Cannabinoid analogs and methods for their preparation

Abstract

To provide novel cannabinoid compounds useful for human therapeutic indications.SOLUTION: Provided herein are cannabinoid analogs, including halogenated cannabinoid analogs, hydroxylated cannabinoid analogs, deuterated cannabinoid analogs, and tritiated cannabinoid analogs. The cannabinoid analogs can be prepared by partial or total expression in modified host cells, such as recombinantly modified yeast cells, optionally in combination with chemical synthetic steps.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 753,708, filed October 31, 2018, and U.S. Provisional Patent Application No. 62 / 767,447, filed November 14, 2018, which applications are incorporated herein by reference in their entireties. [Background technology]

[0002] Background of the Invention Varieties of hemp (Cannabis sativa) are widely cultivated and utilized worldwide for several purposes. The stems, branches, and leaves are used for fiber and fiber-based products; the buds and seeds for food; the seeds for inexpensive oil; the flowers for aromatic, recreational, ceremonial, and medicinal uses; and the flowers and roots for nutritional and additional medical and pharmaceutical uses. Indeed, numerous controlled clinical trials and anecdotal or open-label studies in humans have documented the beneficial effects of both plant extracts and purified hemp plant compounds in a number of human medical conditions. The beneficial activities of compounds from the cannabinoid family described in human studies range from neurological disorders to mood / behavioral disorders, gastrointestinal disorders, and problems with sleep, appetite, and fatigue. Other uses or potential uses include the treatment of various microbial and viral infections and some cancers. Summary of the Invention

[0003] Brief Summary of the Invention Compounds of Formula I TIFF2025170350000002.tif20128, and salts and cannabinoid derivatives thereof, are provided herein: During the ceremony, R 1 is C1~C 20 Haloalkyl, C1-C 20 Hydroxyalkyl, deuterated C1-C 20 Alkyl, tritiated C1-C 20Alkyl, and C2-C 20 alkenyl, R 2 COOR 2a and H, R 2a is selected from the group consisting of C1-C6 alkyl and H; R 3 is selected from the group consisting of H and a prenyl moiety.

[0004] In some embodiments, the cannabinoid derivative is a cannabidiolic acid analog, a cannabidiol analog, a Δ 9 -Tetrahydrocannabinolic acid analogues, Δ 8 -Tetrahydrocannabinolic acid analogues, cannabichromene acid analogues, cannabichromene analogues, cannabinol analogues, cannabinodiol analogues, cannabinolic acid analogues, cannabivarin analogues, cannabivarinic acid analogues, Δ 9 -tetrahydrocannabivarin analogues, Δ 8 -tetrahydrocannabivarin analogues, Δ 9 -tetrahydrocannabivaric acid analogues, Δ 8 - a tetrahydrocannabivarin analog, a cannabigerovarin analog, a cannabigerovarin acid analog, a cannabichromevarin analog, a cannabichromevarinic acid analog, a cannabidivarin analog, a cannabidivarinic acid analog, a cannabiditriol analog, or a cannabicyclol analog.

[0005] Compound of Formula IV Also provided herein are methods for producing TIFF2025170350000003.tif20128, or a salt thereof, In the formula, R 1 is C1~C 20 Haloalkyl, C1-C 20 Hydroxyalkyl, deuterated C1-C 20 Alkyl, tritiated C1-C 20 Alkyl, and C2-C 20 alkenyl, The method comprises reacting a thioester of formula II culturing the modified recombinant host cell in a medium containing TIFF2025170350000004.tif12128; In the formula, R 4 is selected from the group consisting of a coenzyme A (CoA) moiety, a pantetheine moiety, and a cysteamine moiety; the modified recombinant host cell i. thioester of formula II and malonyl-CoA to a tetraketide of formula III a first polynucleotide encoding a synthase that converts ii. A second polynucleotide encoding a 2-alkyl-4,6-dihydroxybenzoate cyclase that converts the tetraketide of formula III to a compound of formula IV. and The modified recombinant host cells are cultured under conditions whereby the products encoded by the first and second polynucleotides are expressed to produce the compound of Formula IV.

[0006] Compounds of formula IV can be converted to several neutral and acidic cannabinoid analogs by the methods described herein. [Brief explanation of the drawings]

[0007] [Figure 1] 1 shows an example of a synthetic route for the preparation of cannabinoid analogs of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0008] Detailed Description of the Invention The present disclosure provides novel cannabinoid compounds useful in several human therapeutic indications, including neurological conditions, mood / behavioral disorders, infectious diseases, and cancer. Methods for the production of pharmaceutical-grade cannabinoids using sustainable, modern biopharmaceutical preparation methods are also provided.

[0009] I. Definition Unless otherwise defined, all technical terms, notations, and other scientific terminology used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this application pertains. In some cases, terms having commonly understood meanings have been defined herein for clarity and / or ease of reference, and the inclusion of these terms herein should not necessarily be construed as representing a substantial difference from the meaning commonly understood in the art.

[0010] As used herein, the terms "cannabinoid," "cannabinoid compound," and "cannabinoid product" are used interchangeably to refer to molecules containing a polyketide moiety, such as olivetolic acid or another 2-alkyl-4,6-dihydroxybenzoic acid, and a terpene-derived prenyl moiety, such as a geranyl group. The geranyl group is derived from the diphosphate ester of geraniol, known as geranyl pyrophosphate or geranyl diphosphate, which can react with olivetolic acid-type compounds to form the acidic cannabinoid cannabigerolic acid (CBGA) and CBGA analogs, as shown in Figure 1. CBGA can be enzymatically converted (e.g., by decarboxylation via in vivo or in vitro enzymatic treatment to form the neutral cannabinoid cannabigerol) and chemically converted (e.g., by heating) to additional bioactive cannabinoids. TIFF2025170350000006.tif38128

[0011] The term cannabinoid includes acidic cannabinoids and neutral cannabinoids. The term "acidic cannabinoid" refers to a cannabinoid that has a carboxylic acid moiety. The carboxylic acid moiety may be in the protonated form (i.e., as -COOH) or in the deprotonated form (i.e., the carboxylate ion -COO - Examples of acidic cannabinoids include cannabigerolic acid, cannabidiolic acid, cannabichromenic acid, and Δ 9The term "neutral cannabinoid" does not include a carboxylic acid moiety (i.e., the moieties -COOH or -COO - Examples of neutral cannabinoids are cannabigerol, cannabidiol, cannabichromene, and Δ 9 -including but not limited to tetrahydrocannabinol.

[0012] The term "2-alkyl-4,6-dihydroxybenzoic acid" refers to a compound having the following structure: TIFF2025170350000007.tif18128, In the formula, R is C1 to C 20 The alkyl group may be halogenated, hydroxylated, deuterated, and / or tritiated as described herein. Examples of 2-alkyl-4,6-dihydroxybenzoic acids include, but are not limited to, olivetolic acid (i.e., 2-pentyl-4,6-dihydroxybenzoic acid; CAS Registry Number 491-72-5) and divalanic acid (i.e., 2-propyl-4,6-dihydroxybenzoic acid; CAS Registry Number 4707-50-0). Olivetolic acid analogs include other 2-alkyl-4,6-dihydroxybenzoic acid derivatives and substituted resorcinols, including, but not limited to, 5-halomethylresorcinol, 5-haloethylresorcinol, 5-halopropylresorcinol, 5-halohexylresorcinol, 5-haloheptylresorcinol, 5-halooctylresorcinol, and 5-halonylresorcinol.

[0013] The term "alkyl," by itself or as part of another substituent, means a straight-chain or branched saturated aliphatic group. Alkyl can have any number of carbons, e.g., C 1~2 , C 1~3 , C 1~4 , C 1~5 , C 1~6 , C 1~7 , C 1~8 , C 1~9 , C1~10 , C 2~3 , C 2~4 , C 2~5 , C 2~6 , C 3~4 , C 3~5 , C 3~6 , C 4~5 , C 4~6 , and C 5~6 For example, C 1~6 Alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl may also refer to alkyl groups having up to 20 carbons, such as, but not limited to, heptyl, octyl, nonyl, decyl, etc.

[0014] The term "alkenyl," by itself or as part of another substituent, means an alkyl group, as defined herein, containing one or more carbon-carbon double bonds. Examples of alkenyl groups include, but are not limited to, vinyl (i.e., ethenyl), crotyl (i.e., but-2-en-1-yl), penta-1,3-dien-1-yl, and the like. Alkenyl moieties may be further substituted, for example, with aryl substituents (e.g., phenyl or hydroxyphenyl in the case of 4-hydroxystyryl).

[0015] The terms "halogen" and "halo," by themselves or as part of another substituent, mean a fluorine, chlorine, bromine, or iodine atom.

[0016] The term "haloalkyl," by itself or as part of another substituent, means an alkyl group in which some or all of the hydrogen atoms have been replaced with halogen atoms. Like alkyl groups, haloalkyl groups can have any suitable number of carbon atoms, e.g., C 1~6For example, haloalkyl includes trifluoromethyl, fluoromethyl, etc. In some cases, the term "perfluoro" is used to define a compound or group in which all hydrogens have been replaced with fluorines. For example, perfluoromethyl means 1,1,1-trifluoromethyl.

[0017] The term "hydroxyalkyl," by itself or as part of another substituent, means an alkyl group in which some or all of the hydrogen atoms have been replaced with hydroxyl groups (i.e., -OH groups). Similar to alkyl and haloalkyl groups, hydroxyalkyl groups can have any suitable number of carbon atoms, e.g., C 1~6 may have.

[0018] The term "deuterated" refers to the substitution of one or more deuterium atoms for one or more hydrogen atoms (i.e. 2 It means a substituent (such as an alkyl group) having a H atom.

[0019] The term "tritiated" refers to the substitution of one or more tritium atoms for one or more hydrogen atoms (i.e. 3 It means a substituent (such as an alkyl group) having a H atom.

[0020] The term "prenyl moiety" refers to a substituent containing at least one methylbutenyl group (e.g., a 3-methylbut-2-en-1-yl group). Prenyl moieties are often biochemically synthesized from isopentenyl pyrophosphate and / or isopentenyl diphosphate to provide terpene natural products and other compounds. Examples of prenyl moieties include, but are not limited to, prenyl (i.e., 3-methylbut-2-en-1-yl), isoprenyl (i.e., 3-methylbut-3-en-1-yl), geranyl, myrcenyl, ocimenyl, farnesyl, and geranylgeranyl.

[0021] The term "geraniol" refers to (2E)-3,7-dimethyl-2,6-octadien-1-ol (CAS Registry Number 106-24-1). The term "geranylation" refers to the covalent attachment of a 3,7-dimethyl-2,6-octadien-1-yl group to a molecule such as 2-alkyl-4,6-hydroxybenzoic acid. As described herein, geranylation can be performed chemically or enzymatically. The term "citral" refers to 3,7-dimethylocta-2,6-dienal.

[0022] "Organic solvent" means a carbon-containing substance that is liquid at ambient temperature and pressure and is substantially free of water. Examples of organic solvents include, but are not limited to, toluene, methylene chloride, ethyl acetate, acetonitrile, tetrahydrofuran, benzene, chloroform, diethyl ether, dimethylformamide, dimethyl sulfoxide, and petroleum ether.

[0023] The term "acid" refers to a substance capable of donating a proton (i.e., hydrogen cation) to form the conjugate base of the acid. Examples of acids include, but are not limited to, mineral acids (e.g., hydrochloric acid, sulfuric acid, etc.), carboxylic acids (e.g., acetic acid, formic acid, etc.), and sulfonic acids (e.g., methanesulfonic acid, p-toluenesulfonic acid, etc.).

[0024] As used herein, the term "treatment" refers to any indication of successful treatment or alleviation of an injury, condition, state, or symptom (e.g., pain), including any objective or subjective parameter, such as remission; relief; reduction of symptoms or an increase in the patient's tolerance of the symptom, injury, condition, or state; or a decrease in the frequency or duration of the symptom or state. Treatment or alleviation of symptoms can be based on any objective or subjective parameter, including, for example, the results of a physical examination.

[0025] As used herein, the term "administering" refers to oral, topical, parenteral, intravenous, intraperitoneal, intramuscular, intralesional, intranasal, subcutaneous, pulmonary, or intrathecal administration to a subject, as well as administration as a suppository or implantation of a slow release device, e.g., a mini-osmotic pump, in a subject.

[0026] The term "effective amount" used herein refers to the dosage that produces the therapeutic effect that it is intended to administer.The exact dosage depends on the purpose of treatment and can be determined by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0027] Throughout this specification and the appended claims, the term "comprise" or variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0028] The term "identical" or "percent identity" in reference to two or more polypeptide sequences refers to two or more sequences or subsequences that, when compared and aligned for maximum correspondence over a comparison window or predetermined region, have a specified percentage of amino acid residues that are the same or identical over a specified region (e.g., at least 70%, at least 75%, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity). Alignment to determine percent amino acid sequence identity can be performed in a variety of ways, including using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or Geneious software. An example of a suitable algorithm for determining percent sequence identity and percent sequence similarity is the BLAST 2.0 algorithm described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990). Thus, BLAST 2.0 can be used with the default parameters described to determine percent sequence identity.

[0029] As used herein, " conservative " substitution refers to the substitution of amino acids that maintain the charge, hydrophobicity, and / or size of side chain group.Exemplary sets of amino acids that can be substituted for each other include: (i) positively charged amino acids such as Lys, Arg, and His; (ii) negatively charged amino acids such as Glu and Asp; (iii) aromatic amino acids Phe, Tyr, and Trp; (iv) nitrogen ring amino acids His and Trp; (v) large aliphatic non-polar amino acids Val, Leu, and Ile; (vi) slightly polar amino acids Met and Cys; (vii) small side chain amino acids Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gln, and Pro; (viii) aliphatic amino acids Val, Leu, Ile, Met, and Cys; and (ix) small hydroxyl amino acids Ser and Thr.In this paragraph, the reference to the charge of amino acid refers to the charge at physiological pH.

[0030] In certain cases, abbreviations are used. For example, the term "CBGA" means cannabigerolic acid. Similarly, "OA" means olivetolic acid; "CBG" means cannabigerol; "CBDA" means cannabidiolic acid; "CBD" means cannabidiol; and "THC" means Δ 9 -Tetrahydrocannabinol (Δ 9 -THC); 8 -THC" is Δ 8 - means tetrahydrocannabinol; "THCA" means Δ 9 -Tetrahydrocannabinolic acid (Δ 9 -THCA); 8 -THCA" is Δ 8 - means tetrahydrocannabinolic acid; "CBCA" means cannabichromenic acid; "CBC" means cannabichromene; "CBN" means cannabinol; "CBND" means cannabinodiol; "CBNA" means cannabinolic acid; "CBV" means cannabivarin; "CBVA" means cannabivarinic acid; "THCV" means Δ9 -Tetrahydrocannabivarin (Δ 9 -THCV); 8 -THCV" is Δ 8 - means tetrahydrocannabivarin; "THCVA" means Δ 9 -Tetrahydrocannabivaric acid (Δ 9 -THCV); 8 -THCVA" is Δ 8 -tetrahydrocannabivaric acid; "CBGV" means cannabigerovarin; "CBGVA" means cannabigerovarinic acid; "CBCV" means cannabichromevarin; "CBCVA" means cannabichromevaric acid; "CBDV" means cannabidivarin; "CBDVA" means cannabidivarin; "MPF" means multiple precursor feeding; "PKS" means polyketide synthase; "GOT" means geranyl pyrophosphate:olivetolic acid geranyltransferase; "YAC" means yeast artificial chromosome; "IRES" or "internal ribosome entry site" means a specialized sequence that directly promotes ribosome binding and mRNA translation independent of the cap structure; "HPLC" means high performance liquid chromatography.

[0031] As used in this specification and the appended claims, the singular forms "a," "and," and "the" include plural referents unless the context clearly dictates otherwise.

[0032] As used herein, the terms "about" and "approximately," when used to modify a particular numerical value, indicate a closed range surrounding that number. For example, if the value is "X," then "about X" or "approximately X" would indicate a value of 0.9X to 1.1X, such as a value of 0.95X to 1.05X, or a value of 0.98X to 1.02X, or a value of 0.99X to 1.01X. Any reference to "about X" or "approximately X" specifically denotes at least the value X, 0.9X, 0.91X, 0.92X, 0.93X, 0.94X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, 1.05X, 1.06X, 1.07X, 1.08X, 1.09X, and 1.1X, as well as values ​​within this range.

[0033] The molecular biology techniques and procedures described or referenced herein, using conventional methodologies such as the widely used molecular cloning methodologies described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, are generally well understood and commonly used by those of skill in the art. Unless otherwise specified, procedures, including the use of commercially available kits and reagents, as appropriate, are generally performed in accordance with manufacturer-defined protocols and / or parameters. Therefore, before describing the present methods, expression systems, and uses, it is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, constructs, and reagents described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is limited only by the appended claims.

[0034] II. Cannabinoid Analogues Compounds of Formula I TIFF2025170350000008.tif20128, and salts and cannabinoid derivatives thereof, are provided herein; During the ceremony, R 1 is C1~C 20 Haloalkyl, C1-C 20 Hydroxyalkyl, deuterated C1-C 10 Alkyl, tritiated C1-C 20 Alkyl, and C2-C 20 alkenyl, R 2 COOR 2a and H, R 2a is selected from the group consisting of C1-C6 alkyl and H; R 3 is selected from the group consisting of H and a prenyl moiety.

[0035] In some embodiments, R 1 is C1~C 20 Haloalkyl (e.g., C1-C 15 Haloalkyl or C1-C 10 haloalkyl). For example, R 1 can be haloethyl (containing 1-5 halogen atoms), halopropyl (containing 1-7 halogen atoms), halobutyl (containing 1-9 halogen atoms), halopentyl (containing 1-11 halogen atoms), halohexyl (containing 1-13 halogen atoms), haloheptyl (containing 1-15 halogen atoms), halooctyl (containing 1-17 halogen atoms), and halonyl (containing 1-19 halogen atoms). Examples of haloalkyl groups include, but are not limited to, chloromethyl, dichloromethyl, trichloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trichloroethyl, 2,2,2-trifluoroethyl, pentachloroethyl, pentafluoroethyl, 1,1,1,3,3,3-hexachloropropyl, 1,1,1,3,3,3-hexafluoropropyl, and the like. In some embodiments, R 1 is C1~C10 Fluoroalkyl, C1-C 10 Chloroalkyl, C1-C 10 Bromoalkyl, and C1-C 10 In some embodiments, R is selected from iodoalkyl. 1 is C1~C 10 Fluoroalkyl, C1-C 10 Chloroalkyl, and C1-C 10 In some embodiments, R is selected from bromoalkyl. 1 is C1~C 10 It is a fluoroalkyl.

[0036] In some embodiments, R 1 is selected from fluoroethyl (containing 1 to 5 fluorine atoms), fluoropropyl (containing 1 to 7 fluorine atoms), fluorobutyl (containing 1 to 9 fluorine atoms), fluoropentyl (containing 1 to 11 fluorine atoms), fluorohexyl (containing 1 to 13 fluorine atoms), fluoroheptyl (containing 1 to 15 fluorine atoms), fluorooctyl (containing 1 to 17 fluorine atoms), and fluorononyl (containing 1 to 19 fluorine atoms).

[0037] In some embodiments, R 1 is selected from 3-fluoropropyl; 3,3,3-trifluoropropyl; 1,1-difluoropropyl; perfluoropropyl; 4-fluorobutyl; 1,1-difluorobutyl; perfluorobutyl; 5-fluoropentyl; 1,1-difluoropentyl; and perfluoropentyl.

[0038] In some embodiments, R 1 is selected from the group consisting of 3-fluoropropyl, 4-fluorobutyl, and 5-fluoropentyl.

[0039] In some embodiments, R 1is selected from the group consisting of 3-chloropropyl, 3-bromopropyl, 3-hydroxypropyl, 4-chlorobutyl, 4-bromobutyl, 4-hydroxybutyl, 5-chloropentyl, 5-bromopentyl, 5-hydroxypentyl, 6-chlorohexyl, 6-bromohexyl, and 6-hydroxyhexyl. 1 is perdeutero-pentyl (i.e., -C5D 11 )

[0040] In some embodiments, R 2 is COOH. R 2 is COOH and R 3 Compounds of formula I where R is H include 1 is halopentyl, hydroxypentyl, deuterated pentyl, or tritiated pentyl.

[0041] In some embodiments, R 2 is H. R 2 is H and R 3 Compounds of formula I where R is H include 1 Olivetol analogues in which is halopentyl, hydroxypentyl, deuterated pentyl, or tritiated pentyl are also included.

[0042] In some embodiments, R 2 COOR 2a and R 2a is C1-C6 alkyl. For example, R 2a can be methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, or branched hexyl.

