WO2019118612A1 - Targeting compounds - Google Patents

Abstract

The disclosure provides, at least in part, liver, intestine and/or kidney-targeting compounds and their use in treating liver, intestine and/or kidney disorders, such as non-alcoholic steatohepatitis, alcoholic steatohepatitis, hepatocellular carcinoma, liver cirrhosis, and hepatitis B; and/or chronic kidney disease, glomerular disease such as IGA nephropathy, lupus nephritis, or polycystic kidney disease. The compounds are contemplated to have activity against methionyl aminopeptidase 2.

Description

TARGETING COMPOUNDS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and priority to, U.S. provisional application serial numbers 62/654,710, filed April 9, 2018; and 62/597,598, filed December 12, 2017; the contents of each of which are hereby incorporated by reference herein in their entirety.

BACKGROUND

[0002] Membrane transporters are found throughout the body and play an important role in drug disposition. The tissue distribution of a drug can have a significant impact on both the agents’ safety and efficacy. Of the over 400 human transporters about 20 of them are important for drug transport (Giacomini KM, Huang SM, Tweedie DJ, Benet LZ, Brouwer KL, Chu X, et al. Membrane transporters in drug development. Nat Rev Drug Discov. 2010; 9(3):2l5-36).

[0003] There are two types of drug transporters; the solute carriers (SLC) and the ATP- binding cassette (ABC) transporters. SLC transporters are expressed in many tissues such as brain, liver, small intestine, kidney and are involved in the uptake of drugs and nutrients into cells. SLC transporters are categorized into three categories (i) the first type transport by facilitated diffusion via a concentration gradient, (ii) the second type utilizes Na⁺ or H⁺ cotransport and (iii) the third type involves exchanging substrates to aid transport. The main SLC transporters include organic anion transporting polypeptides (OATP), organic anion transporters (OAT) and organic cation transporters (OCT).

[0004] The OATPs (SLCOs) represent a superfamily of membrane transport proteins that mediate the sodium-independent transport of a diverse range of amphiphilic organic

compounds. They are expressed in the liver and various other organs and are involved in the distribution of drugs from the blood to tissues. OATP1B1 (SLCOlBl), 1B3 (SLC01B3), and 2B1 (SLC02B1) are important isoforms for drug transport and are localized to the sinusoidal (basolateral) membrane of hepatocytes (Hagenbuch B., & Meier PJ, The superfamily of organic anion transporting polypeptides. Biochim Biophys Acta 2003; 1609(1); 1-18; Fenner KS, Jones HM, Ullah M, Kempshall S, Dickins M, Lai Y, Morgan P, Barton HA. The evolution of the OATP hepatic uptake transport protein family in DMPK sciences: from obscure liver transporters to key determinants of hepatobiliary clearance. Xenobiotica. 2012 Jan; 42(l):28- 45. Epub 2011 Nov 11). [0005] Within the liver, OATP1B1 is expressed uniformly throughout the lobules. The OATP1B1 transporters exhibit broad substrate selectivity that includes anionic (e.g. statins such as pravastatin, pitavastatin, and rosuvastatin), zwitterionic (e.g. rifampicin) and neutral lipophilic (e.g. paclitaxel) drugs. OATP1B1 also transports endogenous substances such as bile acids, thyroid hormones, steroid sulfates, glucuronide conjugates and peptides.

[0006] OATP1B3 is in the sinusoidal membranes of hepatocytes located around the central vein. OATP1B3 has considerable substrate overlap with OATP1B1 but the gastrointestinal peptide Cholecystokinin is reported to be exclusively transported by OATP1B3. Other specific OATP1B3 substrates (amongst the OAT transporters) include docetaxel, digoxin, paclitaxel and the toxin amanitin (Letschert, K., et al., Molecular characterization and inhibition of amanitin uptake into human hepatocytes. Toxicol Sci, 2006. 91(1): p. 140-9). CCK-8 is a specific inhibitor of OATP1B3. Cyclosporine and CI-1034 are inhibitors of the three major OATP isoforms (Rizwan, AN. and Burckhardt, G, Organic anion transporters of the SLC22 family: biopharmaceutical, physiological, and pathological roles. Pharm Res, 2007. 24(3): p. 450-70; Sahi, T, et al., Metabolism and transporter-mediated drug-drug interactions of the endothelin-A receptor antagonist CI-1034. Chem Biol Interact, 2006. 159(2): p. 156-68).

