found 364.1218.
A solution containing BC-17 (515 mg, 0.99 mmol, 1.00 equiv.), NaOH (400 mg, 10.0 mmol, 10.10 equiv.), H2O (13 mL) and 1,4-dioxane (5 mL) was heated at 100° C. for 4 hr. The dark red solution was cooled to 0° C., acidified to pH=4 by the addition of AcOH, and concentrated to dryness in vacuo. Flash column chromatography on C18 using a gradient of 0%-50% ACN in H2O (w/ 0.1% formic acid) afforded BC-18 (157 mg, 47%) as a light brown solid. 1H NMR (600 MHz, DMSO-d6): δ 8.47 (s, 1H), 5.68 (d, J=9.4 Hz, 1H), 5.38 (br s, 1H), 5.28 (br s, 1H), 5.11 (br s, 1H), 4.58-4.50 (m, 1H), 3.84 (d, J=9.2 Hz, 1H), 3.69 (d, J=11.7 Hz, 1H), 3.47-3.42 (m, 1H), 3.37-3.30 (m, 3H), 3.26 (dd, J=9.7, 3.5 Hz, 1H). 13C NMR (126 MHz, DMSO-d6): 163.1, 156.7, 154.5, 143.4, 113.8, 85.0, 80.1, 77.0, 72.0, 69.5, 60.9. HRMS (ESI) m/z: [M+Na]+ calcd for C11H13ClN4O6Na+ 355.0416; found 355.0410. The compound was primarily detected as its in-source fragment: [M+H]+ calcd for C5H4ClN4O+171.0068; found 171.0066.
To BC-18 (51 mg, 0.154 mmol, 1.00 equiv.) in a high-pressure flask was added 40% NHMe2 in H2O (5.1 mL). The flask was sealed and heated to 100° C., at which the resulting solution was stirred for 19 hr. The solution was cooled to room temp., transferred to a round-bottom flask, neutralized with AcOH, and concentrated to dryness in vacuo. Flash column chromatography on C18 using a gradient of 0-100% ACN in H2O (w/ 0.1% formic acid) afforded dmgglu #1 (BC-19, 30 mg, 58%) as an off-white solid. dmgglu #3 was found to be identical to the corresponding peak using HILIC-MS (Method C) in C. elegans wildtype (N2) samples by HPLC-HRMS (see Figure Slc for co-elution data). 1H NMR (500 MHz, DMSO-d6): 10.84 (br s, 1H), 8.20 (s, 1H), 5.57 (d, J=9.3 Hz, 1H), 5.34 (br s, 1H), 5.25 (br s, 1H), 5.09 (br s, 1H), 4.53 (t, J=6.1 Hz, 1H), 3.84 (t, J=9.2 Hz, 1H), 3.68 (d, J=12.0 Hz, 1H), 3.47-3.28 (m, 3H), 3.24 (t, J=9.3 Hz, 1H), 3.03 (s, 6H). 13C NMR (126 MHz, DMSO-d6): δ 159.5, 154.9, 152.7, 142.3, 107.5, 84.7, 79.8, 77.2, 71.8, 69.5, 60.9, 37.9. HRMS (ESI) m/z: [M+Na]+ calcd for C13H19N5O6Na+ 364.1227; found 364.1217. A large fraction of the sample was detected as the in-source fragment: [M+H]+ calcd for C7H10N5O+ 180.0880; found 180.0877.
Under Ar, a suspension of N1-methylguanine (BC-20, 750 mg, 4.54 mmol, 1.36 equiv.), N,O-bis(trimethylsilyl)acetamide (2.4 mL, 9.84 mmol, 3.02 equiv.) and DCE (20 mL) was refluxed for 1 hr until the solution was homogeneous. After cooling to room temp., TMSOTf (1.35 mL, 7.48 mmol, 2.29 equiv.) and alpha-D-glucose pentaacetate (1.27 g, 3.26 mmol, 1.00 equiv.) were added and the resulting solution was refluxed for 36 hr. The resulting orange solution was then concentrated to dryness in vacuo, followed by the addition of NH3/MeOH (7N, 38 mL, 266 mmol, 81 equiv.). The resulting solution was stirred for 7 hr at room temp. and concentrated to dryness in vacuo. Flash column chromatography on C18 using a gradient of 0%-100% ACN in H2O (w/ 0.1% AcOH) was first performed, of which fractions containing products BC-21 and BC-22 and 1-methylguanine (BC-20) were collected (elution at ˜10% ACN). The dried mixture was dissolved in MeOH and filtered, followed by concentration in vacuo with 15 g of silica gel for dry-loading. Flash column chromatography on silica using a gradient of 35%-100% MeOH in DCM was then performed, which afforded BC-22 (256 mg, 24%) and BC-21 (219 mg, 21%), of which could be mostly separated with subsequent chromatography. mgglu #1 (BC-21) and mgglu #5 (BC-22) were compared to isomer peaks (m/z=350.1071) using HILIC-MS in C. elegans wildtype (N2) samples by HILIC-HRMS (Method C) and MS2 (see Figure Slb and S6 for co-elution and MS2 data, respectively). 1H NMR (N9-isomer, BC-21) (600 MHz, D2O): δ 7.98 (s, 1H), 5.47 (d, J=9.4 Hz, 1H), 4.16 (t, J=9.0 Hz, 1H), 3.93 (dd, J=12.4, 1.7 Hz, 1H), 3.82 (dd, J=12.3, 5.0 Hz, 1H), 3.74 (dd, J=9.7, 1.8 Hz, 1H), 3.71 (t, J=8.9 Hz, 1H), 3.66 (t, J=9.3 Hz, 1H), 3.41 (s, 3H). 13C NMR (N9-isomer, BC-21) (126 MHz, D2O): δ 159.2, 155.4, 150.1, 138.6, 116.4, 83.4, 79.3, 76.9, 71.8, 69.6, 61.0, 29.2. HRMS (ESI) m/z: [M+Na]+ calcd for C12H17N5O6Na+ 350.1071; found 350.1060. 1H NMR (N7-isomer, BC-22) (600 MHz, D2O): δ 8.29 (s, 1H), 5.79 (d, J=9.2 Hz, 1H), 4.18 (t, J=9.0 Hz, 1H), 3.94 (dd, J=12.4, 1.7 Hz, 1H), 3.80 (dd, J=12.4, 5.5 Hz, 1H), 3.74 (dd, J=9.5, 1.7 Hz, 1H), 3.71 (t, J=8.9 Hz, 1H), 3.66 (t, J=9.3 Hz, 1H), 3.47 (s, 3H). 13C NMR (N7-isomer, BC-22) (126 MHz, D2O): δ 158.1, 155.9, 155.2, 144.6, 108.2, 85.7, 79.3, 76.8, 72.7, 69.7, 61.2, 29.1. HRMS (ESI) m/z: [M+Na]+ calcd for C12H17N5O6Na+ 350.1071; found 350.1053.
To a solution of BC-22 (120 mg, 0.367 mmol, 1.00 equiv.) in DMF (2 mL) at 0° C. was added TIPDSiCl2 (175 μL, 0.504 mmol, 1.50 equiv.) and imidazole (104 mg, 1.53 mmol, 4.55 equiv.). The reaction mixture was stirred for 15 min at 0° C. and then diluted with DCM, followed by addition of H2O. Organics were extracted 4× with DCM, combined, and then basified using sat. aq. NaHCO3. Organics were then extracted from the aq. layer 3× with DCM, dried using MgSO4, filtered, and concentrated in vacuo. Flash column chromatography on silica using a gradient of 5-50% MeOH in DCM was performed, affording BC-23 (168 mg, 80%) as a white solid. 1H NMR (500 MHz, methanol-d4): δ 8.17 (s, 1H), 5.79 (d, J=9.4 Hz, 1H), 4.16 (dd, J=12.8, 2.1 Hz, 1H), 3.99 (t, J=9.0 Hz, 1H), 3.95 (t, J=9.0 Hz, 1H), 3.90 (dd, J=12.8, 1.0 Hz, 1H), 3.60 (t, J=9.0 Hz, 1H), 3.49 (dt, J=9.5, 1.6 Hz, 1H), 3.45 (s, 3H), 1.25-0.96 (m, 28H). 13C NMR (126 MHz, methanol-d4): δ 158.8, 155.8, 143.3, 136.3, 109.1, 86.7, 80.7, 78.4, 74.0, 70.4, 62. 1, 28.7, 18.0, 17.8, 17.7, 17.6, 14.9, 14.5, 14.0, 13.8.