[0043] In some embodiments, 2,4-dihydroxy-6-perdeuteropentylbenzoic acid, 2,4-dihydroxy-6-(5-fluoropentyl)benzoic acid, 2,4-dihydroxy-6-(4-fluorobutyl)benzoic acid, 6-(4-chlorobutyl)-2,4-dihydroxybenzoic acid, and / or 2,4-dihydroxy-6-(3-fluoropropyl)benzoic acid are provided. In some embodiments, 5-perdeuteropentylbenzene-1,3-diol, 5-(5-fluoropentyl)benzene-1,3-diol, 5-(4-fluorobutyl)benzene-1,3-diol, 5-(4-chlorobutyl)benzene-1,3-diol, and / or 5-(3-fluoropropyl)benzene-1,3-diol are provided.

[0044] In some embodiments, R 3 is the prenyl moiety. For example, R 3 can be prenyl (i.e., 2-methylbut-2-en-1-yl), geranyl (i.e., 3,7-dimethylocta-2,6-dien-1-yl), farnesyl (i.e., 3,7,11-trimethyldodeca-2,6,10-trien-1-yl), or geranylgeranyl (i.e., 3,7,11,15-tetramethylhexadeca-2,6,10,14-tetraen-1-ol). In some embodiments, the prenyl moiety is geranyl. As shown in the non-limiting examples below, the carbon-carbon double bond of the prenyl moiety can be in the cis (Z) or trans (E) configuration, where the wavy line represents the point of attachment of the prenyl moiety to the compound of Formula I. In some embodiments, R 3 is trans-geranyl (i.e., (E)-3,7-dimethylocta-2,6-dien-1-yl). TIFF2025170350000009.tif79140

[0045] In some embodiments, the compound has the structure of Formula Ia: TIFF2025170350000010.tif29128

[0046] R 2Compounds of formula Ia where R is COOH include 1 cannabigerolic acid analogs in which R is halopentyl, hydroxypentyl, deuterated pentyl, or tritiated pentyl. 2 Compounds of formula Ia where R is H include 1 cannabigerol analogs wherein is halopentyl, hydroxypentyl, deuterated pentyl, or tritiated pentyl.

[0047] In some embodiments, 6-(4-chlorobutyl)-3-(3,7-dimethyl-octa-2,6-dienyl)-2,4-dihydroxy-benzoic acid; 3-(3,7-dimethyl-octa-2,6-dienyl)-6-(5-fluoropentyl)-2,4-dihydroxy-benzoic acid; 2-methyl-2-(4-methyl-pent-3-enyl)-7-perdeuteropentyl-2H-chromen-5-ol; 5-hydroxy-2-methyl-2-(4-methyl-pent-3-enyl)-7-perdeuteropentyl-2H-chromene-6-carboxylic acid; 7-(5-fluoropentyl)-2-methyl-2-(4-methyl-pent-3-enyl)-2H-chromen-5-ol; and / or 7-(5-chloropentyl)-2-methyl-2-(4-methyl-pent-3-enyl)-2H-chromen-5-ol.

[0048] In some embodiments, cannabinoid derivatives of the compounds of Formula I and Formula Ia are provided. In some embodiments, the cannabinoid derivatives include halogenated cannabidiolic acids, halogenated cannabidiols, halogenated Δ 9 -Tetrahydrocannabinolic acid, halogenated Δ 8 -Tetrahydrocannabinolic acid, halogenated cannabichromenic acid, halogenated cannabichromene, halogenated cannabinol, halogenated cannabinodioI, halogenated cannabinolic acid, cannabivarin, halogenated cannabivarinic acid, halogenated Δ 9 -Tetrahydrocannabivarin, halogenated Δ 8 -Tetrahydrocannabivarin, halogenated Δ 9-Tetrahydrocannabivaric acid, halogenated Δ 8 - selected from tetrahydrocannabivarin, halogenated cannabigerovarin, halogenated cannabigerovarin, halogenated cannabichromevarin, halogenated cannabichromevarin, halogenated cannabidivarin, halogenated cannabidivarin, halogenated cannabidivarinic acid, halogenated cannabiditriol, and halogenated cannabicyclol.

[0049] In some embodiments, the cannabinoid derivative is a deuterated cannabidiolic acid, a deuterated cannabidiol, a deuterated Δ 9 -Tetrahydrocannabinolic acid, deuterated Δ 8 -Tetrahydrocannabinolic acid, deuterated cannabichromenic acid, deuterated cannabichromene, deuterated cannabinol, deuterated cannabinodiol, deuterated cannabinolic acid, cannabivarin, deuterated cannabivarinic acid, deuterated Δ 9 -Tetrahydrocannabivarin, deuterated Δ 8 -Tetrahydrocannabivarin, deuterated Δ 9 -Tetrahydrocannabivaric acid, deuterated Δ 8 - selected from tetrahydrocannabivarin, deuterated cannabigerovarin, deuterated cannabigerovarinic acid, deuterated cannabichromevarin, deuterated cannabichromevarinic acid, deuterated cannabidivarin, deuterated cannabidivarinic acid, deuterated cannabiditriol, and deuterated cannabicyclol.

[0050] In some embodiments, the cannabinoid derivative is tritiated cannabidiolic acid, tritiated cannabidiol, tritiated Δ 9 -Tetrahydrocannabinolic acid, tritiated Δ 8 -Tetrahydrocannabinolic acid, tritiated cannabichromenic acid, tritiated cannabichromene, tritiated cannabinol, tritiated cannabinodiol, tritiated cannabinolic acid, cannabivarin, tritiated cannabivarinic acid, tritiated Δ 9 -Tetrahydrocannabivarin, tritiated Δ 8-Tetrahydrocannabivarin, tritiated Δ 9 -Tetrahydrocannabivaric acid, tritiated Δ 8 - selected from tetrahydrocannabivarin, tritiated cannabigerovarin, tritiated cannabigerovarinic acid, tritiated cannabichromevarin, tritiated cannabichromevarinic acid, tritiated cannabidivarin, tritiated cannabidivarinic acid, tritiated cannabiditriol, and tritiated cannabicyclol.

[0051] In some embodiments, the cannabinoid derivative is hydroxy-cannabidiolic acid, hydroxy-cannabidiol, hydroxy-Δ 9 -Tetrahydrocannabinolic acid, hydroxy-Δ 8 -Tetrahydrocannabinolic acid, hydroxy-cannabichromene acid, hydroxy-cannabichromene, hydroxy-cannabinol, hydroxy-cannabinodiol, hydroxy-cannabinolic acid, cannabivarin, hydroxy-cannabivarinic acid, hydroxy-Δ 9 -Tetrahydrocannabivarin, hydroxy-Δ 8 -Tetrahydrocannabivarin, hydroxy-Δ 9 -Tetrahydrocannabivaric acid, hydroxy-Δ 8 -tetrahydrocannabivarinic acid, hydroxy-cannabigerovarin, hydroxy-cannabigerovarinic acid, hydroxy-cannabichromevarin, hydroxy-cannabichromevarinic acid, hydroxy-cannabidivarin, hydroxy-cannabidivarinic acid, hydroxy-cannabiditriol, and hydroxy-cannabicyclol.

[0052] Cannabinoid derivatives of the compounds of Formula I and Formula Ia include, but are not limited to, the cannabinoid derivatives set forth in Table 1. As described below, the compounds of Formula I and Formula Ia can be converted to the cannabinoid derivatives enzymatically (e.g., using cannabinoid synthase) or chemically.

[0053] Table 1: Cannabinoid derivatives of compounds of formula I and formula Ia TIFF2025170350000011.tif166157TIFF2025170350000012.tif233157TIFF2025170350 000013.tif240157TIFF2025170350000014.tif233157TIFF2025170350000015.tif91157

[0054] Cannabinoid derivatives of the compounds of Formula I and Formula Ia include CBG, CBDA, CBD, THC, Δ 8 -THC, THCA, Δ 8 -THCA, CBCA, CBC, CBN, CBND, CBNA, CBV, CBVA, THCV, THCVA, Δ 8 Examples include, but are not limited to, analogs of -THCA, CBGV, CBGVA, CBCV, CBCVA, CBDV, and CBDVA. Further examples include cannabichromanone, cannabicoumaronone, cannabiditran, 10-oxo-Δ 6a(10a) -Tetrahydrocannabinol (OTHC), Cannabiglendol, and Δ 7 -Isotetrahydrocannabinol.

[0055] III. Methods for the Enzymatic and Chemoenzymatic Preparation of Cannabinoid Analogues Also provided herein is a method for synthesizing cannabinoid analogs and their intermediates through metabolic pathways in genetically engineered host cells.The term "metabolic pathway" refers to a series of two or more enzymatic reactions in which the product of one enzymatic reaction becomes the substrate for the next enzymatic reaction.At each step of the metabolic pathway, an intermediate compound is formed and used as a substrate for the next step.In some embodiments, each step of the metabolic pathway is carried out in the modified recombinant cells described herein.In some embodiments, at least one step of the metabolic pathway is carried out in the modified recombinant cells described herein, and at least one step of the metabolic pathway is carried out outside the modified recombinant cells, in yeast medium, or in additional co-cultured modified recombinant cells.

[0056] Accordingly, some embodiments of the present disclosure include a compound of formula IV TIFF2025170350000016.tif20128, or a salt thereof, wherein R 1 is C1~C 20 Haloalkyl, C1-C 20 Hydroxyalkyl, deuterated C1-C 20 Alkyl, tritiated C1-C 10 Alkyl, and C2-C 20 alkenyl.

[0057] The method comprises providing a thioester of formula II culturing the modified recombinant host cell in a medium containing TIFF2025170350000017.tif12128; In the formula, R 4 is selected from the group consisting of a coenzyme A (CoA) moiety, a pantetheine moiety, and a cysteamine moiety; The modified recombinant host cell comprises: i. thioester of formula II and malonyl-CoA to a tetraketide of formula III a first polynucleotide encoding a synthase that converts ii. A second polynucleotide encoding a 2-alkyl-4,6-dihydroxybenzoate cyclase that converts the tetraketide of formula III to a compound of formula IV. and The modified recombinant host cells are cultured under conditions whereby the products encoded by the first and second polynucleotides are expressed to produce the compound of Formula IV.

[0058] Olivetolic acid synthase In some embodiments, the synthase is olivetolic acid synthase. In some such embodiments, the host cell is genetically modified to express an exogenous polynucleotide encoding olivetolic acid synthase, or a variant thereof that exhibits polyketide synthase activity, such as a naturally occurring homolog or ortholog, or a non-naturally occurring variant. Olivetolic acid synthase (Taura et al. FEBS Letters 583:2061-2066, 2009), also known as 3,5,7-trioxododecanoyl-CoA synthase, UniProtKB-B1Q2B6, is a type III PKS that catalyzes the condensation of three molecules of acyl-CoA with malonyl-CoA to form the 3,5,7-trioxoalkanoyl-CoA tetraketide shown below: TIFF2025170350000019.tif11128 where "CoA" is coenzyme A and "R" is an alkyl group. When hexanoic acid is used as a starting material for cannabinoid production in natural systems, three molecules of hexanoyl-CoA and malonyl-CoA are condensed to form 3,5,7-trioxododecanoyl-CoA (i.e., "R" is an n-pentyl group). Type III PKSs are homodimeric enzymes that act directly on acyl-CoA substrates (as opposed to acyl carrier protein-bound substrates in Type I and Type II PKSs). Type III PKSs have been well characterized, for example, by Yu et al. (IUBMB Life, 64(4): 285-295, 2012).

[0059] In some embodiments, the olivetolic acid synthase polynucleotide encodes a polypeptide comprising an amino acid sequence exhibiting about 60% or greater identity (e.g., about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the sequence set forth in SEQ ID NO:4. In some embodiments, the olivetolic acid synthase polynucleotide encodes a type III PKS comprising an amino acid sequence that exhibits about 70%, 75%, 80%, 85%, 90%, 95% or more identity to the sequence set forth in SEQ ID NO:4.

[0060] The substrate specificity observed in natural systems is consistent with the R 1 Groups such as C1-C 20 Haloalkyl groups, C1-C 20 Hydroxyalkyl groups, deuterated C1-C 20 Alkyl groups, tritiated C1-C 20 Alkyl groups, and / or C2-C 20 Several starting materials bearing alkenyl groups can be developed. Similarly, the thioesters used in the methods of the present invention are not limited to Coenzyme A (CoA) esters utilized in natural systems. 4 may be a CoA moiety as shown below, where the wavy line represents the point of attachment of the CoA moiety to the sulfur atom in the thioester of formula II. TIFF2025170350000020.tif45128

[0061] Alternatively, R 4 is the pantetheine part TIFF2025170350000021.tif16128 or the cysteamine moiety TIFF2025170350000022.tif5128, where the wavy line represents the R bond to the sulfur atom in the thioester of formula II. 4 represents the attachment point of 4a is H or acetyl (—C(O)CH3). As described below, thioesters of Formula II can be formed enzymatically or prepared chemically.

[0062] 2-Alkyl-4,6-dihydroxybenzoate cyclase The host cell used to produce the compound of Formula IV can be modified to express an exogenous polynucleotide encoding 2-alkyl-4,6-dihydroxybenzoate cyclase. In some embodiments, the 2-alkyl-4,6-dihydroxybenzoate cyclase is a dimeric α+β barrel (DABB) protein domain similar to the DABB polyketide cyclase from Streptomyces. Olivetolate cyclase is described, for example, in Gagne et al. (Proc. Nat. Acad. Sci. USA 109 (31): 12811-12816; 2012). The term "2-alkyl-4,6-dihydroxybenzoate cyclase" includes variants that exhibit cyclase activity, such as truncated or modified polypeptides; and naturally occurring homologs or orthologs. In some embodiments, the 2-alkyl-4,6-dihydroxybenzoate cyclase is olivetolate cyclase (EC number 4.4.1.26) from Cannabis sativa. In some embodiments, the 2-alkyl-4,6-dihydroxybenzoate cyclase produces divalanate (see, e.g., Yang et al., FEBS J. 283:1088-1106, 2016). In some embodiments, the 2-alkyl-4,6-dihydroxybenzoate cyclase is an olivetolate cyclase homolog, such as AtHS1 from Arabidopsis thaliana (Uniprot Q9LUV2), SP1 from Populus tremula (P0A881), At5g22580 from Arabidopsis thaliana (Q9FK81), TcmI cyclase from S. glaucescens (P39890), ActVA-Orf6 from S. coelicolor (Q53908), MLMI from P. reinekei (C5MR76), SnoaB from S. nogalater (O54259), or Mycobacterium tuberculosis (M. The two strains are Rv0793 (O86332) from R. tuberculosis, or PA3566 (Q9HY51) from P. aeruginosa.In some embodiments, the 2-alkyl-4,6-dihydroxybenzoate cyclase comprises a cyclase domain from the benH gene product (B1GSN4) of the benastatin gene cluster of Streptomyces species, e.g., Streptomyces species A2991200, as set forth in SEQ ID NO:9. In some embodiments, the 2-alkyl group of the 2-alkyl-4,6-dihydroxybenzoic acid comprises 1 to 18 carbon atoms. In some embodiments, the 2-alkyl group of the 2-alkyl-4,6-dihydroxybenzoic acid comprises 1 to 12 carbon atoms. In some embodiments, the 2-alkyl group of the 2-alkyl-4,6-dihydroxybenzoic acid comprises 1 to 9 carbon atoms.

[0063] In some embodiments, a polynucleotide encoding a 2-alkyl-4,6-dihydroxybenzoate cyclase encodes a polypeptide exhibiting about 60% or greater identity (e.g., about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the sequence set forth in SEQ ID NO:5, 6, 7, or 9. In some embodiments, the polypeptide exhibits about 70%, 75%, 80%, 85%, 90%, 95% or more identity to the sequence set forth in SEQ ID NO:5, 6, 7, or 9.

[0064] Acyl-CoA synthetase The thioester of Formula II can be enzymatically formed by the host cell or chemically formed prior to cell culture. In some embodiments, the host cell produces a starting material of Formula IIa. and a third polynucleotide encoding an acyl-CoA synthetase that converts TIFF2025170350000023.tif12128 to a thioester of Formula II; and Step (a) comprises culturing the host cell under conditions whereby the product encoded by the third polynucleotide is expressed to produce a thioester of formula II.

[0065] As used herein, the term "acyl-CoA synthetase," which may also be referred to as "acyl-CoA synthase," "acyl-activating enzyme," or "acyl-CoA ligase," refers to an enzyme that converts a carboxylic acid (e.g., the acid starting material of Formula IIa) to an acyl-CoA thioester through a two-step process in which the carboxylic acid and ATP are converted to an enzyme-bound carboxyl-AMP intermediate (called adenylate) with the release of pyrophosphate (PPi). The activated carbonyl carbon of adenylate binds to the thiol of CoA, followed by enzymatic release of the thioester and AMP.

[0066] Any number of acyl-CoA synthetases can be used to form the thioester of Formula II. Acyl-CoA synthetases include, but are not limited to, short-chain acyl-CoA synthetases (EC 6.2.1.1), medium-chain acyl-CoA synthetases (EC 6.2.1.2), long-chain acyl-CoA synthetases (EC 6.2.1.3), and coumarate-CoA ligases (EC 6.2.1.12). Typically, acyl-CoA synthetases contain a 12-amino acid residue domain called the AMP-binding motif (PROSITE PS00455): [LIVMFY]-{E}-{VES}-[STG]-[STAG]-G-[ST]-[STEI]-[SG]-x-[PASLIVM]-[KR]. In a PROSITE sequence, each position in the sequence is separated by a "-" and the symbol "x" means that any residue is allowed at the given position in the sequence. Amino acids that are allowed at a given position are placed between square brackets (e.g., [ST] means that serine or threonine is allowed at the given position in the sequence), while amino acids that are not allowed at the given position are placed between curly brackets (e.g., {VES} indicates that any residue other than valine, glutamic acid, and serine is allowed at the given position in the sequence). The AMP-binding motif has been used to classify polypeptides as acyl-activating enzymes (AAEs) and has helped identify a large superfamily of AAE genes present in Arabidopsis (Shockey et al., 2003, Plant Physiology 132: 1065-1076), Chlamydomonas reinhardtii, Populus trichocharpa, and Physcomitrella patens (Shockey and Browse, 2011, The Plant Journal 66: 143-160).Acyl-CoA synthetases are also described, for example, in WO 2018 / 209143; Black et al. (Biochim Biophys Acta. 1771(3):286-98, 2007); Miyazawa et al. (J. Biol. Chem 290 (45): 26994-27011, 2015); and Stout et al. (Plant J. 71(3):353-365, 2012).

[0067] In some embodiments, the acyl-CoA synthetase is derived from an organism that biosynthesizes resveratrol. In some embodiments, the acyl-CoA synthetase is a coumarate-CoA ligase from Morus or Vitis. In some embodiments, the acyl-CoA synthetase is derived from Ralstonia solanacearum. In some embodiments, the acyl-CoA synthetase from Ralstonia solanacearum is deleted at the N-terminus. See, e.g., SEQ ID NO:8. In some embodiments, the transmembrane domain may be deleted from the acyl-CoA synthetase.

[0068] In some embodiments, the host cell is genetically modified to express an exogenous polynucleotide encoding a revS polypeptide from Streptomyces species (see, e.g., Miyazawa et al., J. Biol. Chem. 290:26994-27001, 2015), or a variant thereof, e.g., a naturally occurring homolog, a naturally occurring ortholog, or a non-naturally occurring variant, that exhibits acyl-CoA synthetase activity. In some embodiments, the polynucleotide encodes a polypeptide exhibiting about 60% or greater identity (e.g., about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the sequence set forth in SEQ ID NO:1. In some embodiments, the polynucleotide encodes a RevS polypeptide that exhibits about 70%, 75%, 80%, 85%, 90%, 95% or more identity to the sequence set forth in SEQ ID NO: 1. In some embodiments, the non-naturally occurring variant contains one or more modifications, e.g., substitutions, such as conservative substitutions, relative to SEQ ID NO: 1, e.g., in a region outside the AMP binding motif or catalytic site.

[0069] In some embodiments, the host cell is genetically modified to express an exogenous polynucleotide encoding a Cannabis sativa-derived acyl-activating enzyme (CsAAE3), or a variant thereof that exhibits acyl-CoA synthetase activity, e.g., a naturally occurring homolog, a naturally occurring ortholog, or a non-naturally occurring variant. In some embodiments, the CsAAE3 polypeptide encoded by the polynucleotide comprises an amino acid sequence exhibiting at least about 60% or more identity (e.g., about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the sequence set forth in SEQ ID NO:2. In some embodiments, the acyl-CoA synthetase polynucleotide encodes CsAAE3, or a homolog or non-naturally occurring variant thereof comprising an amino acid sequence that exhibits about 70%, 75%, 80%, 85%, 90%, 95% or more identity to the sequence set forth in SEQ ID NO: 2. In some embodiments, the non-naturally occurring variant contains one or more modifications, e.g., substitutions, such as conservative substitutions, relative to SEQ ID NO: 2, e.g., within a region outside the AMP binding motif or catalytic site.