[0007] As human OATP1B1 and OAT1B3 are liver specific they may be used for liver targeting to enhance liver concentration and minimize drug exposure to the peripheral tissues (e.g. brain) to reduce toxicity and/or side effects. Some medicinal chemistry design principles of orally active drugs for liver targeting through OATP transporters have been described (Tu M, Mathiowetz AM, Pfefferkom JA, Cameron KO, Dow RL, Litchfield J, Di L, Feng B, Liras S. Medicinal chemistry design principles for liver targeting through OATP transporters. Curr Top Med Chem. 2013; l3(7):857-66). These principles include, e.g., efficient hepatocyte uptake via OATP1B1 and/or OATP1B3 transporters which generally require an acidic function in the molecule, low passive permeability to minimize distribution into peripheral tissues that lack liver-specific OATP transporters, and sufficient bioavailability to support oral

administration.

[0008] Whilst OATP1B1 and 1B3 have been primary human liver-specific transporter targets, OATP2B1 and Sodium Tauracholate Transporting Polypeptide (NCTP1) represent additional human liver uptake transporters which could contribute to a liver targeted approach.

[0009] In addition to the sinusoidal membrane of the hepatocyte, OATP2B1 is widely expressed in tissues including apical membrane of enterocytes, the basal membrane of the syncytiotrophoblasts, the luminal membrane of brain capillary endothelial cells, small arteries and veins of the heart, and the lung. OATP2B1 has broad substrate specificity transporting mostly anionic organic endo- and xenobiotics. It exhibits some pH dependency in that it seems to transport some substrates more efficiently at lower pH. Its substrate specificity appears somewhat more restricted than OATP1B1 and 1B3, however it transports fexofenadine, statins, glibenclamide, glyburide, estrone-3 -sulfate (E3S), dehydroepiandrosterone-3-sulfate (DHEAS), prostaglandin E2, and taurocholate. OATP2B1 in the liver mediates the hepatic uptake of many xenobiotics, including bromosulphophthalein (BSP), benzylpenicillin, fexofenadine, glibenclamide, pravastatin, atorvastatin, rosuvastatin, and fluvastatin. The apparent pH dependency of transport may be important for the gastrointestinal transport of drug substrates such as fexofenadine, as pH varies substantially along the gastro-intestinal tract. OATP2B1 inhibitors include many organic anions, mono- and dicarboxylic acids, steroid hormones and their derivatives, drugs, such as rifamycin SV, pravastatin cyclosporine A and gemfibrozil as well as constituents of citrus juices and herbal extracts.

[0010] OATP family members are poorly conserved evolutionarily and orthologues for human OATPs may not exist in rodents. The significant species differences for the

OATP/Oatp transporters make it challenging to translate pharmacokinetic or pharmacodynamic data from preclinical species to humans.

[0011] Sodium Taurocholate Co-Transporting Polypeptide (NTCP, SLOC10A1) is another liver-specific transporter located on the basolateral membrane domain of hepatocytes in human, rat and other species (Stieger, B., et ah, In situ localization of the hepatocytic Na+/Taurocholate cotransporting polypeptide in rat liver. Gastroenterology, 1994. 107(6): p. 1781-7).