To BC-23 (153 mg, 0.27 mmol, 1.00 equiv.) in DCM (2 mL) and DMF (1 mL) was added dibenzyl N,N-diisopropylphosphoramidite (0.36 mL, 1.07 mmol, 3.96 equiv.), and ImOTf (277 mg, 1.27 mmol, 4.70 equiv.) incrementally over a 2 hr period. Note: this was done to ensure minimization of bis-phosphitylation products. The reaction mixture was then cooled to −78° C. after which mCPBA (77% max, 165 mg, 0.74 mmol, 2.73 equiv.) was added. The resulting mixture was stirred at −78° C. for 20 min. and was then quenched with the addition of sat. aq. NaHCO3 (10 mL) followed by addition of DCM (20 mL). The organic layer was collected and the aqueous layer was extracted an additional 2× with DCM (20 mL each). Combined organics were dried with MgSO4, filtered, and concentrated in vacuo. Flash column chromatography on silica using a gradient of 5-40% MeOH in DCM was performed, affording separable BC-24 (149 mg, 67%) and BC-25 (44 mg, 20%) as white solids. 2′-O isomer (BC-24): 1H NMR (500 MHz, methanol-d4): δ 8.21 (br s, 1H), 7.36-7.13 (m, 8H), 7.14-6.97 (m, 2H), 6.24-5.63 (m, 1H), 5.04-4.89 (m, 2H), 4.78-4.50 (m, 3H), 4.16 (d, J=12.6 Hz, 1H), 4.11-3.95 (m, 1H), 3.91 (d, J=12.6 Hz, 1H), 3.88-3.77 (m, 1H), 3.59-3.47 (m, 1H), 3.25 (br s, 3H), 1.30-0.93 (m, 28H). 2′-O isomer (BC-24): 13C NMR (126 MHz, methanol-d4): δ 156.8, 137.1, 136.8, 129.5, 129.4, 128.9, 109.1, 80.8, 76.9, 70.9, 70.4, 61.9, 28.6, 18.1, 17.8, 17.7, 17.6, 14.8, 14.5, 14.0, 13.8. 3′-O isomer (BC-25): 1H NMR (500 MHz, methanol-d4): δ 8.19 (s, 1H), 7.39-7.28 (m, 10H), 5.77 (d, J=8.5 Hz, 1H), 5.16-5.06 (m, 2H), 5.08-4.96 (m, 2H), 4.54-4.40 (m, 2H), 4.20 (t, J=9.0 Hz, 1H), 4.14 (dd, J=12.8, 1.4 Hz, 1H), 3.93 (dd, J=12.7, 1.7 Hz, 1H), 3.52 (d, J=9.2 Hz, 1H), 3.46 (s, 3H), 1.19-0.82 (m, 28H). 3′-O isomer (BC-25): 13C NMR (126 MHz, methanol-d4): δ 159.0, 155.9, 155.6, 144.0, 137.4 (J=7.0 Hz), 137.2 (d, J=7.4 Hz), 129.7, 129.6, 129.3, 129.0, 109.0, 87.0, 85.6 (d, J=6.8 Hz), 80.1, 72.7, 71.1 (d, J=5.6 Hz), 70.8 (d, J=5.3 Hz), 69.6 (d, J=5.6 Hz), 62.0, 28.7, 18.0, 17.9, 17.8, 17.7, 17.5, 14.4, 14.3, 14.2, 14.1. Note: 2′-O dibenzyl phosphate isomer (BC-24) exhibited extreme line broadening for several signals.
To a solution of BC-24 (90 mg, 0.108 mmol, 1.00 equiv.) in THF (3 mL) at 0° C. was added TBAF (1M in THF, 275 μL, 0.27 mmol, 2.50 equiv.). After 15 min., AcOH (75 uL) was added, and the resulting solution was concentrated in vacuo. Flash column chromatography on silica using a gradient of 5-40% MeOH in DCM was performed, affording BC-26 (37.5 mg, 0.036 mmol, 59%) and BC-27 (11.5 mg, 8 mmol, 18%) of which were mostly separable. 3′-O isomer (BC-26): 1H NMR (500 MHz, methanol-d4): δ 8.23 (s, 1H), 7.39-7.21 (m, 1OH), 5.78 (d, J=9.1 Hz, 1H), 5.15 (d, J=7.5 Hz, 2H), 5.13-5.10 (m, 2H), 4.49 (td, J=9.1, 8.1 Hz, 1H), 4.36 (t, J=9.2 Hz, 1H), 3.90 (dd, J=12.2, 2.3 Hz, 1H), 3.84 (t, J=9.5 Hz, 1H), 3.77 (dd, J=12.2, 5.2 Hz, 1H), 3.63 (ddd, J=9.9, 5.2, 2.3 Hz, 1H), 3.47 (s, 3H). 3′-O isomer (BC-26): 13C NMR (126 MHz, methanol-d4): δ 159.3, 155.9, 155.8, 144.4, 137.5 (d, J=3.5 Hz), 137.4 (d, J=3.5 Hz), 129.6, 129.5, 129.1, 129.0, 109.0, 86.8, 86.0 (d, J=6.9 Hz), 80.5, 72.7 (d, J=3.5 Hz), 70.8 (d, J=5.8 Hz), 69.7 (d, J=3.2 Hz), 62.2, 28.8. 2′-O isomer (BC-27): 1H NMR (600 MHz, methanol-d4): δ 8.28 (s, 1H), 7.40-6.97 (m, 10H), 6.17-5.86 (m, 1H), 5.00-4.93 (m, 1H), 4.67 (dd, J=12.3, 6.7 Hz, 1H), 4.65-4.52 (m, 1H), 3.89 (dd, J=12.2, 1.9 Hz, 1H), 3.78 (t, J=9.0 Hz, 1H), 3.73 (dd, J=12.2, 5.6 Hz, 1H), 3.64 (t, J=9.5 Hz, 1H), 3.63 (ddd, J=9.6, 5.6, 2.0 Hz, 1H), 3.27 (s, 3H).
To a solution of BC-25 (65 mg, 0.078 mmol, 1.00 equiv.) in THF (3 mL) containing AcOH (20 uL) at 0° C. was added TBAF (1M in THF, 200 μL, 0.20 mmol, 2.56 equiv.). The solution was slowly warmed to RT over a 4 hr period, then additional AcOH (40 uL) was added, and the reaction mixture was concentrated in vacuo. Flash column chromatography on silica using a 20 gradient of 15-50% MeOH in DCM was performed, afforded BC-26 (32 mg, 70%).