[0070] In some embodiments, the host cell is genetically modified to express an exogenous polynucleotide encoding the Cannabis sativa-derived acyl-activating enzyme (CsAAE1), or a variant thereof that exhibits acyl-CoA synthetase activity, e.g., a naturally occurring homolog, a naturally occurring ortholog, or a non-naturally occurring variant. In some embodiments, the CsAAE1 polypeptide encoded by the polynucleotide comprises an amino acid sequence exhibiting at least about 60% identity or more (e.g., about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to the sequence set forth in SEQ ID NO:3. In some embodiments, the acyl-CoA synthetase polynucleotide encodes CsAAE1, or a homolog thereof, comprising an amino acid sequence exhibiting about 70%, 75%, 80%, 85%, 90%, 95% or more identity to the sequence set forth in SEQ ID NO:2. In some embodiments, the CsAAE1 polynucleotide encodes a polypeptide lacking the transmembrane domain. In some embodiments, the non-naturally occurring variant contains one or more modifications, e.g., substitutions, such as conservative substitutions, relative to SEQ ID NO:3, e.g., within a region outside the AMP binding motif or catalytic site.

[0071] In some embodiments, R 1 is selected from 4-fluorobutanoic acid; 4,4,4-trifluorobutanoic acid; 2,2-difluorobutanoic acid; perfluorobutanoic acid; 5-fluoropentanoic acid; 2,2-difluoropentanoic acid; perfluoropentanoic acid; 6-fluorohexanoic acid; 2,2-difluorohexanoic acid; and perfluorohexanoic acid.

[0072] In some embodiments, R in the starting material of Formula IIa 1 is selected from the group consisting of 4-fluorobutanoic acid, 5-fluoropentanoic acid, and 6-fluorohexanoic acid.

[0073] In some embodiments, R 1 is selected from the group consisting of 4-chlorobutanoic acid, 4-bromobutanoic acid, 4-hydroxybutanoic acid, 5-chloropentanoic acid, 5-bromopentanoic acid, 5-hydroxypentanoic acid, 6-chlorohexanoic acid, 6-bromohexanoic acid, 6-hydroxyhexanoic acid, 7-chloroheptanoic acid, 7-bromoheptanoic acid, and 7-hydroxyheptanoic acid. 1 is perdeuterohexanoic acid (i.e., D 11 C5COOH).

[0074] Chemical synthesis of thioesters The thioester of formula II is CoA R 4 Part, Pantetheine R 4 Part, or Cysteamine R 4 The thioesters of Formula II can be prepared using acyl-CoA synthetase expressed by the host cells described above, or the thioesters can be synthesized by chemically acylating CoA, pantetheine (i.e., 2,4-dihydroxy-3,3-dimethyl-N-[2-(2-sulfanylethylcarbamoyl)ethyl]butanamide), or cysteamine (i.e., 2-aminoethanethiol) with a carboxylic acid of Formula IIa, or an activated derivative thereof.

[0075] Many suitable carboxylic acids are commercially available or can be found in Fiesers' Reagents for Organic Synthesis Volumes 1-28 (John Wiley & Sons, 2016), March (Advanced Organic Chemistry 6 th Ed. John Wiley & Sons, 2007), and Larock (Comprehensive Organic Transformations 3 rdEd. John Wiley & Sons, 2018). As a non-limiting example, α-halogenated carboxylic acids can be prepared using a halogen (e.g., Br) and catalytic phosphorous acid in the Hell-Volhard-Zelinsky reaction. As another non-limiting example, fluorinated carboxylic acids can be prepared using a catalyst system containing -SELECTFLUOR (chloromethyl-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoroborate)) and a copper(I) bisimine complex as described in Bloom et al. (Angew. Chem. Int. Ed. 2012, 51, 1-5). Hydroxylation of carboxylic acids can be carried out using a platinum catalyst according to the method of Kao and Sen (J. Chem. Soc., Chem. Commun., 1991, 1242-1243). Deuteration of carboxylic acids can be carried out using platinum and rhodium on carbon according to the method of Yamada et al. (RSC Adv., 2015, 5, 13727-13732).

[0076] The carboxylic acid of formula IIa can be used in combination with a coupling agent for acylation of the thiol to be acylated (eg, CoA, pantetheine, or cysteamine). Examples of coupling agents include carbodiimides (e.g., N,N'-dicyclohexylcarbodiimide (DCC), N,N'-dicyclopentylcarbodiimide, N,N'-diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)), phosphonium salts (HOBt, PyBOP, HOAt, etc.), aminium / uronium salts (e.g., pyrimidinium uronium salts such as HATU, tetramethylaminium salts, bispyrrolidinoaminium salts, bispiperidinoaminium salts, imidazolium uronium salts, uronium salts derived from N,N,N'-trimethyl-N'-phenylurea, morpholino-based aminium / uronium coupling reagents, antimonylic acid uronium salts, etc.), organophosphorus reagents (e.g., phosphinic acid derivatives and and phosphoric acid derivatives), organosulfur reagents (e.g., sulfonic acid derivatives), triazine coupling reagents (e.g., 2-chloro-4,6-dimethoxy-1,3,5-triazine, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4 methylmorpholinium chloride, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4 methylmorpholinium tetrafluoroborate, etc.), pyridinium coupling reagents (e.g., Mukaiyama reagent, pyridinium tetrafluoroborate coupling reagent, etc.), polymer-supported reagents (e.g., polymer-bound carbodiimide, polymer-bound TBTU, polymer-bound 2,4,6-trichloro-1,3,5-triazine, polymer-bound HOBt, polymer-bound HOSu, polymer-bound IIDQ, polymer-bound EEDQ, etc.).

[0077] Alternatively, acylation can be carried out using an activated carboxylic acid derivative, such as an acid anhydride, mixed anhydride, acid chloride, or activated ester (e.g., pentafluorophenyl ester or N-hydroxysuccinimidyl ester). Typically, 1 to 10 molar equivalents of the carboxylic acid or activated derivative relative to the thiol are used. For example, 1 to 5 molar equivalents of the acid / acid derivative or 1 to 2 molar equivalents of the acid / acid derivative can be used. In some embodiments, about 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 molar equivalents of the acid / acid derivative relative to the thiol are used to form the thioester of Formula II.

[0078] A base can be used to promote the acylation of a thiol with a carboxylic acid or activated carboxylic acid derivative. Examples of suitable bases include potassium carbonate, sodium carbonate, sodium acetate, Hunig's base (i.e., N,N-diisopropylethylamine), lutidine, including 2,6-lutidine (i.e., 2,6-dimethylpyridine), triethylamine, tributylamine, pyridine, 2,6-di-tert-butylpyridine, 1,8-diazabicycloundec-7-ene (DBU), quinuclidine, and collidine. Combinations of two or more bases can be used. Typically, less than one molar equivalent of base relative to the thiol is used in forming the thioester. For example, 0.05 to 0.9 molar equivalents or 0.1 to 0.5 molar equivalents of base can be used. In some embodiments, about 0.05, 0.1, 0.15, or 0.2 molar equivalents of base relative to the thiol are used in combination with the acid / acid derivative to form the thioester of Formula II.

[0079] Any suitable solvent can be used to form the thioester. Suitable solvents include, but are not limited to, toluene, methylene chloride, ethyl acetate, acetonitrile, tetrahydrofuran, benzene, chloroform, diethyl ether, dimethylformamide, dimethyl sulfoxide, petroleum ether, and mixtures thereof. Typically, the acylation reaction is carried out at a temperature ranging from about 25°C to about 100°C for a period sufficient to form the thioester of Formula II. The reaction can be carried out for a period ranging from a few minutes to several hours or longer, depending on the particular thiol and acid / acid derivative used in the reaction. For example, the reaction can be carried out at about 40°C, or about 50°C, or about 60°C, or about 70°C, or about 80°C for about 10 minutes, or about 30 minutes, or about 1 hour, or about 2 hours, or about 4 hours, or about 8 hours, or about 12 hours.

[0080] In order to prevent undesired side reactions during the acylation step, functional groups such as the primary amine of cysteamine or the hydroxyl group of pantetheine and CoA can be protected.Examples of amine protecting groups include but are not limited to benzyloxycarbonyl; 9-fluorenylmethyloxycarbonyl (Fmoc); tert-butyloxycarbonyl (Boc); allyloxycarbonyl (Alloc); p-toluenesulfonyl (Tos); 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc); 2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl (Pbf); mesityl-2-sulfonyl (Mts); 4-methoxy-2,3,6-trimethylphenylsulfonyl (Mtr); acetamide; phthalimide; etc. Examples of alcohol protecting groups include, but are not limited to, benzyl; tert-butyl; trityl; tert-butyldimethylsilyl (TBDMS; TBS); 4,5-dimethoxy-2-nitrobenzyloxycarbonyl (Dmnb); propargyloxycarbonyl (Poc); and the like. Green and Wuts (Protective Groups in Organic Synthesis, 4 thOther alcohol and amine protecting groups are known to those skilled in the art, including, for example, those described in "Protein-Based Alkyl Groups," Ed. 2007, Wiley-Interscience, New York. The protecting groups can be removed under standard conditions to restore the original functionality after the acylation step.

[0081] prenyltransferase In some embodiments, the recombinant host cell is further modified to express an exogenous polynucleotide encoding a prenyltransferase that catalyzes the attachment of an activated prenyl species (e.g., geranyl pyrophosphate) to a compound of Formula IV or a compound of Formula IVa to form compounds of Formula IV and Formula Va. Examples of prenyltransferases include, but are not limited to, geranyl pyrophosphate:olivetolic acid geranyltransferase (GOT; EC 2.5.1.102) described by Fellermeier and Zenk (FEBS Letters 427:283-285; 1998) and asaprenyltransferases described in WO 2018 / 200888 and WO 2019 / 071000. Streptomyces prenyltransferases, including NphB described by Kumano et al. (Bioorg Med Chem. 16(17): 8117-8126; 2008), may also be used in accordance with the present invention. In some embodiments, the prenyltransferase is fnq26, a flaviolin linalyltransferase from Streptomyces cinnamonensis. In some embodiments, the host cell genetically engineered to express a prenyltransferase can be an engineered host cell described below.

[0082] Thus, some embodiments of the present disclosure involve converting a compound of formula IV above to a compound of formula V TIFF2025170350000024.tif20128 or a salt thereof, wherein R 3 is the prenyl moiety. For example, R 3can be geranyl, farnesyl, or geranylgeranyl, which contain carbon-carbon double bonds in the cis (Z) and / or trans (E) configurations. Some embodiments provide a decarboxylated compound of formula IVa TIFF2025170350000025.tif20128, compound of formula Va TIFF2025170350000026.tif20128, wherein R 3 is the prenyl moiety.

[0083] In some embodiments, the DNA constructs for geranyl pyrophosphate:olivetolic acid geranyltransferase encode wild-type or mutant enzymes with yeast-preferential codons. In some embodiments, DNA constructs encoding bacterial prenyltransferases (e.g., Streptomyces prenyltransferase) that exhibit relaxed substrate specificity are used (Kumano et al., 2008).

[0084] During cultivation and production of prenylated compounds, host cells can be supplied with exogenous prenyl species, such as geraniol. Alternatively, host cells can be cultured in media containing high levels of prenyl precursors, such as prenol, isoprenol, geraniol, etc. In approaches involving multiple precursor feeding (MPF), 5-carbon prenol and isoprenol can be enzymatically converted to the monophosphate level (i.e., dimethylallyl monophosphate and isopentenyl monophosphate) and then to the diphosphate level (i.e., dimethylallyl pyrophosphate and isopentenyl pyrophosphate), which can then be combined to form the 10-carbon geranyl pyrophosphate.

[0085] In some embodiments, the initial phosphorylation event is catalyzed by the enzyme hydroxyethylthiazole kinase. This enzyme has been described in several organisms in which the encoding gene has been derived, including E. coli, Bacillus subtilis, Rhizobium leguminosarum, Pyrococcus horikoshii, S. cerevisiae, and maize species. Further phosphorylation to the diphosphate level can be achieved using the enzymes isoprenyl diphosphate synthase or isopentenyl phosphate kinase, as described in U.S. Patent No. 6,235,514. In some embodiments, a synthetic gene encoding this enzyme, or a more active variant, is derived using the amino acid sequence of Thermoplasma acidophilum, Methanothermobacter thermautotrophicus, Methanocaldococcus jannaschii, peppermint (Mentha x piperita), or mango (Mangifera indica), or other homologous sequences that exhibit kinase activity. The bond to form geranyl pyrophosphate can be catalyzed by a transferase enzyme, such as geranyl pyrophosphate synthase (GPP synthase).

[0086] 10-carbon geranyl pyrophosphate can also be produced by a kinase that phosphorylates geraniol to the monophosphate level, followed by a second kinase to generate geranyl pyrophosphate. In some embodiments, the first kinase event is carried out by the enzyme farnesol kinase (FOLK) (Fitzpatrick, Bhandari, and Crowell, 2011; Plant J. 2011 Jun;66(6):1078-88) or a variant thereof. This kinase enzyme is present in several organisms that exhibit the ability to phosphorylate 5-carbon prenol, including plants (e.g., Arabidopsis thaliana, Camelina sativa, Capsella rubella, Noccaea caerulescens) and fungi (e.g., Candida albicans, Talaromyces atroroseus). Further phosphorylation of geranyl phosphate to geranyl pyrophosphate can be achieved using isopentenyl monophosphate kinase (IPK) or its variants. This kinase enzyme is found in several bacterial and archaeal species, including, but not limited to, Methanocaldococcus jannaschii and Thermoplasma acidophilum. Specific mutations in IPK (Val73, Val130, Ile140) have been reported to enhance geranyl phosphate kinase activity (Mabanglo et al., 2012, ACS Chem. Biol., 7, 7, 1241-1246).

[0087] In some embodiments, the host cell comprises one or more additional exogenous polynucleotides selected from the following three types of exogenous polynucleotides: an exogenous polynucleotide encoding prenol and isoprenol kinase; an exogenous polynucleotide encoding a kinase that produces dimethylallyl pyrophosphate and isopentenyl pyrophosphate when grown in the presence of exogenous prenol and isoprenol; and an exogenous polynucleotide encoding a geranyl pyrophosphate synthase.

[0088] Chemical prenylation In some embodiments, the converting step is performed in vitro. For example, the converting step can include forming a reaction mixture comprising: (1) a compound of Formula IV or Formula IVa; (2) geraniol, activated geraniol (e.g., geranyl bromide, geranyl chloride, geranyl tosylate, geranyl mesylate, etc.), or citral; and (3) an organic solvent under conditions sufficient to produce a compound of Formula V or Formula Va.

[0089] Any suitable organic solvent can be used in the chemical prenylation step provided herein. Suitable solvents include, but are not limited to, toluene, methylene chloride, dichloroethane, ethyl acetate, acetonitrile, tetrahydrofuran, benzene, ethylbenzene, xylene (i.e., m-xylene, o-xylene, p-xylene, or any combination thereof), chloroform, diethyl ether, dimethylformamide, dimethyl sulfoxide, petroleum ether, and mixtures thereof. In some embodiments, the organic solvent is toluene, benzene, ethylbenzene, xylene, or a mixture thereof. In some embodiments, the organic solvent is toluene. In some embodiments, the organic solvent is dichloroethane. Aqueous organic solvent mixtures (i.e., mixtures of water and a water-miscible organic solvent such as tetrahydrofuran or dimethylformamide) can also be used. Generally, the ratio of solvent to compound of Formula IV or Formula IVa ranges from about 1:1 to about 1000:1 by weight. For example, the ratio of solvent to compound of Formula IV or Formula IVa can be about 100:1 by weight, or about 10:1 by weight, or about 5:1 by weight. In certain embodiments, the compound of Formula IV or Formula IVa is present in a yeast mixture (e.g., dried yeast cells or a wet yeast cell pellet recovered from the culture medium). In some such embodiments, the reaction mixture includes host cells (e.g., dried yeast cells). The ratio of solvent to yeast mixture (e.g., dried yeast cells) can range from about 1:1 to about 1000:1 by weight. For example, the ratio of solvent to yeast mixture can be about 100:1 by weight, or about 10:1 by weight, or about 5:1 by weight, or about 2:1 by weight.

[0090] Any suitable amount of geraniol, activated geraniol, or citral can be used in the conversion step. Generally, the reaction mixture contains at least 1 molar equivalent of geraniol, activated geraniol, or citral relative to the compound of Formula IV or Formula IVa. For example, the reaction mixture can contain about 1 molar equivalent to about 10 molar equivalents (e.g., about 1.1 molar equivalents, about 1.2 molar equivalents, or about 2 molar equivalents) of geraniol, activated geraniol, or citral relative to the compound of Formula IV or Formula IVa.

[0091] In some embodiments, including those in which geraniol or activated geraniol is used, the reaction mixture further comprises an acid. Any suitable acid can be used in the converting step. Examples of suitable acids include, but are not limited to, hydrochloric acid, sulfuric acid, nitric acid, formic acid, acetic acid, trifluoroacetic acid, p-toluenesulfonic acid, methanesulfonic acid, and trifluoromethanesulfonic acid. In some embodiments, the acid is a sulfonic acid. In some embodiments, the acid is p-toluenesulfonic acid. Any suitable amount of acid can be used in the converting step. Generally, the reaction mixture comprises about 0.01 molar equivalent to about 10 molar equivalents (e.g., about 0.01 molar equivalent, or about 0.1 molar equivalent, or about 1 molar equivalent) of acid (e.g., p-toluenesulfonic acid) relative to the compound of Formula IV or Formula IVa.

[0092] In some embodiments, including those in which citral is used, the reaction mixture further comprises an amine, such as a diamine (e.g., a 1,2-diamine). Any suitable diamine or other amine can be used in the conversion step. Examples of suitable diamines include, but are not limited to, ethylenediamine, N,N-dimethylethylenediamine, N,N-diethylethylenediamine, N,N'-dimethylethylenediamine, N,N'-diphenylethylenediamine, N,N'-dibenzylethylenediamine, and N,N'-bis(2-hydroxyethyl)ethylenediamine. In some embodiments, the prenylation reaction mixture comprises citral and N,N-dimethylethylenediamine. Any suitable amount of amine can be used in the conversion step. Generally, the reaction mixture comprises about 0.01 molar equivalent to about 10 molar equivalents (e.g., about 0.01 molar equivalent, or about 0.25 molar equivalent, or about 0.1 molar equivalent, or about 1 molar equivalent) of amine (e.g., N,N-dimethylethylenediamine) relative to the compound of Formula IV or Formula IVa.

[0093] In some embodiments, chiral diamines (e.g., (1S,2S)-1,2-di-1-naphthyl-ethylenediamine, (S)-1-[(1-methyl-2-pyrrolidinyl)methyl]piperidine, etc.) can aid in the formation of one or more stereocenters in the prenylated cannabinoid product. For example, the reaction of an olivetolic acid analog (e.g., perdeuteropentyl-olivetolic acid) with citral in the presence of a chiral diamine can stereoselectively afford the corresponding cannabichromene analog. Chiral diamines can be selected to produce specific cannabichromene analog enantiomers (e.g., (S)-2-methyl-2-(4-methylpent-3-en-1-yl)-7-perdeuteropentyl-2H-chromen-5-ol or (R)-2-methyl-2-(4-methylpent-3-en-1-yl)-7-perdeuteropentyl-2H-chromen-5-ol). Chiral diamines can also be selected to produce specific cannabichromene analog enantiomers (e.g., (S ... 1 They may also be used in the stereoselective synthesis of cannabinoids bearing unsubstituted alkyl groups at positions R.1 is C1~C 10 and forming the prenylated cannabinoid product may include reacting a 5-alkyl-resorcinol (e.g., olivetol or divalinol, i.e., 5-propylresorcinol) or a 2-alkyl-4,6-dihydroxybenzoic acid (e.g., olivetolic acid or divalinic acid) with citral in the presence of a chiral diamine to stereoselectively form the prenylated product (e.g., cannabichromene, cannabichromenic acid, or an analog thereof). Alternatively, enantiomeric mixtures (e.g., racemic mixtures) may be prepared and the desired enantiomer isolated by chiral chromatography or selective crystallization.