[0012] NTCP is primarily responsible for the uptake of bile acids from the sinusoids. NTCP is one of the key transporters in the enterohepatic circulation of bile acids, which also include the canalicular efflux transporter BSEP, and ASBT and OSTa/b in the gastrointestinal tract (Doring, B., et ah, The SLC10 carrier family: transport functions and molecular structure. Curr Top Membr, 2012. 70: p. 105-68). Other substrates include sulfated steroids (estrone-3 -sulfate, DHEAS) and thyroid hormones. In comparison to sodium independent organic anion transporters of the SLCO family (i.e. OATPs), NTCP interacts with a relatively narrow panel of drugs and other xenobiotics. A computational model of a substrate pharmacophore has been developed (Dong Z. et al. A Substrate Pharmacophore for the Human Sodium Taurocholate Co-transporting Polypeptide. Int J Pharm. 2015 Jan 15; 478(1): 88-95). NTCP is important in the hepatic uptake of statins, particularly rosuvastatin, and is therefore a risk factor for transporter-mediated DDI for this drug. [0013] Chronic liver diseases such as fatty liver disease and liver fibrosis can be risk factors that may dispose progression to hepatocellular carcinoma, and represent a common and difficult clinical challenge of worldwide importance. For example, liver fibrosis is the excessive accumulation of extracellular matrix proteins including collagen that occurs in most types of chronic liver diseases. Advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension and often requires liver transplantation. Non-alcoholic steatohepatitis (NASH) is a severe form of fatty liver disease that affects 2 to 5 percent of Americans. Many people with NASH have risk factors also associated with HCC, e.g., obesity, diabetes and/or elevated blood cholesterol levels. Alpha-l antitrypsin deficiency is an inherited disorder associated with an increased risk of liver and lung disease. The disorder is associated with the retention of the liver-produced protein alpha-l antitrypsin in the liver and low levels of alpha-l antitrypsin in the serum. Clinical features of alpha-l antitrypsin deficiency include chronic liver inflammation and liver fibrosis, which can progress in advanced forms to chronic hepatitis, cirrhosis and hepatocellular carcinoma. Chronic kidney disease may increase the risk for recurrence and progression of other cancers such as non-muscle invasive bladder cancer, and can also limit the ability to receive and withstand cancer therapies. Other kidney diseases such as lupus nephritis can lead to kidney failure and patients suffering from lupus nephritis are at a higher risk for cancers such as B-cell lymphoma, and for heart and blood vessel problems.

[0014] Methionine aminopeptidase 2 (MetAP2) encodes a protein that functions at least in part by enzymatically removing the amino terminal methionine residue from certain newly translated proteins such as glyceraldehyde-3 -phosphate dehydrogenase. Increased expression of the MetAP2 gene has been historically associated with various forms of cancer. Molecules inhibiting the enzymatic activity of MetAP2 have been identified and have been explored for their utility in the treatment of various tumor types.

SUMMARY

[0015] This disclosure is directed in part to, for example, carboxylic acid (and carboxylate salt) sidechains appended to the 6-position alcohol of fumagillol that are MetAP2 modulators selectively targeting the liver, intestine and/or kidney, their use as medicinal agents, processes for their preparation, and pharmaceutical compositions containing them as an active ingredient both alone or in combination with other agents, as well as provides for their use as

medicaments and/or in the manufacture of medicaments for the inhibition of MetAP2 activity, and for the treatment of a liver, intestinal and/or kidney disorder, in warm-blooded animals such as humans. Also provided are pharmaceutical compositions comprising at least one disclosed compound and a pharmaceutically acceptable carrier. [0016] In an embodiment, disclosed herein is a pharmaceutically acceptable base salt of a compound represented by:

wherein L¹, L² and T are as defined herein.

[0017] Also provided herein are methods of treating or mitigating a liver, intestine and/or kidney disorder in a patient in need thereof, comprising administering an effective amount of a compound described herein to the patient. In some embodiments, the liver disorder may be one or more of, for example, non-alcoholic steatohepatitis, alcoholic steatohepatitis, carcinoma (e.g., hepatocellular carcinoma), liver cirrhosis, and hepatitis B. In other embodiments, the kidney disorder may be one or more of chronic kidney disease, glomerular disease such as iGA nephropathy, lupus nephritis, polycystic kidney disease.