A suspension containing BC-26 (17 mg, 0.020 mmol, 1.00 equiv.), Pd/C (35 mg, 10% w/w), AcOH (300 μL) and MeOH (3 mL) was sparged with Ar for 5 min. then switched to H2, through which the suspension was rapidly stirred under for 4 hr. After sparging with Ar, the reaction mixture was filtered through celite, washed with MeOH/H2O, and the collected filtrate was concentrated to dryness in vacuo affording mgglu #6 (BC-28, 8 mg, 68%) at 92% purity. mgglu #6 was found to be identical to the major isomer peak in C. elegans wildtype (N2) endo-metabolome samples by HILIC-HRMS (see Figures S2c and S8 for co-elution and MS2 data, respectively). Chromatographic Method E was used. mgglu #6-HRMS (ESI) m/z: [M+H]+ calcd for C12H19N5O9P+ 408.0915; found 408.0914.
Phenylacetic acid (8 mg, 0.059 mmol, 4.21 equiv.) and TBTU (19 mg, 0.059 mmol, 4.21 equiv.) were added to a solution of BC-26 (8.2 mg, 0.014 mmol, 1.00 equiv.) in 1 mL dry pyridine. The resulting mixture was stirred for 4 hr at room temp., MeOH (1 mL) was added, transferred to a round-bottom flask, and then concentrated to dryness in vacuo. Flash column chromatography on silica using a gradient of 2.5-40% MeOH in DCM was performed, affording BC-29 (6.0 mg, 61%) as a white solid. Note: co-eluting HOBt was separated by subsequent chromatography. 1H NMR (500 MHz, methanol-d4): δ 8.10 (s, 1H), 7.40-7.15 (m, 15H), 5.73 (d, J=9.1 Hz, 1H), 5.17-5.08 (m, 4H), 4.54 (dd, J=12.1, 1.8 Hz, 1H), 4.47 (q, J=8.7 Hz, 1H), 4.37 (t, J=9.1 Hz, 1H), 4.29 (dd, J=12.1, 4.9 Hz, 1H), 3.84-3.76 (m, 2H), 3.67 (d, J=15.3 Hz, 1H), 3.63 (d, J=15.3 Hz, 1H), 3.44 (s, 3H). 13C NMR (126 MHz, methanol-d4): δ 173.2, 159.3, 155.9, 155.7, 144.2, 137.5 (d, J=2.6 Hz), 137.4 (d, J=2.5 Hz), 135.6, 130.3, 129.6, 129.5, 129.4, 129.0, 128.0, 108.9, 86.7, 85.7, 85.6 (d, J=7.0 Hz), 77.7, 72.4 (d, J=3.2 Hz), 70.8 (d, J=5.9 Hz), 70.0 (d, J=3.4 Hz), 64.4, 41.8, 28.8.
A suspension containing BC-29 (5 mg, 0.0071 mmol, 1.00 equiv.), Pd/C (6.6 mg, 10% w/w), AcOH (47 μL) and MeOH (2 mL) was sparged with Ar for 5 min. then switched to H2, through which the suspension was rapidly stirred under for 1.5 hr. After sparging with Ar, the reaction mixture was filtered through celite using MeOH/H2O and the collected filtrate was concentrated to dryness in vacuo, affording mgglu #51 (BC-30, 3.0 mg, 81%) which was deemed pure enough for no further purification steps. mgglu #51 was found to be identical to the major isomer peak on C18 in C. elegans wildtype (N2),fem-3 (gf), and him-5 endo-metabolome samples by HPLC-HRMS (Method B) and MS2 (see Figures S3c and S10 for co-elution and MS2 data, respectively). mgglu #51—HRMS (ESI) m/z: [M+H]+ calcd for C20H25N5O10P+ 526.1333; found 526.1332.
Benzoic acid (22 mg, 0.18 mmol, 9.49 equiv.) and TBTU (55 mg, 0.14 mmol, 7.64 equiv.) were added to a solution of BC-26 (11 mg, 0.019 mmol, 1.00 equiv.) in 1.5 mL dry pyridine. The resulting mixture was stirred for 10 hr at room temp., MeOH (2 mL) was added, transferred to a round-bottom flask, then concentrated to dryness in vacuo. Flash column chromatography on silica using a gradient of 5-40% MeOH in DCM was performed, affording BC-31 (6.0 mg, 47%) as a white solid. Note: co-eluting HOBt was separated by subsequent chromatography. 1H NMR (500 MHz, methanol-d4): δ 8.19 (s, 1H), 8.06 (d, J=7.2 Hz, 2H), 7.60 (t, J=7.4 Hz, 1H), 7.47 (t, J=7.8 Hz, 2H), 7.39-7.19 (m, 10H), 5.79-5.69 (m, 1H), 5.16 (d, J=7.4 Hz, 2H), 5.14-5.10 (m, 2H), 4.70 (dd, J=12.1, 1.7 Hz, 1H), 4.62-4.46 (m, 3H), 4.03-3.91 (m, 2H), 3.40 (s, 3H). 13C NMR (126 MHz, methanol-d4): δ 167.8, 159.4, 155.9, 155.5, 144.4, 137.5 (d, J=3.1 Hz), 137.4 (d, J=3.3 Hz), 134.3, 131.3, 130.8, 129.6, 129.50, 129.4, 129.0, 108.9, 87.1, 85.7 (d, J=7.1 Hz), 77.8, 72.2 (d, J=3.5 Hz), 70.8 (d, J=6.0 Hz), 69.9 (d, J=3.4 Hz), 64.6, 28.8.
A suspension containing BC-31 (6.0 mg, 0.0087 mmol, 1.00 equiv.), Pd/C (14 mg, 10% w/w), formic acid (50 μL) and MeOH (2 mL) was sparged with Ar for 5 min. then switched to H2, through which the suspension was rapidly stirred under for 3 hr. After sparging with Ar, the reaction mixture was filtered through celite using MeOH/H2O and the collected filtrate was concentrated to dryness in vacuo, affording mgglu #52 (BC-32, 2.5 mg, 57%) which was deemed pure enough for no further purification steps. mgglu #52 was found to be identical to the major isomer peak on C18 in C. elegans wildtype (N2) by HPLC-HRMS (Method A) and MS2 (see Figures S4 and S12 for co-elution and MS2 data, respectively). mgglu #52—HRMS (ESI) m/z: [M+H]+ calcd for C19H22N5O10P+ 512.1177; found 512.1158.
To a solution of BC-21 (70 mg, 0.214 mmol, 1.00 equiv.) in DMF (2 mL) at 0° C. was added TIPDSiCl2 (120 μL, 0.376 mmol, 1.75 equiv.) and imidazole (66 mg, 0.970 mmol, 4.53 equiv.). The reaction mixture was stirred for 45 min at 0° C. and then diluted with DCM, followed by addition of H2O. Organics were extracted 3× with DCM, combined, and then basified using sat. aq. NaHCO3. Organics were then extracted from the aq. layer 3× with DCM, dried using MgSO4, filtered, and concentrated in vacuo. Flash column chromatography on silica using a gradient of 5-40% MeOH in DCM was performed, affording BC-33 (77 mg, 64%) as a white solid. 1H NMR (500 MHz, methanol-d4): δ 7.86 (s, 1H), 5.40 (d, J=9.4 Hz, 1H), 4.16 (dd, J=12.7, 2.2 Hz, 1H), 3.98-3.88 (m, 3H), 3.59 (t, J=9.0 Hz, 1H), 3.47 (dt, J=9.5, 1.5 Hz, 1H), 3.46 (s, 3H), 1.25-0.96 (m, 28H). 13C NMR (126 MHz, methanol-d4): δ 158.9, 156.2, 151.4, 137.6, 116.8, 84.3, 80.8, 78.3, 73.7, 70.4, 62.1, 28.8, 18.0, 17.8, 17.8, 17.8, 17.7, 17.7, 17.6, 14.9, 14.5, 14.0, 13.8.