[0094] The chemical prenylation conversion step can be carried out at any suitable temperature. Typically, the conversion step is carried out at a temperature ranging from about 20°C to about 200°C, e.g., from about 20°C to about 100°C, or from about 20°C to about 80°C, or from about 20°C to about 70°C. The conversion step is carried out for a time sufficient to convert the non-prenylated compound to the prenylated product. The conversion time can range from a few minutes to several hours, depending on factors such as the particular prenyl compound used, the particular solvent used, and the state of the non-prenylated compound (e.g., present in the yeast mixture). In some embodiments, the reaction mixture is maintained at a temperature ranging from about 20°C to about 100°C (e.g., about 60°C) for a time ranging from about 5 minutes to about 360 minutes. In some embodiments, the reaction mixture is maintained at or about 60°C for less than 60 minutes (e.g., about 55 minutes, about 30 minutes, about 15 minutes, or about 10 minutes). In some embodiments, the reaction mixture is maintained at 20°C to 25°C (e.g., about 23°C) for 1 hour or more (e.g., about 60 minutes, or about 2 hours, or about 4 hours, or about 12 hours, or about 18 hours, or about 24 hours).

[0095] host cell In some embodiments, the host cell has been engineered to express an exogenous polynucleotide encoding an acyl-CoA synthetase, such as a revS polypeptide, a CsAAE3, or a CsAAE1 polypeptide; an exogenous polynucleotide encoding an olivetolic acid synthase; and / or an exogenous polynucleotide encoding an above-described 2-alkyl-4,6-dihydroxybenzoate cyclase (e.g., an olivetolic acid cyclase including an embodiment in which the olivetolic acid cyclase is truncated at the amino terminus, the carboxy terminus, or both).

[0096] Any methodology can be used to introduce polynucleotides into host cells. In some embodiments, exogenous polynucleotides encoding two or more enzymes described herein, such as acyl-CoA synthetase, olivetolic acid synthase, e.g., revS or CsAAE3, and 2-alkyl-4,6-dihydroxybenzoate cyclase (e.g., olivetolic acid cyclase or a genetically engineered variant thereof), are present in the same expression construct, e.g., a self-replicating expression vector, and are expressed as a multicistronic RNA, the expression of which is driven by the same promoter. Thus, for example, in some embodiments, an exogenous polynucleotide encoding olivetolic acid synthase and an exogenous polynucleotide encoding 2-alkyl-4,6-dihydroxybenzoate cyclase (e.g., olivetolic acid cyclase) are contained in the same expression construct, e.g., a self-replicating expression vector, separated by an internal ribosome entry site (IRES), and the expression of which is driven by the same promoter to produce a multicistronic mRNA. In some embodiments, the promoter is an alcohol dehydrogenase 2 promoter. In some embodiments, the exogenous polynucleotides are present in the same expression construct, for example, a self-replicating expression vector, and are operably linked to different promoters.In some embodiments, the exogenous polynucleotides are present in two or more expression constructs, for example, a self-replicating expression vector.In some embodiments, the self-replicating expression vector is a yeast artificial chromosome.In some embodiments, one or more exogenous polynucleotides are integrated into the host genome.In some embodiments, multiple exogenous polynucleotides are introduced into host cells by retrotransposon integration.

[0097] In some embodiments, cannabinoid compounds are produced using a compound of Formula IV or Formula IVa expressed in a host cell, and the host cell is further modified to express a polynucleotide encoding a prenyltransferase; a prenol and isoprenol kinase; a kinase that produces dimethylallyl pyrophosphate and isopentenyl pyrophosphate when grown in the presence of exogenous prenol and isoprenol; or a geranyl pyrophosphate synthase as described herein.As described in the previous paragraph, these polynucleotides can be contained in the same or separate expression vectors.

[0098] In some embodiments, the modified recombinant host cell further comprises an exogenous polynucleotide encoding a cannabinoid synthase enzyme that catalyzes the conversion of a first cannabinoid compound intermediate produced in the host cell to form a second cannabinoid compound.

[0099] In some embodiments, the host cell is a yeast or filamentous fungal host cell, such as an Aspergillus host cell. Yeast species that can be used as host cells include, but are not limited to, Saccharomyces, Schizosaccharomyces, Candida, Hansenula, Pichia, Kluyveromyces, Yarrowia, and Phaffia cells. Suitable yeast species include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Hansenula polymorpha, Pichia pastoris, P. canadensis, Kluyveromyces marxianus, Kluyveromyces lactis, Phaffia rhodozyma, and Yarrowia lipolytica.lipolytica). Filamentous fungal genera that can be used as host cells include Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Corynascus, Chaertomium, Cryptococcus, Filobasidium, Fusarium, Gibberella, Humicola, Magnaporthe, Mucor, Myceliophthora, Mucor, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Scytaldiu m, Schizophyllum, Sporotrichum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma cells. Exemplary species of filamentous fungi include Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans ...nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium lucknowense, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Bjerkandera adusta, Ceriporiopsis anneirina aneirina), Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis librosarivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Neurospora intermedia, Penicillium purpurogenum, Penicillium canescens canescens, Penicillium solitum, Penicillium funiculosum, Phanerochaete chrysosporium, Phlebia radiate, Pleurotus eryngii, Talaromyces flavus, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum longibrachiatum, Trichoderma reesei, and Trichoderma viride.

[0100] In some embodiments, the host cell is selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Yarrowia lipolytica, Hansenula polymorpha, and Aspergillus sp.

[0101] In the above embodiments, the gene may be encoded by a chemically synthesized gene encoding a wild-type or mutant enzyme derived from Cannabis sativa, Arabidopsis thaliana, or Pseudomonas species through yeast codon optimization.

[0102] Promoters used to drive transcription of genes in S. cerevisiae and other yeasts are well known in the art and include DNA elements controlled by glucose concentration in the growth medium, such as the alcohol dehydrogenase 2 (ADH2) promoter. If conditional expression is required, other regulatable or inducible promoters are used, such as the promoters driving the expression of the GAL1, MET25, and CUP1 genes. GAL1 and CUP1 are induced by galactose and copper, respectively, while MET25 is induced in the absence of methionine.

[0103] In some embodiments, the one or more exogenous polynucleotides are operably linked to a glucose-regulated promoter, hi some embodiments, expression of the one or more exogenous polynucleotides is driven by an alcohol dehydrogenase 2 promoter.

[0104] Other promoters strongly drive transcription in a constitutive manner, including, but not limited to, regulatory elements for highly expressed yeast glycolytic enzymes such as glyceraldehyde-3-phosphate dehydrogenase (GPD), phosphoglycerate kinase (PGK), pyruvate kinase (PYK), triosephosphate isomerase (TPI), enolase (ENO2), and alcohol dehydrogenase 1 (ADH1). Other strong constitutive promoters that can be used include the promoters derived from the S. cerevisiae transcription elongation factor EF-1α genes (TEF1 and TEF2) (Partow et al., Yeast. 2010, (11):955-64; Peng et al., Microb Cell Fact. 2015, (14):91-102), as well as the high-affinity glucose transporter (HXT7) and chaperonin (SSA1) promoters, which function well under low glucose conditions after the diauxic shift in S. cerevisiae (Peng et al., Microb Cell Fact. 2015, (14):91-102).

[0105] In other embodiments, host cells can increase cannabinoid production by increasing precursor pools, etc. Heterologous natural or chemically synthesized genes for enzymes such as malonyl-CoA synthase (Mutka et al., FEMS Yeast Res. 2006) and acetyl-CoA carboxylase 1 and 2, which are important for PKS biosynthesis, can be upregulated by malonate feeding. Similarly, acetyl-CoA synthase-1 and -2, and other gene products in the mevalonate pathway, such as acetoacetyl-CoA thiolase or the NphT7 gene product from Streptomyces species (Okamura et al., Proc Natl Acad Sci USA. 2010), HMG-CoA synthase, mevalonate kinase, phosphomevalonate kinase, mevalonate diphosphate decarboxylase, isopentenyl diphosphate:dimethylallyl diphosphate isomerase, HMG-CoA reductase, mutant farnesyl pyrophosphate synthase (ERG20; Zhao et al., 2016) from Saccharomyces or other eukaryotic species can also be introduced on a high-level expression plasmid vector or through genomic integration using methods well known to those skilled in the art.These methods can include the use of CRISPR Cas-9 technology, yeast artificial chromosome (YAC), or retrotransposon. Alternatively, if native to the host organism, these genes can be upregulated by methods of genetic element integration known to those skilled in the art.

[0106] In yet another aspect, similar genetic manipulation can be used to reduce the production of natural products that utilize carbon sources, such as ethanol, which leads to a reduction in the use of the carbon source for cannabinoid production.These genes can be completely "knocked out" by deletion from genome, or their activity can be reduced through reducing promoter strength, etc. These genes include the genes for enzymes ADH1 and / or ADH6.Other gene "knockout" includes genes involved in ergosterol pathway, such as ERG9, and the two most promising aromatic decarboxylase genes of yeast, PAD1 and FDC1.

[0107] Further embodiments include genes for accessory enzymes intended to enhance the production of end-product cannabinoids. One such enzyme, catalase, interacts with certain enzymes involved in the oxidative cyclization of CBGA and analogs, such as cannabidiolic acid synthase (Taura et al., 2007, FEBS Lett 581: 2929-2934), Δ 9 It is capable of neutralizing hydrogen peroxide produced by tetrahydrocannabinolic acid synthase (Sirikantaramas et al., 2004, J. Biol. Chem., 279:39767-39774.) and cannabichromene acid synthase (Morimoto et al., 1998, Phytochemistry 49: 1525-1529).

[0108] In a further embodiment, the genetically engineered host cell comprises endogenous or heterologous genes that are up-regulated or down-regulated, for example, to optimize the precursor pool for cannabinoid biosynthesis. Additionally, additional heterologous gene products can be expressed to provide "accessory" functions within the cell. For example, CBDA, Δ 9Overexpressed catalase can be expressed to neutralize the hydrogen peroxide formed in the oxidative cyclization step that yields important acidic cannabinoids such as -THCA and CBCA. "Accessory" genes and their expression products may be obtained through integration into the yeast genome through techniques well known in the art, or may be expressed from plasmids (also known as yeast expression vectors), yeast artificial chromosomes (YACs), or yeast transposons.

[0109] In some embodiments, as further described below, a host cell, such as a yeast strain, transformed or integrated with a plasmid or vector containing the above genes is transformed with a separate expression system for conversion of CBGA or a CBGA analog to a second acidic cannabinoid. In some such embodiments, the expression system is on the same vector, a separate vector, or integrated into the host cell genome.

[0110] The genetically engineered cells of the present invention that produce cannabinoids can be produced by transforming a host cell through genomic integration or by using an episomal plasmid (also called an expression vector, or simply a vector) with at least one nucleotide sequence encoding an enzyme involved in a genetically engineered metabolic pathway. As used herein, the terms "nucleotide sequence," "nucleic acid sequence," and "genetic construct" are used interchangeably and refer to a polymer of single- or double-stranded RNA or DNA, optionally containing synthetic, non-natural, or modified nucleotide bases. The nucleotide sequence may comprise one or more segments of cDNA, genomic DNA, synthetic DNA, or RNA. In some embodiments, the nucleotide sequence is codon-optimized to reflect the normal codon usage of the host cell without altering the polypeptide encoded by the nucleotide sequence. In certain embodiments, the term "codon optimization" or "codon-optimized" refers to modifying the codon content of a nucleic acid sequence to enhance expression in a particular host cell without altering the sequence of the polypeptide encoded by the nucleic acid. In certain embodiments, the term is intended to encompass modifying the codon content of a nucleic acid sequence as a means to regulate the expression level of a polypeptide (e.g., to increase or decrease the expression level). Thus, nucleic acid sequences encoding enzymes involved in the genetic engineering of metabolic pathways are described. In some embodiments, metabolically engineered cells may express one or more polypeptides that exhibit the enzymatic activity required to carry out the steps described below. In some embodiments, nucleotide sequences are synthesized and codon-optimized for expression in yeast according to the methods described in U.S. Pat. No. 7,561,972.

[0111] For example, a particular cell may contain one, two, three, four, five, or more than five nucleic acid sequences, each encoding a polypeptide necessary to produce a cannabinoid compound or a cannabinoid compound intermediate described herein. Alternatively, a single nucleic acid molecule may encode one or more polypeptides. For example, a single nucleic acid molecule may contain nucleic acid sequences encoding two, three, four, or even five different polypeptides. Nucleic acid sequences useful for the inventions described herein can be obtained from a variety of sources, such as amplification of cDNA sequences, DNA libraries, de novo synthesis, and excision of genomic segments. Sequences obtained from these sources can then be modified using standard molecular biology and / or recombinant DNA techniques to generate nucleic acid sequences with desired modifications. Exemplary methods for modifying nucleic acid sequences include, for example, site-directed mutagenesis, PCR mutagenesis, deletion, insertion, substitution, or exchange of portions of a sequence using restriction enzymes, optionally combined with ligation, homologous recombination, site-specific recombination, or various combinations thereof. In other embodiments, the nucleic acid sequence may be a synthetic nucleic acid sequence. Synthetic polynucleotide sequences can be generated using a variety of methods described in U.S. Patent No. 7,323,320 and U.S. Patent Application Publication Nos. 2006 / 0160138 and 2007 / 0269870. Methods for transformation of yeast cells are well known in the art.

[0112] Fermentation conditions Generally, cannabinoid production using the methods provided herein involves culturing a host cell (e.g., yeast or filamentous fungus) genetically engineered to contain the expression system described above. In some embodiments, the carbon source for yeast growth includes, for example, sugars, such as glucose, dextrose, xylose, or other sustainable raw sugars, such as raw sugars derived from cellulose sources. In other embodiments, the carbon source used can be methanol, glycerol, ethanol, or acetate. In some embodiments, the feedstock composition is refined experimentally to optimize yeast growth levels and final cannabinoid production levels, as measured using analytical techniques such as HPLC. In such embodiments, the method involves utilizing a glucose / ethanol or glucose / acetate mixture with a molar ratio of glucose to two carbon sources (ethanol or acetate) ranging from 50 / 50, 60 / 40, 80 / 20, or 90 / 10. The feedstock feed is optimized to induce glucose-regulated promoters and maximize the production of acetyl-CoA and malonyl-CoA precursors in the production strain. In some embodiments, a long-chain hydrocarbon component (e.g., decane, dodecane, oleic acid, methyl oleate, or isopropyl myristate) may be added to the culture (e.g., in an amount ranging from about 1% (w / v) to about 20% (w / v), e.g., 1-10% (w / v)).

[0113] In some embodiments, malonyl-CoA levels can be increased by supplying malonate (sodium salt) and expressing malonyl-CoA synthase (e.g., MatB / C from Rhizobium trifolii or homologs from related organisms such as Streptomyces species; see Biochem. J. (1999) 344: 159-166). In some embodiments, malonyl-CoA levels can be increased by overexpression of acetyl-CoA carboxylase and related pathways in biotin biosynthesis and biotin ligation, as well as pathways that produce acetyl-CoA, the precursor to malonyl-CoA.

[0114] In a further aspect of the present invention, olivetolic acid or its analogs may be obtained by chemical synthesis or biosynthesized in a recombinant production system. In some embodiments, olivetolic acid and its analogs are produced at high levels in the same yeast cell line containing the metabolic pathway for cannabinoid production. High-level production systems for monocyclic polyketide aromatic compounds in yeast are known in the art. See, for example, U.S. Patent No. 9,637,763. In other embodiments, the medium from a yeast strain that produces high levels of olivetolic acid or its analogs can be concentrated and used as a highly compatible feedstock in the MPF method for cannabinoid production.

[0115] Fermentation methods can be tailored to specific yeast strains by differences in carbon utilization pathways or expression regulation. For example, fermentation of Saccharomyces yeasts may require a single glucose feed, a complex nitrogen source (e.g., casein hydrolysate), and multiple vitamin supplements. This contrasts with the methylotrophic yeast Pichia pastoris, which may require glycerol, methanol, and trace mineral sources for optimal growth and expression, but only simple ammonium (nitrogen) salts. See, e.g., Elliott et al. J. Protein Chem. (1990) 9:95-104; U.S. Patent No. 5,324,639; and Fieschko et al. Biotechnol. Bioeng. (1987) 29:1113-1121. Culture media may include components such as yeast extract and peptone. Microorganisms can be cultured in conventional fermentation modes, including, but not limited to, batch, fed-batch, and continuous flow.

[0116] In some embodiments, the rate of glucose addition to the fermentor is adjusted so that the glucose addition rate is approximately equal to the rate of glucose consumption by the yeast. Under these conditions, significant amounts of glucose or ethanol accumulate. The glucose addition rate in this case can depend on factors including, but not limited to, the particular yeast strain, the fermentation temperature, and the physical dimensions of the fermentor.

[0117] In the MPF process, in batch mode, the precursor olivetolic acid (or another olivetolic acid analogue, such as 2-alkyl-4,6-dihydroxybenzoic acid), prenol, isoprenol, or geraniol can be present at a concentration of 0.1-50 grams / L (e.g., 1-10 g / L). In fed-batch mode, the precursors can be slowly fed into the fermentor over a period of 2-20 hours, resulting in a final addition of 1-100 grams / L (e.g., 1-10 grams / L, or 10-100 grams / L) of each required precursor.

[0118] Similarly, carboxylic acid starting materials (including substituted carboxylic acids, e.g., halogenated carboxylic acids, deuterated carboxylic acids, tritiated carboxylic acids, and hydroxylated carboxylic acids), such as substituted hexanoic acid, substituted butanoic acid, substituted pentanoic acid, etc., can be present at a concentration of 0.1 to 50 grams / L (e.g., 1 to 10 g / L). In fed-batch mode, the carboxylic acid can be slowly fed into the fermentor over a period of 2 to 72 hours (e.g., 15 to 60 hours) to achieve a final loading of 1 to 100 grams / L (e.g., 1 to 10 grams / L, or 10 to 100 grams / L) of carboxylic acid.

[0119] Culture conditions, such as expression time, temperature, and pH, can be adjusted to obtain high yields of the desired cannabinoid intermediate (e.g., olivetolic acid analogs) and / or the desired cannabinoid product (e.g., CBGA analogs, CBG analogs). Generally, host cells are cultured in the presence of starting materials, such as hexanoic acid, prenol, or isoprenol, for periods ranging from several hours to one day or more (e.g., 24, 30, 36, or 48 hours), depending on the particular host cell used, at temperatures ranging from about 20°C to about 40°C. For example, S. cerevisiae can be cultured at 25-32°C for 24-40 hours (e.g., 30°C for 30 hours). The pH of the culture medium can be maintained at a specific level by the addition of acids, bases, and / or buffers. In certain embodiments, yeast can be cultured at a pH of 6 or higher to reduce the production of undesirable by-products, such as olivetol. In some embodiments, the pH of the yeast culture ranges from about 6 to about 8. In some embodiments, the pH of the yeast culture is about 6.5. In some embodiments, the pH of the yeast culture is about 7. In some embodiments, the pH of the yeast culture is about 8.

[0120] In some embodiments, the recombinant yeast cells are genetically modified to produce a cannabinoid product or intermediate of interest at a level of at least about 0.1 g / L, at least about 0.25 g / L, at least about 0.5 g / L, at least about 0.75 g / L, at least about 1 g / L, at least about 1.5 g / L, at least about 2 g / L, at least about 2.5 g / L, at least about 3 g / L, at least about 3.5 g / L, at least about 4 g / L, at least about 4.5 g / L, at least about 5 g / L, at least about 5.5 g / L, at least about 6 g / L, at least about 7 g / L, at least about 8 g / L, at least about 9 g / L, or at least 10 g / L when cultured in vivo in a suitable precursor-containing medium as described above. In some embodiments, the recombinant yeast cells are genetically modified to produce the cannabinoid product or intermediate of interest at a level of at least about 20 g / L, at least about 30 g / L, at least about 50 g / L, or at least about 80 g / L when cultured in vivo in a suitable medium.

[0121] Cannabinoid production can be carried out in any vessel that allows cell growth and / or incubation.For example, the reaction mixture can be a bioreactor, a cell culture flask or cell culture plate, a multi-well plate (such as a 96-well, 384-well, 1056-well microtiter plate), a culture flask, a fermenter, or other vessel for cell growth or incubation.The biologically produced product of interest can be isolated from the luminescence medium or cell extract using methods known in the art.For example, solids or cell debris can be removed by centrifugation or filtration.The product of interest can be isolated by, for example, distillation, liquid-liquid extraction, membrane evaporation, adsorption, or other methods.

[0122] Conversion of 2-alkyl-4,6-dihydroxybenzoic acids to cannabinoid products Also provided herein are methods for producing cannabinoid products. In some embodiments, the method includes expressing a cannabinoid starting material in a yeast cell that has been genetically modified to express the cannabinoid starting material, isolating the yeast cell, and converting the cannabinoid starting material into a cannabinoid product in the isolated yeast cell. The cannabinoid starting material can be an acidic cannabinoid, a neutral cannabinoid, or a cannabinoid precursor, such as olivetolic acid or another 2-alkyl-4,6-dihydroxybenzoic acid. The step of converting the cannabinoid starting material can be carried out using the techniques described herein (e.g., chemical or enzymatic geranylation, thermal or enzymatic decarboxylation, etc.), and can be modified according to the identity of the particular cannabinoid starting material or the particular cannabinoid product. The cannabinoid starting material can be expressed, for example, using any of the expression systems described herein. The step of isolating yeast cells can optionally include: collecting yeast cells from culture medium by centrifugation, filtration or other means; washing yeast cells to remove culture medium or other components; removing at least a portion of liquid (e.g., culture medium) from the cells; and / or drying the cells (e.g., by freeze-drying or other means).Isolated yeast cells can be directly subjected to the reaction conditions for forming cannabinoid product.For example, yeast cells can be directly combined with solvent and other reagents as described below.

[0123] In some embodiments, the cannabinoid product is an acidic species, such as a compound of Formula V, or a cannabinoid derivative thereof. In some embodiments, the method further comprises converting the acidic species to a decarboxylated cannabinoid product, such as a compound of Formula Va, or a cannabinoid derivative thereof. Thus, the final cannabinoid product may be a neutral cannabinoid or an acidic cannabinoid. In some embodiments, the conversion of an intermediate compound, such as halogenated CBGA, to another cannabinoid is carried out by a physical or chemical process, such as heating, autoxidation, or ultraviolet light treatment. For example, the method may comprise decarboxylating an acidic cannabinoid in a genetically engineered yeast cell, or after full or partial purification of the cell, through the action of heat, or through the action of a wild-type or mutant decarboxylase enzyme contacting the cannabinoid acid in vivo or in vitro. Decarboxylation of an acidic cannabinoid yields the corresponding neutral cannabinoid. Non-limiting examples include R 1 Decarboxylation of a halogenated CBGA where is haloalkyl gives the corresponding halogenated CBG.

[0124] Cannabinoids formed to create completely unnatural analogs such as esters, ethers, and halogenated derivatives can be further chemically transformed for use as prodrugs or as more active or bioavailable drug substances. In some embodiments, whole yeast cells harboring the biosynthetic cannabinoid substrate can be subjected to this chemical reaction to avoid unnecessary purification steps prior to formation of the desired end product.

[0125] In some embodiments, a first cannabinoid product, which is a compound of Formula V or Formula Va, is converted into a second cannabinoid product through the action of a wild-type or mutant cannabinoid synthase or a wild-type or mutant cannabinoid acid synthase in the same genetically engineered host cell or through the co-culture of two or more recombinant host cell lines, such as yeast strains. For example, the expression system can encode a hemp-THCA synthase, a hemp-CBDA synthase, and / or a hemp-CBCA synthase. In some embodiments, the synthase is a hop-derived homolog, such as a hop-derived CBDA synthase homolog. CBGA analogs can be enzymatically converted into the corresponding CBDA analogs, THCA analogs, and CBCA analogs by the enzymes CBDA synthase, THCA synthase, and CBCA synthase, respectively, and the enzymatic conversion can be performed in vivo or ex vivo.

[0126] In some embodiments, a decarboxylase, such as Aspergillus nidulans orsB decarboxylase, can be used to decarboxylate acidic cannabinoids, such as halogenated CBGA or halogenated CBDA, to form neutral cannabinoid compounds, such as halogenated CBG or halogenated CBD. Alternatively, the acidic cannabinoids can be decarboxylated by maintaining them at elevated temperatures (e.g., about 40°C, 50°C, or 100°C) for times ranging from minutes to hours.

[0127] IV. Pharmaceutical Compositions Also provided herein are pharmaceutical compositions comprising one or more cannabinoid derivatives described above, or one or more pharmaceutically acceptable salts thereof, and one or more pharmaceutically acceptable excipients.

[0128] Pharmaceutical compositions can be prepared by any method known in the field of pharmacy and drug delivery.Generally, the method of preparing this composition comprises the step of combining active ingredients with a carrier containing one or more accessory ingredients.Generally, pharmaceutical compositions are prepared by uniformly and intimately combining active ingredients with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into desired formulation.This composition can be conveniently prepared and / or packaged in unit dosage form.

[0129] Pharmaceutical compositions can be in the form of sterile injectable aqueous or oily solutions and suspensions.Sterile injectable preparations can be formulated using non-toxic parenterally acceptable vehicles, including water, Ringer's solution, and isotonic saline, and acceptable solvents such as 1,3-butanediol.In addition, sterile fixed oils are usually used as solvents or suspension media.For this purpose, any non-irritating fixed oil, including synthetic monoglycerides or diglycerides, can be used.In addition, fatty acids such as oleic acid are used in the preparation of injectable solutions.

[0130] Aqueous suspensions contain a mixture of the active substance with excipients suitable for the manufacture of aqueous suspensions, including, but not limited to, suspending agents such as sodium carboxymethylcellulose, methylcellulose, oily propylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth, and gum acacia; dispersing or wetting agents such as lecithin, polyoxyethylene stearate, and polyethylene sorbitan monooleate; and preservatives such as ethyl, n-propyl, and p-hydroxybenzoates.

[0131] Oily suspensions can be formulated by suspending the active ingredient in vegetable oils, such as peanut oil, olive oil, sesame oil, or coconut oil, or mineral oils, such as liquid paraffin.Oily suspensions can contain thickening agents, such as beeswax, hard paraffin, or cetyl alcohol.These compositions can be preserved by adding antioxidants such as ascorbic acid.

[0132] Dispersible powders and granules (suitable for preparation of an aqueous suspension by the addition of water) may comprise the active ingredient in admixture with a dispersing agent, wetting agent, suspending agent, or combinations thereof. Additional excipients may also be present.

[0133] Pharmaceutical compositions can be in the form of oil-in-water emulsions.The oil phase can be vegetable oil, such as olive oil or peanut oil, or mineral oil, such as liquid paraffin, or their mixture.Suitable emulsifiers can be natural gums such as gum arabic or gum tragacanth; natural phospholipids such as soybean lecithin; esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan monooleate; and condensation products of the partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate.

[0134] Pharmaceutical compositions containing compounds may be in a form suitable for oral administration.Compositions suitable for oral administration include, but are not limited to, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups, elixirs, liquids, buccal patches, oral gels, chewing gums, chewable tablets, effervescent powders, and effervescent tablets.Compositions for oral administration can be formulated according to any method known to those skilled in the art.These compositions may contain one or more agents selected from the group consisting of sweeteners, flavorings, coloring agents, antioxidants, and preservatives to obtain pharmaceutically elegant and palatable formulations.

[0135] Tablets generally contain a mixture of the active ingredient and non-toxic pharmaceutically acceptable excipients, including inert diluents such as cellulose, silicon dioxide, aluminum oxide, calcium carbonate, sodium carbonate, glucose, mannitol, sorbitol, lactose, calcium phosphate, and sodium phosphate; granulating and disintegrating agents such as corn starch and alginic acid; binders such as polyvinylpyrrolidone (PVP), cellulose, polyethylene glycol (PEG), starch, gelatin, and gum arabic; and lubricants such as magnesium stearate, stearic acid, and talc. Tablets may be uncoated, or may be enteric-coated or otherwise coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby achieving a sustained action over a longer period of time. For example, a time-delay material such as glyceryl monostearate or glyceryl distearate may be used. Tablets may be coated with a semipermeable membrane and an optional polymeric osmogen according to known techniques to form osmotic pump compositions for controlled release.

[0136] Compositions for oral administration can be formulated as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent (e.g., calcium carbonate, calcium phosphate, or kaolin), or as soft gelatin capsules in which the active ingredient is mixed with water or an oil medium (e.g., peanut oil, liquid paraffin, or olive oil).

[0137] Pulmonary compositions also include, but are not limited to, dry powder compositions containing the cannabinoid derivatives described herein. Pulmonary compositions can be inhaled from any suitable dry powder inhaler known to those skilled in the art. In certain cases, the compositions can be conveniently delivered in the form of an aerosol spray from a pressurized pack or nebulizer, using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of pressurized aerosols, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges, such as gelatin, for use in an inhaler or insufflator can be formulated, containing a powder mixture of the compound and a suitable powder base, such as lactose or starch.

[0138] The cannabinoid derivatives provided herein may be administered topically as solutions, ointments, creams, gels, suspensions, eye drops, and the like. Additionally, transdermal delivery of the cannabinoid derivatives can be achieved by iontophoretic patches and the like. The cannabinoid derivatives may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, and therefore melts in the rectum to release the drug. These materials include cocoa butter and polyethylene glycol.

[0139] V. Methods of Treating Cannabinoid Receptor-Mediated Diseases Also provided herein are methods for treating diseases, conditions, and / or disorders, including those mediated by cannabinoid receptor activity, comprising administering to a subject in need thereof an effective amount of a cannabinoid derivative as described above. The method is useful for treating pain; skin conditions; muscle conditions, including but not limited to muscular dystrophy; metabolic syndromes, such as type 2 diabetes, dyslipidemia, and obesity; eating disorders; gastrointestinal disorders; allergies; asthma; chronic obstructive pulmonary disorder; glaucoma; cardiovascular diseases or disorders, such as hypertension, congestive heart failure, cardiac hypertrophy, peripheral arterial disease, atherosclerosis, stroke, myocardial infarction, and chemotherapy-related cardiotoxicity; fatty liver disease (steatohepatitis) and non-alcoholic fatty liver disease; kidney disease; diseases or disorders characterized by an addiction component, such as smoking addiction or withdrawal, alcohol addiction or withdrawal, and drug addiction or withdrawal; bone diseases or disorders, such as osteoporosis, Paget's disease of bone, and bone cancer; cancer, including but not limited to breast cancer; inflammatory or autoimmune diseases, such as rheumatoid arthritis, inflammatory bowel disease, and psoriasis; Tourette's syndrome; It can be used to treat several conditions, including, but not limited to, psychiatric diseases or disorders such as depression, anxiety, mania, schizophrenia, etc.; sleep disorders (e.g., insomnia); fatigue; disorders or diseases associated with memory loss and / or cognitive function loss, such as Parkinson's disease, Alzheimer's disease, and dementia; multiple sclerosis; epilepsy; spinal cord injury; and infectious diseases, such as bacterial, fungal, and viral infections.

[0140] The compound can be administered in any suitable dose in the present method. Generally, the compound is administered at a dose ranging from about 0.1 milligrams to about 1000 milligrams per kilogram of subject body weight (i.e., about 0.1-1000 mg / kg). The dose of the compound can be, for example, about 0.1-1000 mg / kg, or about 1-500 mg / kg, or about 25-250 mg / kg, or about 50-100 mg / kg. The dose can be about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 mg / kg.

[0141] Dosage can vary depending on the patient's needs, the severity of the disorder being treated, and the specific formulation being administered.The dosage administered to a patient should be sufficient to obtain a beneficial therapeutic response in the patient.The size of the dosage will also be determined by the existence, nature, and extent of any adverse side effects associated with the administration of the drug in a specific patient.Determining the dosage appropriate for a specific situation is within the skill of an ordinary medical practitioner.The total dosage can be divided and administered in several portions over a suitable time period to treat the condition or disorder.

[0142] Administration can be carried out over a period of time, which varies depending on the nature of specific disorders, their severity, and the patient's overall condition.Administration can be carried out twice a day, including every hour, every 2 hours, every 3 hours, every 4 hours, every 6 hours, every 8 hours, or every 12 hours, or at intermediate intervals therebetween.Administration can be carried out once a day, once every 36 hours or every 48 hours, or once every month or several months.After treatment, the patient can be monitored for changes in the patient's condition and the alleviation of the symptoms of the disorder.If the patient does not respond significantly to a specific dosage level, the dosage can be increased, and if the alleviation of the symptoms of the disorder is observed, or if the disorder is eliminated, or if unacceptable side effects are observed at a specific dosage, the dosage can be reduced.

[0143] A therapeutically effective amount of a cannabinoid derivative can be administered to a subject in a treatment regimen comprising an interval of at least 1 hour, 6 hours, 12 hours, 24 hours, 36 hours, or 48 hours between administrations. Administration can be performed at intervals of at least 72 hours, 96 hours, 120 hours, 168 hours, 192 hours, 216 hours, or 240 hours, or an equivalent number of days. The administration regimen can consist of two or more different interval sets. For example, the first part of the administration regimen can be administered to a subject multiple times a day, once a day, once every two days, or once every three days. The administration regimen can begin by administering the administration to a subject once every two days, once every three days, once a week, once every two weeks, or once a month. The first part of the administration regimen can be administered, for example, for up to 30 days, for example, for 7 days, 14 days, 21 days, or 30 days. A subsequent second portion of the dosing regimen may follow, with different intervals, such as once a week, once every 14 days, or once a month, lasting from 4 weeks to up to 2 years or more, for example, 4, 6, 8, 12, 16, 26, 32, 40, 52, 63, 68, 78, or 104 weeks. Alternatively, if the disorder goes into remission or gradually improves, the dosage may be maintained or kept below the maximum amount. If the condition or disorder recurs, the first dosing regimen may be resumed until improvement is observed, and the second dosing regimen may be administered again. This cycle may be repeated multiple times as needed.

[0144] Additional active agents or therapeutic agents can be co-administered with cannabinoid derivatives or otherwise combined.Suitable additional active agents and therapeutic agents for use in this method include but are not limited to compounds used in the treatment of type 2 diabetes and obesity, such as insulin and insulin analogues, dipeptidyl peptidase 4 (DPP-4) inhibitors, glucagon-like peptide 1 analogues, hypoglycemic agents such as α-glucosidase inhibitors, biguanides, sulfonylureas, thiazolidinediones, weight loss therapeutic agents such as appetite suppressants, serotonin reuptake inhibitors, noradrenaline reuptake inhibitors, β3-adrenergic receptor agonists and lipase inhibitors. The compound used in the treatment of cardiovascular disease and cardiovascular dysfunction can be used in this method, including but not limited to diuretics, angiotensin converting enzyme (ACE) inhibitors, angiotensin II antagonists, beta-blockers, calcium antagonists such as nifedipine, HMG-CoA reductase inhibitors such as statins, digoxin, aldosterone antagonists and organic nitrates.Other lipid-regulating agents can be used in this method, including but not limited to fibrates and bile acid-binding resins.Cannabinoid derivatives can also be used with the compound used to support smoking cessation, including but not limited to norepinephrine-dopamine reuptake inhibitors such as bupropion.

[0145] In this method, compounds used in the treatment of bone disease and bone disorders can be used in combination with cannabinoid derivatives.These compounds include but are not limited to anti-resorptive agents, such as bisphosphonates, anabolic agents, such as parathyroid hormone, RANKL inhibitors, such as denosumab; and estrogen replacement and selective estrogen receptor modulators, such as raloxifene.Cancer treatment agents can be used in combination with cannabinoid derivatives.Examples of anti-cancer agents include but are not limited to chemotherapeutic agents (such as carboplatin, paclitaxel, pemetrexed, etc.), tyrosine kinase inhibitors (such as erlotinib, crizotinib, osimertinib, etc.), and immunotherapeutic agents (such as pembrolizumab, nivolumab, durvalumab, atezolizumab, etc.).

[0146] Compounds used in the treatment of diseases or disorders with an inflammatory or autoimmune component may be used in combination with cannabinoid derivatives. These compounds include nonsteroidal anti-inflammatory drugs (NSAIDs); disease-modifying antirheumatic drugs, such as immunosuppressants; anti-TNF agents, such as infliximab, etanercept, and adalimumab; and anti-B cell therapeutics, such as rituximab.

[0147] The method may involve the use of compounds used in the treatment of psychiatric diseases and disorders in combination with cannabinoid derivatives. These compounds include GABA A Modulators, e.g., benzodiazepines; 5HT 1AReceptor agonists, such as buspirone; beta-blockers; antipsychotics, such as dopamine receptor blockers, and other drugs that regulate monoamine receptors, monoamine transporters, or monoamine metabolism, such as tricyclic antidepressants, serotonin reuptake inhibitors, and monoamine oxidase inhibitors; lithium; and antiepileptic drugs, such as sodium channel blockers, T-type calcium channel blockers, or GABA transaminase or GABA reuptake blockers, including phenytoin, carbamazepine, valproic acid, and vigabatrin.The compound used in the treatment of the disease or disorder characterized by memory impairment and / or cognitive function loss, including but not limited to these dopamine agonists and anticholinesterase agents, can be used in this method.

[0148] Examples of antibiotics that can be used in conjunction with cannabinoid derivatives in the treatment of bacterial infections include quinolones (e.g., moxifloxacin, gemifloxacin, ciprofloxacin, ofloxacin, trovafloxacin, sitafloxacin, etc.), beta-lactams (e.g., penicillins, e.g., amoxicillin, amoxicillin-clavulanate, piperacillin-tazobactam, penicillin G, etc.); as well as cephalosporins (e.g., ceftriaxone, etc.), macrolides (e.g., erythromycin, azithromycin, clarithromycin, etc.), aminoglycosides (e.g., amikacin, gentamicin, kanamycin, neomycin, netilmicin, tobramycin, etc.), monobactams (e.g., aztreonam, etc.), carbapenems (e.g., doripenem, imipenem, meropenem, ertapenem, etc.), thiazolides (e.g., tizoxanidine, nitazoxanidine, RM 4807, RM 4809, etc.), tetracyclines (e.g., tetracycline, minocycline, doxycycline, eravacycline, etc.), lincosamides (e.g., lincomycin, clindamycin, etc.), sulfonamides (e.g., trimethoprim, sulfamethoxazole, etc.), and nitroimidazoles (e.g., metronidazole, satranidazole, etc.). Examples of antiviral agents that can be used in conjunction with cannabinoid derivatives in the treatment of viral infections include, but are not limited to, viral uncoating inhibitors (e.g., amantadine, rimantadine, etc.), neuraminidase inhibitors (e.g., oseltamivir, zanamivir, laninamivir, and peramivir), reverse transcriptase inhibitors (e.g., tenofovir, stavudine, zidovudine, zalcitabine, emtricitabine, lamivudine, etc.), and protease inhibitors (e.g., ritonavir, indinavir, boceprivir, etc.), and polynucleotide synthesis inhibitors (e.g., sofosbuvir, dasabuvir, etc.). [Example]

[0149] VI. Working Examples Example 1 Formation of 2,4-dihydroxy-6-perdeuteropentylbenzoic acid and 5-perdeuteropentylbenzene-1,3-diol in S. cerevisiae The S. cerevisiae ADH2 promoter was chemically synthesized and fused to a synthetic gene encoding a mutant asa acyl-activating enzyme 1 (CsAAE1ΔTM) lacking the transmembrane domain coding sequence (amino acids 245–267). The S. cerevisiae ADH2 transcription termination sequence was also fused to the gene sequence adjacent to the synthetic stop codon. The expression cassette was cloned into a yeast expression vector containing a URA3 selectable marker. Similarly, the synthetic genes for the acyl-activating enzyme CsAAE3 (derived from asa) and revS (medium-chain fatty acid acyl-CoA ligase from Streptomyces sp. SN-593) were cloned into separate URA3 vectors. Each URA3-based vector was transformed into competent Saccharomyces cerevisiae InvSc1 (MATa / alpha his3D1 leu2 trp1-289 ura3-52) cells (Invitrogen). These cells had been transformed with a selectable marker LEU2-based vector containing several individually mutated asaolivetolic acid cyclase genes and an asaolivetolic acid synthase / tetraketide synthase (OAS / TKS) gene fused to a synthetic gene encoding the Arabidopsis cyclase enzyme AtHS1 via an S. cerevisiae p150 internal ribosome entry site (IRES) and the human ubiquitin gene.

[0150] Transformants were plated onto minimal agar plates containing amino acids for selection based on uracil and leucine prototrophy (6.7 g / L yeast nitrogen base (DIFCO) without amino acids or ammonium sulfate, 20 g / L glucose, 20 g / L agar). Transformants were removed and grown for 24 hours in minimal medium lacking uracil and leucine. Plasmid DNA was isolated from the transformants and analyzed by restriction digestion to confirm identity.

[0151] Successful transformants for each strain were used to inoculate 2 mL of minimal medium lacking uracil and leucine, which was grown overnight at 30°C in an orbital shaker. A 500 μL aliquot of this culture was used to inoculate 50 mL of the same medium, and the culture was grown for 24 hours at 30°C in an orbital shaker. Cultures were similarly inoculated into 300 mL of the same medium and, after overnight growth, transferred to an aerobic, stirred, 7-liter fermentor (Eppendorf) containing 1.2 L of 2X YEP medium (Wobbe, in Current Protocols in Molecular Biology, Supplement 34:13.0.1-13.13.9 (Wiley, 1996)) (20 g / L yeast extract, 40 g / L peptone).

[0152] Approximately 16 hours after seeding, after all residual glucose was consumed, perdeuterated hexanoic acid (D 11 0.2 grams of α-HA) was added directly to the fermenter, and the culture was supplemented with 14.3% glucose, 3.5% sodium acetate, and D 11 2X YEPD containing 0.8 grams of -HA was fed over an elapsed fermentation time of 72 hours.