To a suspension of BC-33 (74 mg, 0.130 mmol, 1.00 equiv.) and DCM (1 mL)/DMF (3 mL) at room temp. was added dibenzyl N,N-diisopropylphosphoramidite (0.13 mL, 0.39 mmol, 3.00 equiv.), and ImOTf (85 mg, 0.39 mmol, 3.00 equiv.). The reaction mixture was stirred at room temp. for 15 min. at which a homogenous solution formed and then cooled to −78° C. after which 1 mL of DCM and mCPBA (77% max, 87 mg, 0.39 mmol, 3.00 equiv.) were added. The resulting mixture was stirred at −78° C. for 10 min. and was then quenched with the addition of sat. aq. NaHCO3 (3 mL) followed by the addition of H2O (10 mL) and DCM (20 mL). The organic layer was collected and the aqueous layer was extracted 3× with DCM (20 mL each). Combined organics were dried with MgSO4, filtered, and concentrated in vacuo. Flash column chromatography on silica using a gradient of 2.5-20% MeOH in DCM was performed, affording BC-34 (94 mg, 87%). 1H NMR (600 MHz, methanol-d4): δ 7.88 (s, 1H), 7.33-7.20 (m, 8H), 7.08-7.02 (m, 2H), 5.68 (d, J=9.3 Hz, 1H), 4.93 (dd, J=11.5, 7.5 Hz, 1H), 4.87 (dd, J=11.1, 8.3 Hz, 1H), 4.68 (dd, J=11.7, 6.2 Hz, 1H), 4.58 (dd, J=11.7, 8.3 Hz, 1H), 4.17 (dd, J=12.8, 2.1 Hz, 1H), 3.99 (t, J=9.2 Hz, 1H), 3.93 (dd, J=12.9, 1.5 Hz, 1H), 3.87 (t, J=9.0 Hz, 1H), 3.54 (dt, J=9.5, 1.5 Hz, 1H), 3.26 (s, 3H), 1.27-0.97 (m, 28H). 13C NMR (126 MHz, methanol-d4): δ 158.6, 156.1, 151.3, 137.6, 137.1 (d, J=7.1 Hz), 136.7 (d, J=7.1 Hz), 129.5, 129.4, 128.9, 128.3, 116.8, 82.3, 80.9, 80.1, 76.9, 70.9 (d, J=5.9 Hz), 70.6, 70.3 (d, J=5.3 Hz), 61.9, 28.8, 18.0, 17.8, 17.7, 17.6, 14.8, 14.5, 14.1, 13.8.
To a solution of BC-34 (96 mg, 0.116 mmol, 1.00 equiv.) in THF (4 mL) at 0° C. was added TBAF (1M in THF, 300 μL, 0.30 mmol, 2.59 equiv.). After 10 min., AcOH (100 μL) was added, and the resulting solution was concentrated in vacuo. Due to poor solubility of the resulting 3′-O product in DCM/MeOH, the crude was dissolved in a mixture of ACN and minimal H2O and dry-loaded with 2 gram silica. Flash column chromatography on silica using a gradient of 5-40% MeOH in DCM was performed, affording pure 3′-O isomer (BC-35, 34 mg, 50%) and several mixed fractions containing 4 mg of 3′-O isomer (6%) and 16 mg of 2′-O isomer (BC-36, 23%). 2′-O isomer (BC-36): 1H NMR (500 MHz, methanol-d4): δ 7.99 (s, 1H), 7.38-7.20 (m, 8H), 7.10-6.99 (m, 2H), 5.69 (d, J=9.3 Hz, 1H), 4.97 (dd, J=11.8, 7.4 Hz, 1H), 4.92 (dd, J=11.9, 8.2 Hz, 1H), 4.66 (dd, J=11.9, 6.2 Hz, 1H), 4.53 (dd, J=11.9, 7.9 Hz, 1H), 3.89 (dd, J=12.4, 1.8 Hz, 1H), 3.80 (d, J=8.7 Hz, 1H), 3.74 (dd, J=12.1, 4.8 Hz, 1H), 3.65-3.51 (m, 2H), 3.26 (s, 3H). 2′-O isomer (BC-36): 13C NMR (126 MHz, methanol-d4): δ 158.6, 156.2, 151.3, 137.1 (d, J=7.3 Hz), 136.7 (d, J=7.8 Hz), 129.6, 129.5, 129.3, 128.9, 128.1, 82.0, 81.3, 79.8, 77.3, 71.2, 70.9 (d, J=5.9 Hz, 1H), 70.21 (d, J=5.8 Hz, 1H), 62.2, 28.8. 3′-O isomer (BC-35): 1H NMR (500 MHz, DMSO-d6): δ 7.94 (s, 1H), 7.43-7.28 (m, 8H), 7.09 (s, 2H), 5.87 (d, J=6.6 Hz, 1H), 5.60 (d, J=7.3 Hz, 1H), 5.30 (d, J=9.3 Hz, 1H), 5.12 (d, J=6.9 Hz, 2H), 5.10-5.03 (m, 2H), 4.74 (t, J=5.9 Hz, 1H), 4.39 (q, J=8.9 Hz, 1H), 4.23 (td, J=9.2, 6.5 Hz, 1H), 3.72 (dd, J=11.2, 5.7 Hz, 1H), 3.60-3.47 (m, 2H), 3.43 (ddd, J=11.2, 5.8, 1.8 Hz, 1H), 3.32 (s, 3H). 3′-O isomer (BC-35): 13C NMR (126 MHz, DMSO-d6): δ 156.4, 154.3, 149.7, 136.5 (d, J=3.6 Hz), 136.4 (d, J=3.8 Hz), 136.1, 128.4, 128.2, 128.1, 127.7, 115.5, 84.3 (d, J=6.7 Hz), 81.9, 79.4, 69.9, 68.5, 68.4, 68.3, 60.4, 28.1.
A suspension containing BC-35 (8 mg, mmol, 1.00 equiv.), Pd/C (13 mg, 10% w/w), AcOH (100 μL) H2O/THF (4 mL, 1:1) was sparged with Ar for 5 min. then switched to H2, through which the suspension was rapidly stirred under for hr. After sparging with Ar, the reaction mixture was filtered through celite using MeOH/H2O and the collected filtrate was concentrated to dryness in vacuo affording mgglu #2 (BC-37, 5.5 mg, quant). mgglu #X was compared to the corresponding peak in C. elegans wildtype (N2) endo-metabolome samples by HILIC-HRMS (see Figure S2c for co-elution data). Chromatographic Method E was used.
Phenylacetic acid (9.0 mg, 0.066 mmol, 2.64 equiv.) and TBTU (21 mg, 0.065 mmol, 2.60 equiv.) were added to a solution of BC-35 (15 mg, 0.025 mmol, 1.00 equiv.) in 1 mL dry pyridine. The resulting mixture was stirred for 4 hr at room temp., MeOH (1 mL) and DCM (2 mL) was added, the solution was transferred to a round-bottom flask, and then concentrated to dryness in vacuo ensuring all pyridine was removed. Flash column chromatography on silica using a gradient of 2.5-40% MeOH in DCM was performed, affording BC-38 (12.8 mg, 73%) as a white solid. 1H NMR (500 MHz, methanol-d4): δ 7.77 (s, 1H), 7.40-7.16 (m, 15H), 5.44 (d, J=9.4 Hz, 1H), 5.17-5.08 (m, 4H), 4.50 (dd, J=12.0, 1.9 Hz, 1H), 4.45 (q, J=8.7 Hz, 1H), 4.29 (dd, J=12.0, 5.6 Hz, 1H), 4.24 (t, J=9.3 Hz, 1H), 3.78 (ddd, J=10.0, 5.6, 2.2 Hz, 1H), 3.71 (t, J=9.4 Hz, 1H), 3.66 (d, J=15.2 Hz, 1H), 3.62 (d, J=15.2 Hz, 1H), 3.46 (s, 3H). 13C NMR (126 MHz, methanol-d4): δ 173.2, 158.9, 156.2, 151.4, 137.8, 137.5 (d, J=2.6 Hz), 137.4 (d, J=2.6 Hz), 135.6, 130.4, 129.6, 129.5, 129.0, 128.0, 116.9, 85.7 (d, J=6.9 Hz), 85.6, 83.9, 77.8, 71.9 (d, J=3.6 Hz), 70.8, 70.1 (d, J=3.6 Hz), 64.4, 41.8, 28.8.