[0153] Cells were collected by centrifugation of 500 μL aliquots of cultures harvested after 24, 48, and 72 hours of growth and lysed by boiling for approximately 2 minutes in 50 μL of 2X SDS gel loading buffer. Cell lysates were analyzed by loading onto a 4-20% SDS-PAGE gel. A band corresponding to the predicted size of the encoded enzyme was observed.

[0154] For further quantification and analog confirmation, cells were separated from the medium by centrifugation, the medium was acidified with glacial acetic acid, and the deuterated product was extracted using ethyl acetate. The product was further purified by column chromatography or by using a Sep-Pak C18 cartridge with acetonitrile / formic acid elution and subjected to NMR and mass spectrometry.

[0155] TIFF2025170350000027.tif191282,4-Dihydroxy-6-perdeuteropentylbenzoic acid (1). TIFF2025170350000028.tif19153

[0156] TIFF2025170350000029.tif191285-Perdeuteropentylbenzene-1,3-diol (2). LC-MS / ESI: Calculated value [C 11 H5D 11 O2] 191.18; Found [M+H] 192.35.

[0157] High levels of the analogs were observed to be biosynthesized using various acyl-activating enzymes: revS (>40 mg / L); CsAAE3 (approximately 20-30 mg / L); and CsAAE1ΔTM (3-4 mg / L). The product distribution from olivetolic acid to olivetol analogs varied depending on the actual length of the mutant cyclase used, with AtHS1 cyclase yielding essentially exclusively olivetol analogs (5-perdeuteropentylbenzene-1,3-diol).

[0158] Example 2 Formation of 2,4-dihydroxy-6-(5-fluoropentyl)-benzoic acid, 2,4-dihydroxy-6-(4-fluorobutyl)-benzoic acid, 5-(5-fluoropentyl)-benzene-1,3-diol, and 5-(4-fluorobutyl)-benzene-1,3-diol in S. cerevisiae The strain described in Example 1 expressing the acyl-activating enzymes revS and CsAAE3 was grown in 4 mL of selective medium at 30°C for 24 hours and then inoculated into 1X YPD to obtain a total cell culture volume of 40 mL. After 30 hours of growth at 30°C, 6-fluorohexanoic acid or 5-fluoropentanoic acid was added to the culture to obtain a total concentration of 2 mM, and the culture was grown for an additional 48 hours at 30°C. Analog production was monitored by HPLC, and purification was achieved as described above. The yield of 2,4-dihydroxy-6-(4-fluorobutyl)-benzoic acid was approximately 5 mg / L, while the yield of 2,4-dihydroxy-6-(5-fluoropentyl)-benzoic acid was approximately 60 mg / L.

[0159] 2,4-Dihydroxy-6-(4-fluorobutyl)-benzoic acid. LC-MS / ESI: Calculated [C 11 H 13 FO4] 228.08; measured values ​​[MH] 226.95, [MH-CO2] 183.05.

[0160] 2,4-Dihydroxy-6-(5-fluoropentyl)-benzoic acid. LC-MS / ESI: Calculated [C 12 H 15 FO4] 242.10; measured values ​​[MH] 240.95, [MH-CO2] 197.05, [2M-H] 482.80.

[0161] Example 3 Formation of 2,4-dihydroxy-6-(5-fluoropentyl)-benzoic acid and 5-(5-fluoropentyl)-benzene-1,3-diol in S. cerevisiae The strain described in Example 1 expressing the acyl-activating enzymes revS and CsAAE3 is grown in 4 mL of selective medium at 30°C for 24 hours and then inoculated into 1X YPD to give a total cell culture volume of 40 mL. After 30 hours of growth at 30°C, 6-fluorohexanoic acid is added to the culture to a total concentration of 2 mM, and the culture is grown for an additional 48 hours at 30°C. Analog production is monitored by HPLC, and purification is achieved as described above.

[0162] Example 4 Formation of 2,4-dihydroxy-6-(3-fluoropropyl)-benzoic acid and 5-(3-fluoropropyl)-benzene-1,3-diol in S. cerevisiae The strains described in Example 1 expressing revS and CsAAE3 were grown in 4 mL of selective medium at 30°C for 24 hours and then inoculated into 1× YPD to obtain a total cell culture of 40 mL. After 30 hours of growth at 30°C, 4-fluorobutanoic acid was added to the culture to a total concentration of 2 mM, and the culture was grown for an additional 48 hours at 30°C. Analog production was monitored by HPLC, and purification was achieved as described above. Unlike perdeutero analog production, the 3-fluoropropyl divalinic acid analog was only observed in strains expressing revS. Strains expressing CsAAE3 did not produce the analog at levels above the detection limit by HPLC. As described above, a complete change in the ratio of the acid analog to the decarboxylated fluoro-divalinol analog was observed using the truncated (95 amino acid) asa cyclase or the AtHS1 cyclase.

[0163] Example 5 Formation of 6-(4-chlorobutyl)-2,4-dihydroxybenzoic acid and 5-(4-chlorobutyl)-benzene-1,3-diol in S. cerevisiae The strain described in Example 1 expressing revS was grown in 50 mL of selective medium directly from the selective culture plate at 30°C for 24 hours and then inoculated into 2X YEPD to give a total cell culture volume of 500 mL. After 30 hours of growth at 30°C, 5-chloropentanoic acid was added to the culture to a total concentration of 2 mM, and the culture was grown for an additional 48 hours at 30°C. Analog production was monitored by the appearance of a reverse-phase HPLC peak in the predicted region, and purification was achieved as described above. The yield of 6-(4-chlorobutyl)-2,4-dihydroxybenzoic acid in the culture was approximately 30 mg / L.

[0164] 6-(4-chlorobutyl)-2,4-dihydroxybenzoic acid. LC-MS / ESI: Calculated value [C 11 H 13 ClO4] 244.05; found [MH] 242.95.

[0165] In all of the above examples, media containing various amounts of acid analogs and olivetol / divalinol analogs could be quantitatively converted to the decarboxylated analogs by removing the yeast cells by centrifugation, if desired, and heating the remaining analog-containing media at 100°C for 1 hour.

[0166] Example 6 Use of an organic phase overlay to reduce the toxicity of starting materials and products Perdeuterohexanoic acid, 6-fluorohexanoic acid, 4-fluorobutanoic acid, 5-chloropentanoic acid, hexanoic acid, and butanoic acid were individually fed to the yeast strains described above in Examples 1-3. Cultivation of the cells proceeded as described in Example 3, except that 10% by volume of oleyl alcohol was added to the culture along with the aliphatic acid or aliphatic acid analog at the 30 hour time point. This procedure resulted in increased levels of the desired product.

[0167] Example 7 Direct production of CBGA analogues in S. cerevisiae The deutero-, fluoro-, and chloroaliphatic acid analogs, along with hexanoic and butanoic acids, were fed to yeast strains grown as described in Examples 1-3 above, except that they had already been modified by integrative transformation of genes involved in upregulating the yeast mevalonate pathway to produce high levels of geranyl diphosphate. The strains also carried integrated genes expressing various prenyltransferases for the conversion of olivetolic acid analogs to CBGA analogs. The resulting CBGA analogs were isolated from centrifuged yeast cells by solvent extraction using methanol, ethanol, or ethyl acetate and characterized by mass spectrometry and NMR analysis. 6-(4-chlorobutyl)-3-(3,7-dimethyl-octa-2,6-dienyl)-2,4-dihydroxybenzoic acid was prepared using this method. LC-MS / ESI: Calculated [C 21 H 29 ClO4] 380.18; found [M+H] 380.95 (Cl), [MH] 378.85 (Cl). TIFF2025170350000030.tif18128

[0168] 3-(3,7-Dimethyl-octa-2,6-dienyl)-6-(5-fluoropentyl)-2,4-dihydroxy-benzoic acid (5-fluoro-cannabigerolic acid) was also prepared using this method. LC-MS / ESI: Calculated [C 22 H 31 FO4] 278.22; measured values ​​[M+H] 379.05, [MH] 376.90. TIFF2025170350000031.tif18128

[0169] Example 8 Chemical transformation of olivetol / olivetolic acid analogues into CBC / CBCA analogues. CBCA and CBC analogs were prepared as follows. To a solution of 35 mg (0.2 mmol) of perdeuteropentyl-olivetolic acid or perdeuteropentyl-olivetolic acid in 0.5 mL of dichloroethane, 0.085 mL (approximately 2.5 equivalents) of E / Z-citral was added, followed by 0.005 mL (25 mol%) of N,N-dimethylethylenediamine. The reaction was initiated at 23 °C. The reaction was monitored by quantitative RP-HPLC; no substrate remained after 18 h. The reaction mixture was purified directly by a single injection onto a Gilson preparative C18 RP-HPLC automated system using a steep linear gradient of water / MeOH / 0.1% formic acid (2.5 mL / min). Fractions were monitored by UV (230 nm), and appropriate fractions were combined, concentrated under reduced pressure, and reconcentrated in MeOH to remove residual water, affording the products in molar yields ranging from 65% to 73%. The CBCA and CBC analogs were characterized by mass spectrometry and NMR analysis.

[0170] 2-Methyl-2-(4-methyl-pent-3-enyl)-7-perdeuteropentyl-2H-chromen-5-ol (perdeuteropentyl-CBC). LC-MS / ESI: calculated [C 21 H 19 D 11O2] 325.29; Found: [M+H] 326.25.

[0171] 5-Hydroxy-2-methyl-2-(4-methyl-pent-3-enyl)-7-perdeuteropentyl-2H-chromene-6-carboxylic acid (perdeuteropentyl-CBCA). LC-MS / ESI: Calculated [C 22 H 19 D 11 O4] 369.28; Measured values: [MH] 367.90, [MH-CO2] 324.00.

[0172] Fluorinated and chlorinated CBC / CBCA analogs are prepared as described above for the perdeuterated analogs.

[0173] Example 9 Preparation and Use of N-Acetylcysteine ​​Amides and Pantetheine Thioesters To a stirred solution of 5.5 mmol of carboxylic acid containing 5 mmol of succinic acid and 5 mmol of N-acetylcysteamine (or pantetheine) in 4 mL of dichloromethane (DCM) at 0 °C, 1 mL of a cold solution of 5.25 mmol of dicyclohexylcarbodiimide (DCC) in DCM was added. The solution was warmed to 23 °C and stirred for 18 h. After cooling to 0 °C, the resulting insoluble N,N'-dicyclohexylurea was filtered off and washed with cold DCM. The solvent was removed, and the residue was redissolved in DCM and, after refiltration as necessary, extracted with 1 N HCl, followed by 5% NaHCO3 in saturated aqueous NaCl. The organic layer was dried over NaSO4 and concentrated to give the thioester in high yield. The product was further purified by recrystallization, distillation, or chromatography. Specific examples include thioesters prepared from hexanoic acid, butanoic acid, pentanoic acid, heptanoic acid, octanoic acid, and fluorine- or deuterium-substituted alkyl and alkenyl acids, such as 4-fluorobutanoic acid, 5-fluoropentanoic acid, 6-fluorohexanoic acid, and perdeuterohexanoic acid.

[0174] The thioesters are fed to a level of 2 mM in cultures such as those described in Examples 1 and 2, and samples are taken for HPLC analysis after 30 hours and after an additional 46 hours of growth. The yield of each acid product is determined to be greater than 40 mg / L.

[0175] Example 10 Chemical transformation of olivetolic acid analogues into CBGA analogues To a suspension of 20 mg of deutero-, fluoro-, or chloroolivetolic acid analogs in 0.25 mL of toluene, add 2.6 mg of p-toluenesulfonic acid and 18 μL of geraniol. The suspension is heated to 60 °C and monitored by reverse-phase HPLC (Kinetex 5 μm-XB, 50 x 4.6 mm, 100 A column, linear gradient from 20% 50 mM ammonium formate / acetonitrile to 100% acetonitrile, 2.5 mL / min over 6 min). The corresponding CBGA analogs reach maximum yield after approximately 50 min and are identified and characterized by mass spectrometry and NMR.

[0176] Example 11 Formation of 5-fluorocannabichromene and 5-chlorocannabichromene 2,4-Dihydroxy-6-(5-fluoropentyl)-benzoic acid was prepared according to Example 2, decarboxylated, and converted to 5-fluorocannabichromene. A 37.5 mg sample of 2,4-dihydroxy-6-(5-fluoropentyl)-benzoic acid was taken up in 1 mL of 95% ethanol and heated at 80° C. for 18 hours to completely decarboxylate, yielding 5-(5-fluoropentyl)-benzene-1,3-diol. LC-MS / ESI: Calculated [C 11 H 15 FO2] 198.11; found [M+H] 199.25, [MH] 197.05. The resulting solution was concentrated to dryness (under reduced pressure) and subjected to reaction conditions similar to those outlined in Example 8. 11.1 mg of 7-(5-fluoropentyl)-2-methyl-2-(4-methyl-pent-3-enyl)-2H-chromen-5-ol (5-fluorocannabichromene) was obtained. TIFF2025170350000032.tif34158

[0177] Similarly, 5-chlorocannabichromene was prepared from 2,4-dihydroxy-6-(4-chlorobutyl)-benzoic acid (see Example 5). TIFF2025170350000033.tif26159

[0178] Example 12 CB2 receptor agonist activity of 5-fluorocannabichromene The activity of 5-fluorocannabichromene (5F-CBC) was tested in AtT20 cells stably expressing HA-tagged human CB1 and CB2 receptors as described by Udoh et al. ("Cannabichromene is a cannabinoid CB2 receptor agonist." British Journal of Pharmacology, 2019, doi: 10.1111 / bph.14815). The EC2 of 5F-CBC at CB2 was 50 The observed value was 2.1 μM.

[0179] VII. Illustrative Embodiments Exemplary embodiments provided in accordance with the subject matter disclosed herein include, but are not limited to, the following embodiments and the accompanying claims. 1. Compounds of Formula I TIFF2025170350000034.tif20128, or a salt or cannabinoid derivative thereof: During the ceremony, R 1 is C1~C 20 Haloalkyl, C1-C 20 Hydroxyalkyl, deuterated C1-C 20 Alkyl, tritiated C1-C 20 Alkyl, and C2-C 20 alkenyl, R 2 COOR 2a and H, R 2a is selected from the group consisting of H and C1-C6 alkyl; R3 is selected from the group consisting of a prenyl moiety and H. 2. R 1 However, C1~C 10 Haloalkyl, C1-C 10 Hydroxyalkyl, deuterated C1-C 10 Alkyl, tritiated C1-C 10 Alkyl, and C2-C 10 The compound of embodiment 1, or a salt or cannabinoid derivative thereof, selected from the group consisting of: alkenyl 3. R 1 However, C1~C 10 Haloalkyl, C1-C 10 Hydroxyalkyl, deuterated C1-C 10 Alkyl and tritiated C1-C 10 The compound of embodiment 1, or a salt or cannabinoid derivative thereof, wherein the compound is selected from the group consisting of alkyl. 4. R 1 C1~C 10 The compound of embodiment 1, or a salt or cannabinoid derivative thereof, wherein: 5. R 1 The compound of embodiment 4, or a salt or cannabinoid derivative thereof, wherein is selected from the group consisting of fluoropentyl, fluoroethyl, fluoropropyl, fluorobutyl, fluorohexyl, fluorooctyl, and fluorononyl. 6. R 1 The compound of embodiment 4, or a salt or cannabinoid derivative thereof, wherein is selected from the group consisting of 5-fluoropropyl, 4-fluorobutyl, and 3-fluoropentyl. 7. R 1 C1~C 10 Bromoalkyl or C1-C 10 The compound of embodiment 4, or a salt or cannabinoid derivative thereof, which is chloroalkyl. 8. R 1 C1~C 10 The compound of embodiment 1, or a salt or cannabinoid derivative thereof, wherein: 9. R 1 Deuterated C1~C10 Alkyl or tritiated C1-C 10 The compound of embodiment 1, or a salt or cannabinoid derivative thereof, wherein: 10. R 2 The compound of any of embodiments 1-9, or a salt or cannabinoid derivative thereof, wherein is selected from the group consisting of COOH and H. 11. R 2 11. The compound of any one of aspects 1-10, or a salt or cannabinoid derivative thereof, wherein 12. R 2 11. The compound of any one of embodiments 1 to 10, or a salt or cannabinoid derivative thereof, wherein 13. R 3 13. The compound of any one of aspects 1 to 12, or a salt or cannabinoid derivative thereof, wherein 14. R 3 13. The compound of any of embodiments 1-12, or a salt or cannabinoid derivative thereof, wherein is a prenyl moiety. 15. The compound of any one of aspects 1-12 and 14, or a salt or cannabinoid derivative thereof, wherein the prenyl moiety is 3,7-dimethylocta-2,6-dien-1-yl. 16. The compound has the structure of Formula Ia The compound of embodiment 1, or a salt or cannabinoid derivative thereof, having TIFF2025170350000035.tif28128. 17. A cannabinoid derivative or a salt thereof of the compound of any one of Aspects 1 to 16. 18. Halogenated cannabidiolic acids, halogenated cannabidiols, halogenated Δ 9 -Tetrahydrocannabinolic acid, halogenated Δ 8 -Tetrahydrocannabinolic acid, halogenated cannabichromenic acid, halogenated cannabichromene, halogenated cannabinol, halogenated cannabinodioI, halogenated cannabinolic acid, cannabivarin, halogenated cannabivarinic acid, halogenated Δ 9 -Tetrahydrocannabivarin, halogenated Δ 8-Tetrahydrocannabivarin, halogenated Δ 9 -Tetrahydrocannabivaric acid, halogenated Δ 8 18. The cannabinoid derivative of embodiment 17, or a pharmaceutically acceptable salt thereof, selected from the group consisting of tetrahydrocannabivarin, halogenated cannabigerovarin, halogenated cannabigerovarin acid, halogenated cannabichromevarin, halogenated cannabichromevarinic acid, halogenated cannabidivarin, halogenated cannabidivarinic acid, halogenated cannabiditriol, and halogenated cannabicyclol. 19. A pharmaceutical composition comprising the cannabinoid derivative of embodiment 17 or embodiment 18 and a pharmaceutically acceptable excipient. 20. A method for treating a disease or condition mediated by cannabinoid receptor activity, comprising administering to a subject in need thereof an effective amount of the cannabinoid derivative of embodiment 17 or embodiment 18, or a pharmaceutically acceptable salt thereof, or an effective amount of the composition of embodiment 19. 21. Compound of Formula IV 1. A method for producing TIFF2025170350000036.tif20128, or a salt thereof, comprising: In the formula, R 1 is C1~C 20 Haloalkyl, C1-C 20 Hydroxyalkyl, deuterated C1-C 20 Alkyl, tritiated C1-C 20 Alkyl, and C2-C 20 alkenyl, The method comprises reacting a thioester of formula II culturing the modified recombinant host cell in a medium containing TIFF2025170350000037.tif12128; In the formula, R 4 is selected from the group consisting of a coenzyme A (CoA) moiety, a pantetheine moiety, and a cysteamine moiety; the modified recombinant host cell iii. ester of formula II and malonyl-CoA to form a tetraketide of formula III a first polynucleotide encoding a synthase that converts the nucleotide sequence of ... iv. A second polynucleotide encoding a 2-alkyl-4,6-dihydroxybenzoate cyclase that converts the tetraketide of formula III to a compound of formula IV. and the modified recombinant host cell is cultured under conditions whereby the products encoded by the first and second polynucleotides are expressed to produce the compound of Formula IV; method. 22. R 1 However, C1~C 10 Haloalkyl, C1-C 10 Hydroxyalkyl, deuterated C1-C 10 Alkyl, tritiated C1-C 10 Alkyl, and C2-C 10 22. The method of embodiment 21, wherein the alkyl group is selected from the group consisting of alkenyl. 23. The method of embodiment 21 or embodiment 22, wherein the synthase is olivetolic acid synthase. 24. The method of any one of aspects 21 to 23, wherein the 2-alkyl-4,6-dihydroxybenzoate cyclase is a truncated olivetolic acid cyclase. 25. R 4 is a CoA moiety. 26. The host cell is a starting material of formula IIa and wherein step (a) comprises culturing the host cell under conditions in which a product encoded by the third polynucleotide is expressed to produce the thioester of Formula II. The method of any one of aspects 21 to 25. 27. The method of embodiment 26, wherein the acyl-CoA synthetase is revS or CsAAE3. 28. R in the thioester of formula II 4is a pantetheine moiety or a cysteamine moiety. 29. A compound of formula IV is reacted with a compound of formula V TIFF2025170350000040.tif20128 or a salt thereof, wherein R 3 The method of any of embodiments 21-28, wherein 30. Decarboxylate the compound of formula V to give the compound of formula Va. The method of embodiment 29, further comprising obtaining TIFF2025170350000041.tif20128. 31. Decarboxylation of a compound of formula IV to give a compound of formula IVa The method of any of embodiments 21 to 28, further comprising obtaining TIFF2025170350000042.tif20128. 32. Reacting a compound of formula IVa with a compound of formula Va TIFF2025170350000043.tif20128, wherein R 3 is a prenyl moiety. 33. The method of any of embodiments 29, 30, and 32, wherein the host cell further comprises a fourth polynucleotide encoding a prenyltransferase enzyme that converts the compound of formula IV to the compound of formula V, and wherein converting step (b) comprises culturing the host cell under conditions wherein the product encoded by the fourth polynucleotide is expressed to produce the compound of formula V. 34. The method of embodiment 32, wherein the host cell further comprises a fourth polynucleotide encoding a prenyltransferase enzyme that converts the compound of formula IVa to a compound of formula Va, and wherein converting step (b) comprises culturing the host cell under conditions wherein a product encoded by the fourth polynucleotide is expressed to produce the compound of formula Va. 35. The method of embodiment 33 or embodiment 34, wherein the prenyltransferase is geranyl pyrophosphate:olivetolic acid geranyltransferase. 36. The method of embodiment 29 or embodiment 30, wherein converting step (b) comprises forming a reaction mixture comprising (1) a compound of formula IV, (2) geraniol, activated geraniol, or citral, and (3) an organic solvent, and maintaining the reaction mixture under conditions sufficient to produce a compound of formula V. 37. The method of embodiment 32, wherein the converting step (b) comprises forming a reaction mixture comprising (1) a compound of formula IVa, (2) geraniol, activated geraniol, or citral, and (3) an organic solvent, and maintaining the reaction mixture under conditions sufficient to produce a compound of formula Va. 38. Cannabidiolic acid analogs, cannabidiol analogs, Δ 9 -Tetrahydrocannabinolic acid analogues, Δ 8 -Tetrahydrocannabinolic acid analogues, cannabichromene acid analogues, cannabichromene analogues, cannabinol analogues, cannabinodiol analogues, cannabinolic acid analogues, cannabivarin analogues, cannabivarinic acid analogues, Δ 9 -tetrahydrocannabivarin analogues, Δ 8 -tetrahydrocannabivarin analogues, Δ 9 -tetrahydrocannabivaric acid analogues, Δ 8 - The compound prepared according to any of embodiments 29, 30, and 32-37, wherein the compound is a tetrahydrocannabivarinic acid analog, a cannabigerovarin analog, a cannabigerovarinic acid analog, a cannabichromevarin analog, a cannabichromevarinic acid analog, a cannabidivarin analog, a cannabidivarinic acid analog, a cannabidivarinic acid analog, a cannabiditriol analog, or a cannabicyclol analog.

[0180] The invention illustratively described herein can suitably be practiced in the absence of any element or elements, or any limitation or limitations not specifically disclosed herein. Thus, for example, in each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" can be replaced with either of the other two terms. Thus, for example, some embodiments may encompass host cells "comprising" some components, other embodiments will encompass host cells "consisting essentially of" the same components, and still other embodiments will encompass host cells "consisting of" the same components. The terms and expressions used are used as terms of description rather than as terms of limitation, and in using these terms and expressions, there is no intention to exclude any equivalents of the features shown and described, or portions thereof. Rather, it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, while the invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that those skilled in the art can make modifications and variations of the concepts disclosed herein, and that these modifications and variations are considered to be within the scope of the invention as defined by the appended claims.

[0181] The foregoing written description is believed to be sufficient to enable one skilled in the art to practice the invention. The following examples are offered for illustrative purposes only and are not intended to limit the scope of the invention in any way. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

[0182] Where references are made herein to patents, other external documents, or other sources of information, this is generally for the purpose of providing a context for describing the features of the present invention. Unless specifically stated otherwise, references to these external documents should not be construed as an admission that these documents or these sources are prior art or form part of the common general knowledge in the art to any extent. All patents, patent applications, and references cited herein are incorporated herein by reference in their entirety.

[0183] Exemplary Sequences SEQ ID NO: 1 exemplary RevS polypeptide sequence GenBank BAK64635.1 TIFF2025170350000044.tif77160

[0184] SEQ ID NO:2 Exemplary AsaCSAAE3 polypeptide sequence; GenBank AFD33347.1 TIFF2025170350000045.tif70160

[0185] SEQ ID NO:3 Exemplary AsaCSAAE1 polypeptide sequence; GenBank AFD33345.1 Transmembrane domain is underlined TIFF2025170350000046.tif92160

[0186] SEQ ID NO:4 Exemplary olivetolic acid synthase polypeptide sequence; UniProtKB / Swiss-Prot: B1Q2B6.1 TIFF2025170350000047.tif47159

[0187] SEQ ID NO: 5 Exemplary olivetolate cyclase polypeptide sequence; UniProtKB / Swiss-Prot: I6WU39.1 TIFF2025170350000048.tif11158

[0188] SEQ ID NO:6: Olivetolate cyclase polypeptide sequence lacking the N-terminal methionine and C-terminal lysine compared to SEQ ID NO:5 TIFF2025170350000049.tif11159

[0189] SEQ ID NO:7 A truncated cyclase 95 aa lacking the N-terminal methionine and the 5 amino acid sequence YTPRK at the C-terminus compared to SEQ ID NO:5 TIFF2025170350000050.tif11159

[0190] SEQ ID NO: 8 Amino acid sequence of the 415 amino acid C-terminal domain of Ralstonia solanacearum acyl-CoA synthase TIFF2025170350000051.tif55159

[0191] SEQ ID NO:9 Amino acid sequence of the cyclase domain from the benH gene product of the benastatin gene cluster of Streptomyces sp. TIFF2025170350000052.tif19159

[0192] Sequence information SEQUENCE LISTING <110> BAYMEDICA, INC. <120> CANNABINOID ANALOGS AND METHODS FOR THEIR PREPARATION <150> US 62 / 767,447 <151> 2018-11-14 <150> US 62 / 753,708 <151> 2018-10-31 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 579 <212> PRT <213> Streptomyces sp. <400> 1 Met Glu Leu Ala Leu Pro Ala Glu Leu Ala Pro Thr Leu Pro Glu Ala 1 5 10 15 Leu Arg Leu Arg Ser Glu Gln Gln Pro Asp Thr Val Ala Tyr Val Phe 20 25 30 Leu Arg Asp Gly Glu Thr Pro Glu Glu Thr Leu Thr Tyr Gly Arg Leu 35 40 45 Asp Arg Ala Ala Arg Ala Arg Ala Ala Ala Leu Glu Ala Ala Gly Leu 50 55 60 Ala Gly Gly Thr Ala Val Leu Leu Tyr Pro Ser Gly Leu Glu Phe Val 65 70 75 80 Ala Ala Leu Leu Gly Cys Met Tyr Ala Gly Thr Ala Gly Ala Pro Val 85 90 95 Gln Val Pro Thr Arg Arg Arg Gly Met Glu Arg Ala Arg Arg Ile Ala 100 105 110 Asp Asp Ala Gly Ala Lys Thr Ile Leu Thr Thr Thr Ala Val Lys Arg 115 120 125 Glu Val Glu Glu His Phe Ala Asp Leu Leu Thr Gly Leu Thr Val Ile 130 135 140 Asp Thr Glu Ser Leu Pro Asp Val Pro Asp Asp Ala Pro Ala Val Arg 145 150 155 160 Leu Pro Gly Pro Asp Asp Val Ala Leu Leu Gln Tyr Thr Ser Gly Ser 165 170 175 Thr Gly Asp Pro Lys Gly Val Glu Val Thr His Ala Asn Phe Arg Ala 180 185 190 Asn Val Ala Glu Thr Val Glu Leu Trp Pro Val Arg Ser Asp Gly Thr 195 200 205 Val Val Asn Trp Leu Pro Leu Phe His Asp Met Gly Leu Met Phe Gly 210 215 220 Val Val Met Pro Leu Phe Thr Gly Val Pro Ala Tyr Leu Met Ala Pro 225 230 235 240 Gln Ser Phe Ile Arg Arg Pro Ala Arg Trp Leu Glu Ala Ile Ser Arg 245 250 255 Phe Arg Gly Thr His Ala Ala Ala Pro Ser Phe Ala Tyr Glu Leu Cys 260 265 270 Val Arg Ser Val Ala Asp Thr Gly Leu Pro Ala Gly Leu Asp Leu Ser 275 280 285 Ser Trp Arg Val Ala Val Asn Gly Ala Glu Pro Val Arg Trp Thr Ala 290 295 300 Val Ala Asp Phe Thr Glu Ala Tyr Ala Pro Ala Gly Phe Arg Pro Gln 305 310 315 320 Ala Met Cys Pro Gly Tyr Gly Leu Ala Glu Asn Thr Leu Lys Leu Ser 325 330 335 Gly Ser Pro Glu Asp Arg Pro Pro Thr Leu Leu Arg Ala Asp Ala Ala 340 345 350 Ala Leu Gln Asp Gly Arg Val Val Pro Leu Thr Gly Pro Gly Thr Asp 355 360 365 Gly Val Arg Leu Val Gly Ser Gly Val Thr Val Pro Ser Ser Arg Val 370 375 380 Ala Val Val Asp Pro Gly Thr Gly Thr Glu Gln Pro Ala Gly Arg Val 385 390 395 400 Gly Glu Ile Trp Ile Asn Gly Pro Cys Val Ala Arg Gly Tyr His Gly 405 410 415 Arg Pro Ala Glu Ser Ala Glu Ser Phe Gly Ala Arg Ile Ala Gly Gln 420 425 430 Glu Ala Arg Gly Thr Trp Leu Arg Thr Gly Asp Leu Gly Phe Leu His 435 440 445 Asp Gly Glu Val Phe Val Ala Gly Arg Leu Lys Asp Val Val Ile His 450 455 460 Gln Gly Arg Asn Phe Tyr Pro Gln Asp Ile Glu Leu Ser Ala Glu Val 465 470 475 480 Ser Asp Arg Ala Leu His Pro Asn Cys Ala Ala Ala Phe Ala Leu Asp 485 490 495 Asp Gly Arg Thr Glu Arg Leu Val Leu Leu Val Glu Ala Asp Gly Arg 500 505 510 Ala Leu Arg Asn Gly Gly Ala Asp Ala Leu Arg Ala Arg Val His Asp 515 520 525 Ala Val Trp Asp Arg Gln Arg Leu Arg Ile Asp Glu Ile Val Leu Leu 530 535 540 Arg Arg Gly Ala Leu Pro Lys Thr Ser Ser Gly Lys Val Gln Arg Arg 545 550 555 560 Leu Ala Arg Ser Arg Tyr Leu Asp Gly Glu Phe Gly Pro Ala Pro Ala 565 570 575 Arg Glu Ala <210> 2 <211> 543 <212> PRT <213> Cannabis sativa <400> 2 Met Glu Lys Ser Gly Tyr Gly Arg Asp Gly Ile Tyr Arg Ser Leu Arg 1 5 10 15 Pro Pro Leu His Leu Pro Asn Asn Asn Asn Leu Ser Met Val Ser Phe 20 25 30 Leu Phe Arg Asn Ser Ser Ser Tyr Pro Gln Lys Pro Ala Leu Ile Asp 35 40 45 Ser Glu Thr Asn Gln Ile Leu Ser Phe Ser His Phe Lys Ser Thr Val 50 55 60 Ile Lys Val Ser His Gly Phe Leu Asn Leu Gly Ile Lys Lys Asn Asp 65 70 75 80 Val Val Leu Ile Tyr Ala Pro Asn Ser Ile His Phe Pro Val Cys Phe 85 90 95 Leu Gly Ile Ile Ala Ser Gly Ala Ile Ala Thr Thr Ser Asn Pro Leu 100 105 110 Tyr Thr Val Ser Glu Leu Ser Lys Gln Val Lys Asp Ser Asn Pro Lys 115 120 125 Leu Ile Ile Thr Val Pro Gln Leu Leu Glu Lys Val Lys Gly Phe Asn 130 135 140 Leu Pro Thr Ile Leu Ile Gly Pro Asp Ser Glu Gln Glu Ser Ser Ser 145 150 155 160 Asp Lys Val Met Thr Phe Asn Asp Leu Val Asn Leu Gly Gly Ser Ser 165 170 175 Gly Ser Glu Phe Pro Ile Val Asp Asp Phe Lys Gln Ser Asp Thr Ala 180 185 190 Ala Leu Leu Tyr Ser Ser Gly Thr Thr Gly Met Ser Lys Gly Val Val 195 200 205 Leu Thr His Lys Asn Phe Ile Ala Ser Ser Leu Met Val Thr Met Glu 210 215 220 Gln Asp Leu Val Gly Glu Met Asp Asn Val Phe Leu Cys Phe Leu Pro 225 230 235 240 Met Phe His Val Phe Gly Leu Ala Ile Ile Thr Tyr Ala Gln Leu Gln 245 250 255 Arg Gly Asn Thr Val Ile Ser Met Ala Arg Phe Asp Leu Glu Lys Met 260 265 270 Leu Lys Asp Val Glu Lys Tyr Lys Val Thr His Leu Trp Val Val Pro 275 280 285 Pro Val Ile Leu Ala Leu Ser Lys Asn Ser Met Val Lys Lys Phe Asn 290 295 300 Leu Ser Ser Ile Lys Tyr Ile Gly Ser Gly Ala Ala Pro Leu Gly Lys 305 310 315 320 Asp Leu Met Glu Glu Cys Ser Lys Val Val Pro Tyr Gly Ile Val Ala 325 330 335 Gln Gly Tyr Gly Met Thr Glu Thr Cys Gly Ile Val Ser Met Glu Asp 340 345 350 Ile Arg Gly Gly Lys Arg Asn Ser Gly Ser Ala Gly Met Leu Ala Ser 355 360 365 Gly Val Glu Ala Gln Ile Val Ser Val Asp Thr Leu Lys Pro Leu Pro 370 375 380 Pro Asn Gln Leu Gly Glu Ile Trp Val Lys Gly Pro Asn Met Met Gln 385 390 395 400 Gly Tyr Phe Asn Asn Pro Gln Ala Thr Lys Leu Thr Ile Asp Lys Lys 405 410 415 Gly Trp Val His Thr Gly Asp Leu Gly Tyr Phe Asp Glu Asp Gly His 420 425 430 Leu Tyr Val Val Asp Arg Ile Lys Glu Leu Ile Lys Tyr Lys Gly Phe 435 440 445 Gln Val Ala Pro Ala Glu Leu Glu Gly Leu Leu Val Ser His Pro Glu 450 455 460 Ile Leu Asp Ala Val Val Ile Pro Phe Pro Asp Ala Glu Ala Gly Glu 465 470 475 480 Val Pro Val Ala Tyr Val Val Arg Ser Pro Asn Ser Ser Leu Thr Glu 485 490 495 Asn Asp Val Lys Lys Phe Ile Ala Gly Gln Val Ala Ser Phe Lys Arg 500 505 510 Leu Arg Lys Val Thr Phe Ile Asn Ser Val Pro Lys Ser Ala Ser Gly 515 520 525 Lys Ile Leu Arg Arg Glu Leu Ile Gln Lys Val Arg Ser Asn Met 530 535 540 <210> 3 <211> 720 <212> PRT <213> Cannabis sativa <400> 3 Met Gly Lys Asn Tyr Lys Ser Leu Asp Ser Val Val Ala Ser Asp Phe 1 5 10 15 Ile Ala Leu Gly Ile Thr Ser Glu Val Ala Glu Thr Leu His Gly Arg 20 25 30 Leu Ala Glu Ile Val Cys Asn Tyr Gly Ala Ala Thr Pro Gln Thr Trp 35 40 45 Ile Asn Ile Ala Asn His Ile Leu Ser Pro Asp Leu Pro Phe Ser Leu 50 55 60 His Gln Met Leu Phe Tyr Gly Cys Tyr Lys Asp Phe Gly Pro Ala Pro 65 70 75 80 Pro Ala Trp Ile Pro Asp Pro Glu Lys Val Lys Ser Thr Asn Leu Gly 85 90 95 Ala Leu Leu Glu Lys Arg Gly Lys Glu Phe Leu Gly Val Lys Tyr Lys 100 105 110 Asp Pro Ile Ser Ser Phe Ser His Phe Gln Glu Phe Ser Val Arg Asn 115 120 125 Pro Glu Val Tyr Trp Arg Thr Val Leu Met Asp Glu Met Lys Ile Ser 130 135 140 Phe Ser Lys Asp Pro Glu Cys Ile Leu Arg Arg Asp Asp Ile Asn Asn 145 150 155 160 Pro Gly Gly Ser Glu Trp Leu Pro Gly Gly Tyr Leu Asn Ser Ala Lys 165 170 175 Asn Cys Leu Asn Val Asn Ser Asn Lys Lys Leu Asn Asp Thr Met Ile 180 185 190 Val Trp Arg Asp Glu Gly Asn Asp Asp Leu Pro Leu Asn Lys Leu Thr 195 200 205 Leu Asp Gln Leu Arg Lys Arg Val Trp Leu Val Gly Tyr Ala Leu Glu 210 215 220 Glu Met Gly Leu Glu Lys Gly Cys Ala Ile Ala Ile Asp Met Pro Met 225 230 235 240 His Val Asp Ala Val Val Ile Tyr Leu Ala Ile Val Leu Ala Gly Tyr 245 250 255 Val Val Val Ser Ile Ala Asp Ser Phe Ser Ala Pro Glu Ile Ser Thr 260 265 270 Arg Leu Arg Leu Ser Lys Ala Lys Ala Ile Phe Thr Gln Asp His Ile 275 280 285 Ile Arg Gly Lys Lys Arg Ile Pro Leu Tyr Ser Arg Val Val Glu Ala 290 295 300 Lys Ser Pro Met Ala Ile Val Ile Pro Cys Ser Gly Ser Asn Ile Gly 305 310 315 320 Ala Glu Leu Arg Asp Gly Asp Ile Ser Trp Asp Tyr Phe Leu Glu Arg 325 330 335 Ala Lys Glu Phe Lys Asn Cys Glu Phe Thr Ala Arg Glu Gln Pro Val 340 345 350 Asp Ala Tyr Thr Asn Ile Leu Phe Ser Ser Gly Thr Thr Gly Glu Pro 355 360 365 Lys Ala Ile Pro Trp Thr Gln Ala Thr Pro Leu Lys Ala Ala Ala Asp 370 375 380 Gly Trp Ser His Leu Asp Ile Arg Lys Gly Asp Val Ile Val Trp Pro 385 390 395 400 Thr Asn Leu Gly Trp Met Met Gly Pro Trp Leu Val Tyr Ala Ser Leu 405 410 415 Leu Asn Gly Ala Ser Ile Ala Leu Tyr Asn Gly Ser Pro Leu Val Ser 420 425 430 Gly Phe Ala Lys Phe Val Gln Asp Ala Lys Val Thr Met Leu Gly Val 435 440 445 Val Pro Ser Ile Val Arg Ser Trp Lys Ser Thr Asn Cys Val Ser Gly 450 455 460 Tyr Asp Trp Ser Thr Ile Arg Cys Phe Ser Ser Ser Gly Glu Ala Ser 465 470 475 480 Asn Val Asp Glu Tyr Leu Trp Leu Met Gly Arg Ala Asn Tyr Lys Pro 485 490 495 Val Ile Glu Met Cys Gly Gly Thr Glu Ile Gly Gly Ala Phe Ser Ala 500 505 510 Gly Ser Phe Leu Gln Ala Gln Ser Leu Ser Ser Phe Ser Ser Gln Cys 515 520 525 Met Gly Cys Thr Leu Tyr Ile Leu Asp Lys Asn Gly Tyr Pro Met Pro 530 535 540 Lys Asn Lys Pro Gly Ile Gly Glu Leu Ala Leu Gly Pro Val Met Phe 545 550 555 560 Gly Ala Ser Lys Thr Leu Leu Asn Gly Asn His His Asp Val Tyr Phe 565 570 575 Lys Gly Met Pro Thr Leu Asn Gly Glu Val Leu Arg Arg His Gly Asp 580 585 590 Ile Phe Glu Leu Thr Ser Asn Gly Tyr Tyr His Ala His Gly Arg Ala 595 600 605 Asp Asp Thr Met Asn Ile Gly Gly Ile Lys Ile Ser Ser Ile Glu Ile 610 615 620 Glu Arg Val Cys Asn Glu Val Asp Asp Arg Val Phe Glu Thr Thr Ala 625 630 635 640 Ile Gly Val Pro Pro Leu Gly Gly Gly Pro Glu Gln Leu Val Ile Phe 645 650 655 Phe Val Leu Lys Asp Ser Asn Asp Thr Thr Ile Asp Leu Asn Gln Leu 660 665 670 Arg Leu Ser Phe Asn Leu Gly Leu Gln Lys Lys Leu Asn Pro Leu Phe 675 680 685 Lys Val Thr Arg Val Val Pro Leu Ser Ser Leu Pro Arg Thr Ala Thr 690 695 700 Asn Lys Ile Met Arg Arg Val Leu Arg Gln Gln Phe Ser His Phe Glu 705 710 715 720 <210> 4 <211> 385 <212> PRT <213> Cannabis sativa <400> 4 Met Asn His Leu Arg Ala Glu Gly Pro Ala Ser Val Leu Ala Ile Gly 1 5 10 15 Thr Ala Asn Pro Glu Asn Ile Leu Leu Gln Asp Glu Phe Pro Asp Tyr 20 25 30 Tyr Phe Arg Val Thr Lys Ser Glu His Met Thr Gln Leu Lys Glu Lys 35 40 45 Phe Arg Lys Ile Cys Asp Lys Ser Met Ile Arg Lys Arg Asn Cys Phe 50 55 60 Leu Asn Glu Glu His Leu Lys Gln Asn Pro Arg Leu Val Glu His Glu 65 70 75 80 Met Gln Thr Leu Asp Ala Arg Gln Asp Met Leu Val Val Glu Val Pro 85 90 95 Lys Leu Gly Lys Asp Ala Cys Ala Lys Ala Ile Lys Glu Trp Gly Gln 100 105 110 Pro Lys Ser Lys Ile Thr His Leu Ile Phe Thr Ser Ala Ser Thr Thr 115 120 125 Asp Met Pro Gly Ala Asp Tyr His Cys Ala Lys Leu Leu Gly Leu Ser 130 135 140 Pro Ser Val Lys Arg Val Met Met Tyr Gln Leu Gly Cys Tyr Gly Gly 145 150 155 160 Gly Thr Val Leu Arg Ile Ala Lys Asp Ile Ala Glu Asn Asn Lys Gly 165 170 175 Ala Arg Val Leu Ala Val Cys Cys Asp Ile Met Ala Cys Leu Phe Arg 180 185 190 Gly Pro Ser Glu Ser Asp Leu Glu Leu Leu Val Gly Gln Ala Ile Phe 195 200 205 Gly Asp Gly Ala Ala Ala Val Ile Val Gly Ala Glu Pro Asp Glu Ser 210 215 220 Val Gly Glu Arg Pro Ile Phe Glu Leu Val Ser Thr Gly Gln Thr Ile 225 230 235 240 Leu Pro Asn Ser Glu Gly Thr Ile Gly Gly His Ile Arg Glu Ala Gly 245 250 255 Leu Ile Phe Asp Leu His Lys Asp Val Pro Met Leu Ile Ser Asn Asn 260 265 270 Ile Glu Lys Cys Leu Ile Glu Ala Phe Thr Pro Ile Gly Ile Ser Asp 275 280 285 Trp Asn Ser Ile Phe Trp Ile Thr His Pro Gly Gly Lys Ala Ile Leu 290 295 300 Asp Lys Val Glu Glu Lys Leu His Leu Lys Ser Asp Lys Phe Val Asp 305 310 315 320 Ser Arg His Val Leu Ser Glu His Gly Asn Met Ser Ser Ser Thr Val 325 330 335 Leu Phe Val Met Asp Glu Leu Arg Lys Arg Ser Leu Glu Glu Gly Lys 340 345 350 Ser Thr Thr Gly Asp Gly Phe Glu Trp Gly Val Leu Phe Gly Phe Gly 355 360 365 Pro Gly Leu Thr Val Glu Arg Val Val Val Arg Ser Val Pro Ile Lys 370 375 380 Tyr 385 <210> 5 <211> 101 <212> PRT <213> Cannabis sativa <400> 5 Met Ala Val Lys His Leu Ile Val Leu Lys Phe Lys Asp Glu Ile Thr 1 5 10 15 Glu Ala Gln Lys Glu Glu Phe Phe Lys Thr Tyr Val Asn Leu Val Asn 20 25 30 Ile Ile Pro Ala Met Lys Asp Val Tyr Trp Gly Lys Asp Val Thr Gln 35 40 45 Lys Asn Lys Glu Glu Gly Tyr Thr His Ile Val Glu Val Thr Phe Glu 50 55 60 Ser Val Glu Thr Ile Gln Asp Tyr Ile Ile His Pro Ala His Val Gly 65 70 75 80 Phe Gly Asp Val Tyr Arg Ser Phe Trp Glu Lys Leu Leu Ile Phe Asp 85 90 95 Tyr Thr Pro Arg Lys 100 <210> 6 <211> 99 <212> PRT <213> Cannabis sativa <400> 6 Ala Val Lys His Leu Ile Val Leu Lys Phe Lys Asp Glu Ile Thr Glu 1 5 10 15 Ala Gln Lys Glu Glu Phe Phe Lys Thr Tyr Val Asn Leu Val Asn Ile 20 25 30 Ile Pro Ala Met Lys Asp Val Tyr Trp Gly Lys Asp Val Thr Gln Lys 35 40 45 Asn Lys Glu Glu Gly Tyr Thr His Ile Val Glu Val Thr Phe Glu Ser 50 55 60 Val Glu Thr Ile Gln Asp Tyr Ile Ile His Pro Ala His Val Gly Phe 65 70 75 80 Gly Asp Val Tyr Arg Ser Phe Trp Glu Lys Leu Leu Ile Phe Asp Tyr 85 90 95 Thr Pro Arg <210> 7 <211> 95 <212> PRT <213> Cannabis sativa <400> 7 Ala Val Lys His Leu Ile Val Leu Lys Phe Lys Asp Glu Ile Thr Glu 1 5 10 15 Ala Gln Lys Glu Glu Phe Phe Lys Thr Tyr Val Asn Leu Val Asn Ile 20 25 30 Ile Pro Ala Met Lys Asp Val Tyr Trp Gly Lys Asp Val Thr Gln Lys 35 40 45 Asn Lys Glu Glu Gly Tyr Thr His Ile Val Glu Val Thr Phe Glu Ser 50 55 60 Val Glu Thr Ile Gln Asp Tyr Ile Ile His Pro Ala His Val Gly Phe 65 70 75 80 Gly Asp Val Tyr Arg Ser Phe Trp Glu Lys Leu Leu Ile Phe Asp 85 90 95 <210> 8 <211> 415 <212> PRT <213> Ralstonia solanacearum <400> 8 Met Ala Phe Asn Glu Arg Val Val Asp Trp Gln Gln Val Ala Gly Ala 1 5 10 15 Gln Pro Asp Ala Ser Pro Glu Arg Met Ser Ala Asp Asp Pro Phe Met 20 25 30 Ile Ile Tyr Thr Ser Gly Thr Thr Gly Lys Pro Lys Gly Thr Val His 35 40 45 Thr His Gly Ser Phe Pro Met Lys Ile Ala His Asp Ser Ala Ile His 50 55 60 Phe Asn Val Ser Pro Lys Asp Val Phe Cys Trp Pro Ala Asp Met Gly 65 70 75 80 Trp Val Ala Gly Thr Leu Val Met Ser Cys Ala Leu Leu Arg Gly Ala 85 90 95 Thr Leu Val Cys Tyr Asp Gly Ala Pro Asp Phe Pro Asp Trp Ser Arg 100 105 110 Met Ser Arg Leu Ile Glu Arg His Arg Val Thr His Phe Gly Ser Ala 115 120 125 Pro Thr Leu Ile Arg Gly Leu Ala Ser Asn Glu Ala Ile Ala Thr Gln 130 135 140 Gly Asp Val Ser Ser Val Lys Leu Leu Ile Thr Ala Gly Glu Gly Ile 145 150 155 160 Asp Pro Glu His Phe Leu Trp Phe Gln Lys Ala Phe Gly Gly Gly His 165 170 175 Arg Pro Val Ile Asn Tyr Thr Gly Gly Thr Glu Val Ser Gly Ala Leu 180 185 190 Leu Ser Ser Val Val Ile Lys Pro Ile Ser Pro Ala Gly Phe Asn Thr 195 200 205 Ala Ser Pro Gly Val Ala Thr Asp Val Val Asp Ala Glu Gly His Ser 210 215 220 Val Thr Gly Glu Val Gly Glu Leu Ala Ile Arg Lys Pro Phe Ile Gly 225 230 235 240 Met Thr Arg Ser Phe Trp Gln Asp Asp Glu Arg Tyr Leu Asp Ser Tyr 245 250 255 Trp Arg Thr Ile Pro Gly Ile Trp Val His Gly Asp Leu Ala Met Arg 260 265 270 Arg Glu Asp Gly Met Trp Phe Met Met Gly Arg Ser Asp Asp Thr Ile 275 280 285 Lys Leu Ala Gly Lys Arg Leu Gly Pro Ala Glu Ile Glu Asp Val Leu 290 295 300 Leu Glu Leu Pro Glu Ile Ala Glu Ala Ala Ala Ile Gly Val Glu Asp 305 310 315 320 Pro Val Lys Gly Gln Lys Leu Val Val Phe Val Val Ala Ser Lys Ala 325 330 335 Ser Thr Ala Ser Ala Asp Ala Leu Ala Ser Val Ile Gly Lys His Val 340 345 350 Asp Leu Arg Leu Gly Arg Pro Phe Arg Pro Ser Val Val His Val Val 355 360 365 Ala Gln Leu Pro Lys Thr Arg Ser Ser Lys Ile Met Arg Arg Val Ile 370 375 380 Arg Ser Val Tyr Thr Gly Lys Pro Ala Gly Asp Leu Ser Ser Leu Asp 385 390 395 400 Asn Pro Leu Ala Leu Asp Glu Ile Arg Ser Ala Ala Ala Val Ser 405 410 415 <210> 9 <211> 146 <212> PRT <213> Streptomyces sp. <400> 9 Ala Gly Arg Thr Asp Asn Ser Val Val Ile Asp Ala Pro Val Gln Leu 1 5 10 15 Val Trp Asp Met Thr Asn Asp Val Ser Gln Trp Ala Val Leu Phe Glu 20 25 30 Glu Tyr Ala Glu Ser Glu Val Leu Ala Val Asp Gly Asp Thr Val Arg 35 40 45 Phe Arg Leu Thr Thr Gln Pro Asp Glu Asp Gly Lys Gln Trp Ser Trp 50 55 60 Val Ser Glu Arg Thr Arg Asp Leu Glu Asn Arg Thr Val Thr Ala Arg 65 70 75 80 Arg Leu Asp Asn Gly Leu Phe Glu Tyr Met Asn Ile Arg Trp Glu Tyr 85 90 95 Thr Glu Gly Pro Asp Gly Val Arg Met Arg Trp Ile Gln Glu Phe Ser 100 105 110 Met Lys Pro Ser Ala Pro Val Asp Asp Ser Gly Ala Glu Asp His Leu 115 120 125 Asn Arg Gln Thr Val Lys Glu Met Ala Arg Ile Lys Lys Leu Ile Glu 130 135 140 Glu Ala 145 <210> 10 <211> 5 <212> PRT <213> Cannabis sativa <400> 10 Tyr Thr Pro Arg Lys 1 5