A suspension containing BC-38 (12.5 mg, 0.018 mmol, 1.00 equiv.), Pd/C (17 mg, 10% w/w), AcOH (117 μL) and MeOH (2.5 mL) was sparged with Ar for 5 min. then switched to H2, through which the suspension was rapidly stirred under for 1.5 hr. After sparging with Ar, the reaction mixture was filtered through celite using MeOH/H2O and the collected filtrate was concentrated to dryness in vacuo. The dried solution was loaded onto celite in H2O and flash column chromatography on C18 using a gradient of 0-100% ACN (w/ 0.1% formic acid) in H2O (w/ 0.1% formic acid) was performed, affording mgglu #11 (BC-39, 5.0 mg, 55%) of which was found to be identical to a minor isomer peak on C18 in C. elegans wildtype (N2) and fem-3 (OE) and him-5 endo-metabolome samples by HPLC-HRMS (see Figure S3c for co-elution data). Chromatographic Method B was used.
To a solution of BC-15 (95 mg, 0.290 mmol, 1.00 equiv.) in DMF (2 mL) at 0° C. was added TIPDSiCl2 (140 μL, 0.435 mmol, 1.50 equiv.) and imidazole (93 mg, 1.36 mmol, 4.70 equiv.). The reaction mixture was stirred for 45 min at 0° C. and then diluted with DCM, followed by addition of H2O. Organics were extracted 3× with DCM, combined, and then basified using sat. aq. NaHCO3. Organics were then extracted from the aq. layer 3× with DCM, dried using Na2SO4, filtered, and concentrated in vacuo. Flash column chromatography on silica using a gradient of 5-40% MeOH in DCM was performed, affording BC-40 (70 mg, 42%) as a white solid. 1H NMR (600 MHz, methanol-d4): δ 7.84 (s, 1H), 5.42 (d, J=9.3 Hz, 1H), 4.17 (dd, J=12.6, 2.1 Hz, 1H), 4.11 (t, J=9.2 Hz, 1H), 3.97-3.88 (m, 2H), 3.61 (t, J=9.0 Hz, 1H), 3.49 (d, J=9.4 Hz, 1H), 2.94 (s, 3H), 1.30-0.90 (m, 28H). 13C NMR (126 MHz, methanol-d4): δ 159.5, 154.8, 153.5, 138.1, 117.4, 85.2, 80.8, 78.3, 73.36, 70.4, 62.1, 28.2, 18.0, 17.8, 17.8, 17.7, 17.6, 14.9, 14.5, 13.9, 13.8.
Benzylchloroformate (75 μL, 0.526 mmol, 4.28 equiv.) and DMAP (52.5 mg, 0.430 mmol, 3.41 equiv.) were added portion wise to a solution of BC-40 (72 mg, 0.126 mmol, 1.00 equiv.) in 4 mL DCM at 0° C. over a 45 min period. The resulting solution was stirred up to room temp. and stirred at that temp. for 15 min. The reaction mixture was then diluted with DCM and quenched with the addition of sat. aq. NaHCO3. The organic layer was collected and additional organics were extracted 3× with DCM. The combined organics were dried using Na2SO4, filtered, and concentrated in vacuo. Flash column chromatography on silica using a gradient of 3-30% MeOH in DCM afforded BC-41 (73 mg, 85%) as a white solid. 1H NMR (600 MHz, methanol-d4): δ 7.81 (s, 1H), 7.30-7.23 (m, 3H), 7.11-7.05 (m, 2H), 5.65 (d, J=9.4 Hz, 1H), 5.22 (br m, 1H), 5.05 (d, J=12.3 Hz, 1H), 4.91 (d, J=12.3 Hz, 1H), 4.18 (dd, J=12.8, 2.3 Hz, 1H), 4.01 (t, J=9.3 Hz, 1H), 3.95 (d, J=11.4 Hz, 1H), 3.86 (t, J=9.1 Hz, 1H), 3.57 (dt, J=9.5, 1.4 Hz, 1H), 2.89 (s, 3H), 1.24-1.00 (m, 28H). 13C NMR (126 MHz, methanol-d4): δ 159.3, 155.7, 154.8, 153.1, 137.8, 136.7, 129.6, 129.5, 128.8, 117.1, 80.9, 78.6, 75.7, 70.7, 70.4, 62.0, 28.2, 18.0, 17.8, 17.7, 17.5, 14.8, 14.5, 14.0, 13.8.
To an inhomogeneous solution of BC-41 (73 mg, 0.104 mmol, 1.00 equiv.) in DCM (3 mL) was added dibenzyl N,N-diisopropylphosphoramidite (70 μL, 0.208 mmol, 2.00 equiv.), and ImOTf (45 mg, 0.208 mmol, 2.00 equiv.). After 1 hr, an additional portion of dibenzyl N,N-diisopropylphosphoramidite (27.5 μL, 0.082 mmol, 0.78 equiv.), and ImOTf (11 mg, 0.050 mmol, 2.48 equiv.) were added and stirred for another hr. The resulting solution was cooled to 78° C. after which mCPBA (77% max, 95 mg, 0.425 mmol, 4.09 equiv.) was added. The resulting mixture was stirred up to 0° C. over a 1 hr period and was then diluted in DCM and quenched with the addition of sat. aq. NaHCO3 (10 mL). The organic layer was collected and the aqueous layer was extracted 3× with DCM (15 mL each). Combined organics were dried with Na2SO4, filtered, and concentrated in vacuo. Flash column chromatography on silica using a gradient of 3-30% MeOH in DCM was performed, affording BC-42 (88 mg, 87%) as a white solid. 1H NMR (600 MHz, methanol-d4): δ 7.86 (s, 1H), 7.38-7.30 (m, 1OH), 7.24-7.19 (m, 3H), 7.02-6.97 (m, 2H), 5.75 (d, J=8.9 Hz, 1H), 5.72-5.60 (br m, 1H), 5.02-4.95 (m, 4H), 4.94 (d, J=12.2 Hz, 1H), 4.76 (q, J=8.8 Hz, 1H), 4.66 (d, J=12.3 Hz, 1H), 4.24 (t, J=9.2 Hz, 1H), 4.17 (dd, J=12.9, 2.1 Hz, 1H), 3.98 (dd, J=12.9, 1.6 Hz, 1H), 3.64 (dt, J=9.5, 1.9 Hz, 1H), 2.90 (s, 3H), 1.16-0.87 (m, 28H). 13C NMR (126 MHz, methanol-d4): 159.3, 155.2, 154.8, 153.0, 138.0, 137.1 (d, J=6.7 Hz), 136.9 (d, J=6.5 Hz), 136.5, 129.8, 129.7, 129.6, 129.5, 129.4, 129.3, 128.8, 82.2, 82.1, 80.1, 76.3, 71.2 (d, J=5.8 Hz), 71.0 (d, J=5.7 Hz), 70.9, 69.9 (d, J=5.0 Hz), 61.8, 28.2, 18.0, 17.9, 17.8, 17.7, 17.5, 14.4, 14.3, 14.2, 14.1. HRMS (ESI) m/z: [M+H]+ calcd for C46H63N5O12PSi2+ 964.3744; found 964.3733.