Claims

1. Compounds of Formula I or a salt or cannabinoid derivative thereof: During the ceremony, R 1 is C 5 ~C 20 Haloalkyl, C 1 ~C 4 Haloalkyl, C 1 ~C 20 Hydroxyalkyl, deuterated C 1 ~C 20 Alkyl, tritiated C 1 ~C 20 Alkyl, and C 2 ~C 20 alkenyl, R 2 COOR 2a and H, R 2a H and C 1 ~C 6 is selected from the group consisting of alkyl, R 3 is selected from the group consisting of a prenyl moiety and H.

2. R 1 But C 5 ~C 10 Haloalkyl, C 1 ~C 4 Haloalkyl, C 1 ~C 10 Hydroxyalkyl, deuterated C 1 ~C 10 Alkyl, tritiated C 1 ~C 10 Alkyl, and C 2 ~C 10 2. The compound of claim 1, or a salt or cannabinoid derivative thereof, selected from the group consisting of alkenyl.

3. R 1 But C 5 ~C 10 Haloalkyl, C 1 ~C 4 Haloalkyl, C 1 ~C 10 Hydroxyalkyl, deuterated C 1 ~C 10 Alkyl and tritiated C 1 ~C 10 2. The compound of claim 1, or a salt or cannabinoid derivative thereof, selected from the group consisting of alkyl.

4. R 1 C 5 ~C 10 Haloalkyl or C 1 ~C 4 2. The compound of claim 1, or a salt or cannabinoid derivative thereof, which is haloalkyl.

5. R 1 5. The compound of claim 4, or a salt or cannabinoid derivative thereof, wherein is selected from the group consisting of fluoropentyl, fluoroethyl, fluoropropyl, fluorobutyl, fluorohexyl, fluorooctyl, and fluorononyl.

6. R 1 5. The compound of claim 4, or a salt or cannabinoid derivative thereof, wherein is selected from the group consisting of 5-fluoropropyl, 4-fluorobutyl, and 3-fluoropentyl.

7. R 1 C 1 ~C 10 Bromoalkyl or C 1 ~C 10 5. The compound of claim 4, or a salt or cannabinoid derivative thereof, which is chloroalkyl.

8. R 1 C 1 ~C 10 2. The compound of claim 1, or a salt or cannabinoid derivative thereof, which is hydroxyalkyl.

9. R 1 Deuterated C 1 ~C 10 Alkyl or tritiated C 1 ~C 10 2. The compound of claim 1, or a salt or cannabinoid derivative thereof, wherein:

10. R 2 2. The compound of claim 1, or a salt or cannabinoid derivative thereof, wherein is selected from the group consisting of COOH and H.

11. R 2 2. The compound of claim 1, or a salt or cannabinoid derivative thereof, wherein is COOH.

12. R 2 2. The compound of claim 1, or a salt or cannabinoid derivative thereof, wherein

13. R 3 2. The compound of claim 1, or a salt or cannabinoid derivative thereof, wherein

14. R 3 2. The compound of claim 1, or a salt or cannabinoid derivative thereof, wherein is a prenyl moiety.

15. 2. The compound of claim 1, or a salt or cannabinoid derivative thereof, wherein the prenyl moiety is 3,7-dimethylocta-2,6-dien-1-yl.

16. The compound has the structure of formula Ia 2. The compound of claim 1, or a salt or cannabinoid derivative thereof, having the formula:

17. A cannabinoid derivative of the compound of claim 1 or a salt thereof.

18. Halogenated cannabidiolic acids, halogenated cannabidiols, halogenated Δ 9 -Tetrahydrocannabinolic acid, halogenated Δ 8 -Tetrahydrocannabinolic acid, halogenated cannabichromenic acid, halogenated cannabichromene, halogenated cannabinol, halogenated cannabinodioI, halogenated cannabinolic acid, cannabivarin, halogenated cannabivarinic acid, halogenated Δ 9 -Tetrahydrocannabivarin, halogenated Δ 8 -Tetrahydrocannabivarin, halogenated Δ 9 -Tetrahydrocannabivaric acid, halogenated Δ 8 18. The cannabinoid derivative or pharmaceutically acceptable salt thereof of claim 17, wherein the cannabinoid derivative is selected from the group consisting of tetrahydrocannabivarin, halogenated cannabigerovarin, halogenated cannabigerovarinic acid, halogenated cannabichromevarin, halogenated cannabichromevarinic acid, halogenated cannabidivarin, halogenated cannabidivarinic acid, halogenated cannabiditriol, and halogenated cannabicyclol.

19. 19. A pharmaceutical composition comprising a cannabinoid derivative according to claim 17 or claim 18 and a pharmaceutically acceptable excipient.

20. A method for treating a disease or condition mediated by cannabinoid receptor activity, comprising administering to a subject in need thereof an effective amount of a cannabinoid derivative or a pharmaceutically acceptable salt thereof as described in claim 17 or claim 18, or an effective amount of a composition as described in claim 19.

21. Compound of Formula IV or a salt thereof, comprising the steps of: In the formula, R 1 is C 1 ~C 20 Haloalkyl, C 1 ~C 20 Hydroxyalkyl, deuterated C 1 ~C 20 Alkyl, tritiated C 1 ~C 20 Alkyl, and C 2 ~C 20 alkenyl, The method comprises reacting a thioester of formula II Culturing the modified recombinant host cell in a medium comprising In the formula, R 4 is selected from the group consisting of a coenzyme A (CoA) moiety, a pantetheine moiety, and a cysteamine moiety; the modified recombinant host cell iii. ester of formula II and malonyl-CoA to form a tetraketide of formula III a first polynucleotide encoding a synthase that converts iv. A second polynucleotide encoding a 2-alkyl-4,6-dihydroxybenzoate cyclase that converts the tetraketide of formula III to a compound of formula IV. and the modified recombinant host cell is cultured under conditions whereby the products encoded by the first and second polynucleotides are expressed to produce the compound of Formula IV; method.

22. R 1 But C 1 ~C 10 Haloalkyl, C 1 ~C 10 Hydroxyalkyl, deuterated C 1 ~C 10 Alkyl, tritiated C 1 ~C 10 Alkyl, and C 2 ~C 10 22. The method of claim 21, wherein the alkyl group is selected from the group consisting of alkenyl.

23. 22. The method of claim 21, wherein the synthase is olivetolic acid synthase.

24. 22. The method of claim 21, wherein the 2-alkyl-4,6-dihydroxybenzoate cyclase is a truncated olivetolic acid cyclase.

25. R 4 is a CoA moiety.

26. The host cell is a starting material of formula IIa to a thioester of formula II, and step (a) comprises culturing the host cell under conditions in which a product encoded by the third polynucleotide is expressed to produce a thioester of formula II.

22. The method of claim 21.

27. 27. The method of claim 26, wherein the acyl-CoA synthetase is revS or CsAAE3.

28. R in the thioester of formula II 4 22. The method of claim 21, wherein is a pantetheine moiety or a cysteamine moiety.

29. Compound of formula IV is reacted with compound of formula V or a salt thereof, wherein R 3 22. The method of claim 21, wherein is a prenyl moiety.

30. Decarboxylation of the compound of formula V to give a compound of formula Va 30. The method of claim 29, further comprising obtaining:

31. Decarboxylation of a compound of formula IV to give a compound of formula IVa 22. The method of claim 21, further comprising obtaining:

32. Compound of formula IVa is reacted with compound of formula Va wherein R 3 32. The method of claim 31, wherein is a prenyl moiety.

33. 30. The method of claim 29, wherein the host cell further comprises a fourth polynucleotide encoding a prenyltransferase enzyme that converts the compound of formula IV to the compound of formula V, and wherein converting step (b) comprises culturing the host cell under conditions in which the product encoded by the fourth polynucleotide is expressed to produce the compound of formula V.

34. 33. The method of claim 32, wherein the host cell further comprises a fourth polynucleotide encoding a prenyltransferase enzyme that converts the compound of formula IVa to the compound of formula Va, and wherein converting step (b) comprises culturing the host cell under conditions in which a product encoded by the fourth polynucleotide is expressed to produce the compound of formula Va.

35. 34. The method of claim 33, wherein the prenyltransferase is geranyl pyrophosphate:olivetolic acid geranyltransferase.

36. 30. The method of claim 29, wherein the converting step (b) comprises forming a reaction mixture comprising: (1) a compound of formula IV; (2) geraniol, activated geraniol, or citral; and (3) an organic solvent; and maintaining the reaction mixture under conditions sufficient to produce a compound of formula V.

37. 33. The method of claim 32, wherein the converting step (b) comprises forming a reaction mixture comprising: (1) a compound of formula IVa; (2) geraniol, activated geraniol, or citral; and (3) an organic solvent; and maintaining the reaction mixture under conditions sufficient to produce a compound of formula Va.

38. Cannabidiolic acid analogs, cannabidiol analogs, Δ 9 -Tetrahydrocannabinolic acid analogues, Δ 8 -Tetrahydrocannabinolic acid analogues, cannabichromene acid analogues, cannabichromene analogues, cannabinol analogues, cannabinodiol analogues, cannabinolic acid analogues, cannabivarin analogues, cannabivarinic acid analogues, Δ 9 -tetrahydrocannabivarin analogues, Δ 8 -tetrahydrocannabivarin analogues, Δ 9 -tetrahydrocannabivaric acid analogues, Δ 8 30. The compound prepared according to claim 29, which is a tetrahydrocannabivarin analog, a cannabigerovarin analog, a cannabigerovarin acid analog, a cannabichromevarin analog, a cannabichromevarinic acid analog, a cannabidivarin analog, a cannabidivarin acid analog, a cannabidivarinic acid analog, a cannabiditriol analog, or a cannabicyclol analog.

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