To a solution of BC-42 (88 mg, 0.091 mmol, 1.00 equiv.) in THF (3.5 mL) containing AcOH (15 uL, 0.26 mmol) at 0° C. was added TBAF (1M in THF, 270 μL, 0.270 mmol, 2.97 equiv.). After stirring for 5 hr cold (cooling with an ice-water bath), additional TBAF (50 μL, 0.050 mmol, 0.55 equiv) and AcOH (5 μL) was added, and the resulting solution stirred for an additional 2 hr up to room temp. until majority of starting material was consumed. An additional portion of AcOH was added (100 μL) and the solution was then concentrated to remove THF. Flash column chromatography on silica using a gradient of 5-40% MeOH in DCM was performed, affording BC-43 (48 mg, 76%). Samples contained residual TBAF fragments (10% w/w) which were unable to completely separate with subsequent purification. 1H NMR (600 MHz, methanol-d4): δ 7.97 (s, 1H), 7.42-7.26 (m, 10H), 7.25-7.17 (m, 3H), 7.00-6.94 (m, 2H), 5.78 (d, J=9.2 Hz, 1H), 5.55-5.44 (br m, 1H), 5.11 (d, J=7.6 Hz, 1H), 5.10 (d, J=7.9 Hz, 1H), 5.00 (d, J=7.6 Hz, 2H), 4.86 (d, J=12.3 Hz, 1H), 4.75 (q, J=9.0 Hz, 1H), 4.58 (d, J=12.3 Hz, 1H), 3.95-3.88 (m, 2H), 3.80 (dd, J=12.4, 5.1 Hz, 1H), 3.69 (ddd, J=9.8, 5.1, 2.1 Hz, 1H), 2.89 (s, 3H). 13C NMR (126 MHz, methanol-d4): δ 159.3, 155.2, 154.9 153.0, 137.8, 137.2 (d, J=7.3 Hz), 137.1 (d, J=7.3 Hz), 136.3, 129.6, 129.5, 129.1, 128.9, 128.8, 116.9, 82.7 (d, J=6.8 Hz), 80.7, 76.8, 71.1 (d, J=6.0 Hz), 70.9, 69.7 (d, J=3.7 Hz), 61.9, 28.2.
Phenylacetic acid (15 mg, 0.110 mmol, 4.78 equiv.) and TBTU (32 mg, 0.100 mmol, 4.35 equiv.) were added to a solution containing BC-43 (16.5 mg, 0.023 mmol, 1.00 equiv.) and 1 mL dry pyridine. The resulting mixture was stirred for 6 hr at room temp., DCM (2 mL) and MeOH (0.5 mL) were added, the solution was transferred to a round-bottom flask, and then concentrated to dryness in vacuo ensuring all pyridine was removed. Flash column chromatography on silica using a gradient of 2.5-30% MeOH in DCM was performed, affording BC-44 (12 mg, 64%) as a colorless solid. Samples contained some residual phenylacetic acid. 1H NMR (600 MHz, methanol-d4): δ 7.70 (s, 1H), 7.39-6.91 (m, 25H), 5.73 (d, J=9.2 Hz, 1H), 5.44-5.29 (br m, 1H), 5.10 (dd, J=7.9, 3.6 Hz, 2H), 4.99 (d, J=7.6 Hz, 2H), 4.85 (m, 1H), 4.73 (q, J=8.9 Hz, 1H), 4.58 (d, J=12.3 Hz, 1H), 4.53 (dd, J=12.3, 2.1 Hz, 1H), 4.33 (dd, J=12.2, 5.3 Hz, 1H), 3.91-3.86 (m, 1H), 3.84 (t, J=9.4 Hz, 1H), 3.68 (d, J=14.9 Hz, 1H), 3.64 (d, J=14.9 Hz, 1H), 2.89 (s, 3H).
A suspension containing BC-44 (12 mg, 0.014 mmol, 1.00 equiv.), Pd/C (18 mg, 10% w/w), formic acid (200 μL) and MeOH (4 mL) was sparged with Ar for 5 min. then switched to H2, through which the suspension was rapidly stirred under for 2 hr. After sparging with Ar, the reaction mixture was filtered through celite and washed with MeOH/H2O and the collected filtrate was concentrated almost to dryness in vacuo and the resulting solution was loaded on celite. Flash column chromatography on C18 using a gradient of 1-100% ACN in H2O (w/ 0.1% formic acid) afforded mgglu #31 (BC-45, 3.5 mg, 46%). mgglu #31 was found as an isomeric peak on C18 in C. briggsae endo-metabolome samples form/z=526.1333 by HPLC-HRMS (see Figures S3c and S11 for co-elution and MS2 data, respectively). Chromatographic Method B was used.
Tyglu synthesis is achieved by coupling N-Boc-tyramine with α-D-fluoroglucose, for selective preparation of the O-linked tyramine-glucoside, as reported for the sngl syntheses, and followed by 4,6-di-O-protection using 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane. Wadzinski, et. al., Nature Chemistry 10, 644-652 (2018); Yu, J. et al. “Parallel pathways for serotonin biosynthesis and metabolism in C. elegans.” Nat Chem Biol (Accepted). 2-O-acylated tyglu derivatives is prepared via esterification (e.g. using EDC/DMAP) with different carboxylic acids, which results in preferential acylation of the 2-position, followed by 3-O-phosphorylation (e.g. as described for the syntheses of sngl #4) and subsequently deprotection (e.g. using tetrabutylammonium fluoride) to furnish the target tyglu MOGLs. 6-O-acylated tyglu is synthesized by first protecting the 2-OH in the 4,6-diprotected intermediate above with benzyl chloroformate, then 3-O-phosphate is installed using similar procedure as above. Next 6-O-esterification is achieved using esterification on the 4,6-deprotected precursor, followed by subsequent deprotections steps to furnish the target tyglu compounds.
Oglu synthesis is achieved by coupling the phenolic OH of N—O-di-Boc protected octopamine (tert-butyl (S)-(2-((tert-butoxycarbonyl)oxy)-2-(4-hydroxyphenyl)ethyl)carbamate) with α-D-fluoroglucose as above. 2-O-acylated and 6-O-acylated oglu can be produced using procedures analogous to those outlined above for the synthesis of tyglu MOGLs.
The synthesis of angl #7 is achieved by coupling the unprotected precursor angl #1 and 2-methylbutanoic acid. The synthesis of angl #8 is achieved using a procedure analogous to that outlined above for 6-O-acylated tyglu MOGLs.
To selectively synthesize angl #6, N-Boc-anthranilic acid is coupled with glucose, then the protected product is 4,6-di-O-protected using 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane. 2-OH is protected with benzyl chloroformate, followed by installation of methoxybenzyl-protected 3-O-phosphate. A deprotection/esterification sequence analogous to the above examples is performed to achieve the final product angl #6.
The following compounds have also been synthesized in a manner similar to the ones shown in the Examples above, confirmed by NMR and mass spectrometry. The structures of each are shown in Table S5.
Exemplary synthetic compounds confirmed by HRMS.
Interaction of MOGLs with the Proteasome
To investigate the cellular interactors of MOGLs, two independent approaches were applied: thermal proteome profiling (TPP) and limited proteolysis-coupled mass spectrometry (LiP-MS), to uncover binding events upon compound treatment of C. elegans lysates using MOGLs of the sngl-class as an example. Overlap of positive hits in the two assays revealed that thermal stability (as measured by TPP) and tendency toward proteolysis (as measured by LiP-MS) of proteasome alpha and beta subunits were changed upon incubation with the MOGLs sngl #1 and sngl #2 (
To obtain mechanistic insight into the binding of proteasome with sngl #1/sngl #2, LiP-MS data were analyzed to reveal peptides around sub-structures of proteins where the binding occurs. We used AlphaFold to predict structures and found that the differential peptides generally resided in or close to the two center layers of beta sheets in a single proteasome subunit, which is likely to affect proper folding of these proteins (
C. elegans lysate preparation and compound incubation. Synchronized pah-1(syb3596); tph-1(mg280) double knockout C. elegans were grown with ΔtnaA E. coli (JW3686-7) for two generations; growth conditions and harvesting procedures were as described in the ‘Nematode cultures’ section above. Worms were lysed by sonication with lysis buffer (25 mM Tris pH 7.5, 150 mM NaCl, 1.5 mM MgCl2.1 mM phenylmethylsulfonyl fluoride, 1× protease inhibitor cocktail (Roche), 1 mM sodium fluoride) for 5 min (3 s on/off pulse cycle at Amp 100). Protein concentration was determined by Bradford assay (MilliporeSigma) and diluted to 1 mg/mL by lysis buffer. To each aliquot of lysate was added compound (sngl #1, sngl #2, N-acetylserotonin, and solvent (ethanol) control (Mock)) to a final concentration of 0.1 mM and the mixtures were shaken for 15 min at room temperature.
Thermal proteome profiling (TPP) sample preparation. Methods were modified from published protocol (Franken, H., T. et al. Nature Protocols 10(10): 1567-1593). Lysate-compound mixture were aliquoted into 0.2-mL PCR tubes, heated at specified temperatures for 3 min using a Thermal Cycler (BioRad), centrifuged at 16,000 g at 4° C. for 5 min, and the supernatant were transferred into Eppendorf tubes. Four volumes of prechilled (−20° C.) acetone were added to each sample for precipitation of proteins overnight. Samples were centrifuged at 16,000 g at 4° C. for 20 min, washed once by methanol, and the resulting protein pellets were dissolved in urea buffer (50 mM ammonium bicarbonate, 2 M thiourea, 6 M urea). To digest proteins for mass spectrometry analysis, proteins were first reduced by dithiothreitol (final concentration 5 mM) for 30 min at room temperature, alkylated by iodoacetamide (final concentration 15 mM) for 30 min at room temperature in the dark, digested by LysC/Trypsin Mix (Promega) (enzyme/substrate=1/30, w/w) based on manufacturer's instructions. Samples were acidified with trifluoroacetic acid to pH ˜3, and desalted in a C18 96-well Plate (Sep-Pak, Waters), dried by a Speedvac. Peptides were measured by nanoHPLC-MS/MS by standard methods.
Limited proteolysis-coupled mass spectrometry (LiP-MS) sample preparation. Methods were modified from published protocol (Schopper, S., A. et al. Nature Protocols 12(11): 2391-2410). Lysate-compound mixture were aliquoted into 0.2-mL PCR tubes, proteinase K (enzyme/substrate=1/100, w/w) was added, incubated at 25° C. for 5 min in a Thermal Cycler (BioRad), heated at 98° C. for 5 min for irreversible denaturation of proteinase K, and stored in −20° C. overnight. Procedures for protein digestion and peptide desalting for mass spectrometry analysis were the same as described in ‘Thermal proteome profiling (TPP) sample preparation’ section above.
As described herein, MOGL biosynthesis is strongly upregulated during starvation. When testing whether MOGL production via CEST-1.2 is required for starvation survival, the results show that lifespan of starved cest-1.2 mutant adults was significantly reduced compared to wildtype. (
This experiment found that MOGL production via CEST-1.2 is required for normal lifespan, which suggest that MOGL production protects regulates kinase-dependent stress response pathways, such as oxidative stress, thermal stress, e.g. via binding to kinases and other components of these signaling pathways (
We further found that mutants lacking MOGL production via CEST-1.2 or CEST-2.1 are more sensitive to the oxidant juglone (tested at 300 μM) compared to wildtype, suggesting that the modular glucosides are protective by modulating the oxidative stress response. When one family of MOGLs, the indole-containing glucosides (iglu's), were tested it was found that C. elegans fed a diet of E. coli that is unable to produce indole (ΔtnaA)—and thus is unable to produce iglu-style MOGLs—is indeed more sensitive to 300 μM juglone exposure, suggesting that iglu-type MOGLs modulate the oxidative stress response in C. elegans. For the purpose of this experiment, all animals were grown on normal media seeded with either E. coli K12 or ΔtnaA bacteria until reaching adulthood (adult day 1). The assay was performed on freshly made media supplemented with 300 μM juglone (Sigma #H47003) and seeded with E. coli OP50 bacteria. Animals were scored as dead or alive every 2 hours, for a total of 10 hours. Death was measured as a failure to respond to respond to a gentle touch. See
To evaluate the effects of MOGLs in primary human tissues and to demonstrate the relative activities of the MOGLs described herein for the treatment of specific human diseases, pure samples of each compound in Table S5 are synthesized according to the procedures described herein and evaluated using the BioMAP® Phenotypic Profiling Assay system. See for example: Kim, et. al., Cell Chemical Biology, 27:6, 698-707 (2020).
MOGLs are screened in vitro against using a panel of 12 human primary cell-based co-culture systems (venular endothelial cells, lung fibroblasts, and peripheral blood mononuclear cells, PBMCs) that model various tissues and diseases. Protein biomarker readouts in these mixed cell systems are used to quantify the effects of the MOGLs.
Screening is conducted with the BioMAP® Diversity PLUS assay performed by DiscoverX. Human primary cells in BioMAP systems are used at early passage (passage 4 or earlier) to minimize adaptation to cell culture conditions and preserve physiological signaling responses. All cells are from a pool of multiple donors (n=2-6), commercially purchased and handled according to the recommendations of the manufacturers. Human blood derived CD14+ monocytes are differentiated into macrophages in vitro before being added to the/Mphg system. Abbreviations are used as follows: Human umbilical vein endothelial cells (HUVEC), Peripheral blood mononuclear cells (PBMC), Human neonatal dermal fibroblasts (HDFn), B cell receptor (BCR), T cell receptor (TCR) and Toll-like receptor (TLR). Cell ty pes and stimuli used in each system are as follows: 3C system [HUVEC+(IL-1β, TNFα and IFNγ)], 4H system [HUVEC+(IL-4 and histamine)], LPS system [PBMC and HUVEC+LPS (TLR4 ligand)], SAg system [PBMC and HUVEC+TCR ligands (1×)], BT system [CD19+ B cells and PBMC+(α-IgM and TCR ligands (0.001×)], BF4T system [bronchial epithelial cells and HDFn+(TNFα and IL-4)], BE3C system [bronchial epithelial cells +(IL-1β, TNFα and IFNγ)], CASM3C system [coronary artery smooth muscle cells +(IL-1β, TNFα and IFNγ)], HDF3CGF system [HDFn+(IL-1β, TNF∘, IFNγ, EGF, bFGF and PDGF-BB)], KF3CT system [keratinocytes and HDFn+(IL-1β, TNFα and IFNγ)], MyoF system [differentiated lung myofibroblasts+(TNFα and TGFβ)] and /Mphg system [HUVEC and M1 macrophages+Zymosan (TLR2 iigand)]. Systems are derived from either single cell types or co-culture systems. Adherent cell types are cultured in 96- or 384-well plates until confluence, followed by the addition of PBMC (SAg and LPS systems). The BT system consists of CD19+B cells co-cultured with PBMC and stimulated with a BCR activator and low levels of TCR stimulation. MOGLs are prepared in DMSO (final concentration ≤0.1%) and added at a final concentration of 21 μM, 1 h before stimulation and remain in culture for 24 h (48 h: MyoF system; 72 h: BT system (soluble readouts); 168 h: BT system (secreted IgG)). Each plate contains drug controls, negative controls (e.g., non-stimulated conditions) and vehicle controls (e.g., 0.1% DMSO) appropriate for each system. Direct ELISA is used to measure biomarker levels of cell-associated and cell membrane targets. Soluble factors from supernatants are quantified using either HTRF@R detection, bead-based multiplex irnmunoassay or capture ELISA. Overt adverse effects of test agents on cell proliferation and viability (cytotoxicity) are detected by sulforhodamine B (SRB) staining, for adherent cells, and alamarBlue® reduction for cells in suspension. For proliferation assays, individual cell types are cultured at sub-confluence and measured at time points optimized for each system (48 h: 3C and CASM3C systems; 72 h: BT and HDF3CGF systems; 96 h: SAg system). Cytotoxicity for adherent cells is measured by SRB (24 h: 3C, 4H, LPS, SAg, BF4T, BE3C, CASM3C, HDF3CGF, KF3CT, /Mphg systems; 48 h: MyoF sy stem), and by alamarBlue staining for cells in suspension (24 h: SAg system; 42 h: BT system) at the time points indicated.
Results from the MOGL screening assays described above are analyzed as follows: Biomarker measurements in a MOGL-treated sample are divided by the average of control samples (at least 6 vehicle controls from the same plate) to generate a ratio that is then log 10 transformed. Significance prediction envelopes are calculated using historical vehicle control data at a 95% confidence interval. The results are further interpreted through Profile-, Benchmark-, Similarity- and Cluster Ananlyses as described below:
Profile Analysis. Bioactivities are confirmed when 2 or more consecutive MOGL concentrations change in the same direction relative to vehicle controls, are outside of the significance envelope, and have at least one concentration with an effect size >20% (log 10 ratiol >0.1). Biomarker key activities are described as modulated if these activities increase in some systems, but decrease in others. Cytotoxic conditions are noted when total protein levels decrease by more than 50% (log 10 ratio of SRB or alamarBlue levels <−0.3). A MOGL is considered to have broad cytotoxicity when cytotoxicity is detected in 3 or more systems. Concentrations of MOGLs with detectable broad cytotoxicity are excluded from biomarker activity annotation and downstream benchmarking, similarity search and cluster analysis. Antiproliferative effects of tested MOGLs are defined by an SRB or alamarBlue log 10 ratio value <−0.1 from cells plated at a lower density. Cytotoxicity and antiproliferative arrows only require one concentration to meet the indicated threshold for profile annotation.
Benchmark Analysis. Common biomarker readouts are noted when the readout for both profiles are outside of the significance envelope with an effect size >20% in the same direction. Differentiating biomarkers are annotated when one profile has a readout outside of the significance envelope with an effect size >20%, and the readout for the other profile is either inside the envelope or in the opposite direction.
Similarity Analysis. Common biomarker readouts are noted when the readout for both profiles is outside of the significance envelope with an effect size >20% in the same direction.
Concentrations of MOGLs that have 3 or more detectable systems with cytotoxicity are excluded from similarity analysis. Concentrations of MOGLs that have 1-2 systems with detectable cytotoxicity are included in the similarity search analysis, along with an overlay of the database match with the top concentration of the test agent.
Cluster Analysis. Cluster analysis (function similarity map) uses the results of pairwise correlation analysis to project the “proximity” of MOGL activity profiles from multi-dimensional space into two dimensions. Functional clustering of the MOGL profiles are generated during this analysis using Pearson correlation values for pairwise comparisons of the profiles for each agent at each concentration, and then subjects the pairwise correlation data to multidimensional scaling. MOGLs that do not cluster with one another are interpreted as mechanistically distinct.
To assess the activity of MOGLs in inhibiting proteasome activity, an assay is undertaken to measure the accumulation of undegradable undegradable polyubiquitinated proteins in by measuring the size and/or abundance of nuclear aggregations of ubiquitinated proteins termed “aggresomes”, using a cell- and imaging-based screening system adapted from a method reported in Marine Drugs, 2018 October; 16(10): 395. DOI:10.3390/mnd16100395.
Synthetic samples of each of the MOGLs in Table S5 are diluted at 10-fold intervals between 10 nM and 1 mM.
The controls and MOGLs are diluted and dispensed into culture plates at the concentrations noted above along with two known proteasome inhibitors as positive controls (Bortezomib, 13.1 nM and 7.76 nM; and MG132, 0.97 μM and 1.65 μM).
HEK293T cells transiently expressing EGFP-UL76 are seeded at 1×106 cells onto 6-cm culture dishes one day before transfection. Then, 3 μg of plasmid DNA pEGFP-UL76 is transfected into HEK293T cells mediated by Lipofectanine Plus and Lipofectamine (Thermo Fisher Scientific, Waltham, MA, USA). After 3 h of transfection, the transfected cells are trypsinized and dispensed into black glass-bottom 96-well plates at 1×104 cells per well in a volume of 200 μL per well, including the indicated compound at each concentration with three repeats. The culture plates containing the cells and tested compounds are incubated at 5% CO2 and 37° C. for 48 h. Subsequently, the cells are fixed in 1% paraformaldehyde for 10 min and simultaneously permeabilized with 0.1% IGEPAL® CA-630, then stained with 1.5 μg/mL DAPI on ice for 30 min. After extensive washing with PBS, the cells are submerged in PBS, sealed in the dark, and stored at 4 CC.
Image acquisition is accomplished using an ImageXpress Micro Widefield HTCJS system (Molecular Device, San Jose, CA, USA) under an objective magnification of 20×Ph1. Each well is acquired in 25 consecutive images in 5×5 sites with 38% well area coverage. Two modules of MetaExpress, Cell Scoring and Multi-Wavelength Cell Scoring, are employed to analyze the high-content measurements. Cell Scoring is configured to define nuclei marked by 4′ 6-diamidino-2-phenylindole (DAPI) staining with diameters of 8 to 15 μm, whereas EGFP-UL76 aggresomes have diameters of 1 to 50 μm. The intensity of the above background was determined according to the manufacturer's instructions. Multi-Wavelength Cell Scoring was configured to classify aggresomes by size into pit and vesicle categories. The pit category contained aggresomes with diameters of 1 μm to 5 μm, whereas the vesicle category contained aggresomes with diameters of 5 μm to 50 μm. The data are compiled into cell-by-cell and site-by-site measurements. The relative ratios are calculated by normalization to the value of the control without MOGL treatment.
Ratios are calculated by comparison of the aggresome characteristics of MOGL treated cells to the control value obtained without MOGL treatment. 1 MOGL treatments showing statistically-relevant lose-dependent increases the number and/or size of aggresomes relative to the negative control are confirmed to have proteasome inhibitory activity.
Tables S4a and S4b list differential metabolites from C. elegans and C. briggsae that are more than 50-fold reduced or abolished in Cel-cest-1.2 or Cbr-cest-2 mutants compared to C. elegans wildtype (N2) or C. briggsae wildtype (AF 16), respectively. Columns include: m/z detected in both ESI− and ESI+ mode, retention time, small molecule identifier ((SMID) at www.SMID-DB.org), predicted molecular formula, detected MS/MS fragments in ESI− and ESI+ mode, the putative molecular moieties attached to the hexose core (all entries in the list contain a putative phosphate moiety), and the abundances of each metabolite in Cel-cest1. 2 or Cbr-cest-2 compared to C. elegans wildtype (N2) or C. briggsae wildtype (AF16), respectively (“Fold over wildtype”).
While we have described a number of embodiments of this invention, it is apparent that our basic examples may be altered to provide other embodiments that utilize the compounds and methods of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example.
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The present application claims priority to U.S. Provisional Application No. 63/237,811, filed Aug. 27, 2021, which is incorporated herein by reference in its entirety.
This invention was made with Government support under Grant Nos. R35GM131877 and U2CES030167 awarded by the National Institutes of Health. The United States Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US22/41757 | 8/26/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63237811 | Aug 2021 | US |