[0018] For example, disclosed herein are methods of treating, or preventing the development of, hepatic preneoplastic lesions in patient in need thereof, comprising administering an effective amount of a compound disclosed herein. In some embodiments, for example, the patient may be at risk for but not yet suffering from NASH. In some embodiments, the patient may have at least one of, for example: minimal liver fibrosis, minimal hepatic steatosis, lobular inflammation, and/or balloon degeneration. In some embodiments, the patient may suffer from type 2 diabetes and/or is obese. In some embodiments the patient may suffer from alpha- 1 antitrypsin deficiency. In some embodiments, provided herein are methods of treating hepatocellular carcinoma in a patient in need thereof, comprising administering to the patient an effective amount of a disclosed compound.

[0019] Contemplated methods of treating may also include administering a disclosed compound in combination with one or more other agents (and/or in combination with weight loss surgery and/or weight loss therapy). For example, contemplated methods herein can include administering one or more additional agents, e.g. as disclosed herein, for example, an anti diabetic medication (metformin), a modulator of glucagon -like peptide -1 receptor such as exenatide and liraglutide, obeticholic acid, a PPAR agonist, and/or a thiazolidinedione such as pioglitazone. DETAILED DESCRIPTION

[0020] The features and other details of the disclosure will now be more particularly described. Before further description of the present invention, certain terms employed in the specification, examples and appended claims are collected here. These definitions should be read in light of the remainder of the disclosure and as understood by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art.

Definitions

[0021] “Treating” includes any effect, e.g., lessening, reducing, modulating, or eliminating, that results in the improvement of the condition, disease, disorder and the like.

[0022] The term“alkenyl” as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon double bond. Exemplary alkenyl groups include, but are not limited to, a straight or branched group of 2-6 or 3-4 carbon atoms, referred to herein as C_2-6alkenyl, and C_3-4alkenyl, respectively. Exemplary alkenyl groups include, but are not limited to, vinyl, allyl, butenyl, pentenyl, etc.

[0023] The term“alkoxy” as used herein refers to a straight or branched alkyl group attached to oxygen (alkyl-O-). Exemplary alkoxy groups include, but are not limited to, alkoxy groups of 1-6 or 2-6 carbon atoms, referred to herein as Ci_-6alkoxy, and C_2-6alkoxy, respectively. Exemplary alkoxy groups include, but are not limited to methoxy, ethoxy, isopropoxy, etc.

[0024] The term“alkoxyalkyl” as used herein refers to a straight or branched alkyl group attached to oxygen, attached to a second straight or branched alkyl group (alkyl -O-alkyl-). Exemplary alkoxyalkyl groups include, but are not limited to, alkoxyalkyl groups in which each of the alkyl groups independently contains 1-6 carbon atoms, referred to herein as Ci. ₆alkoxy-Ci-₆alkyl. Exemplary alkoxyalkyl groups include, but are not limited to

methoxym ethyl, 2-methoxyethyl, 1 -methoxy ethyl, 2-methoxypropyl, ethoxym ethyl, 2- isopropoxy ethyl etc.

[0025] The term“alkyoxycarbonyl” as used herein refers to a straight or branched alkyl group attached to oxygen, attached to a carbonyl group (alkyl -O-C(O)-). Exemplary

alkoxycarbonyl groups include, but are not limited to, alkoxycarbonyl groups of 1-6 carbon atoms, referred to herein as Ci_-6alkoxycarbonyl. Exemplary alkoxycarbonyl groups include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, t-butoxycarbonyl, etc.

[0026] The term“alkenyloxy” used herein refers to a straight or branched alkenyl group attached to oxygen (alkenyl-O-). Exemplary alkenyloxy groups include, but are not limited to, groups with an alkenyl group of 3-6 carbon atoms, referred to herein as C3_-6alkenyloxy.

Exemplary“alkenyloxy” groups include, but are not limited to allyloxy, butenyloxy, etc.

[0027] The term“alkynyloxy” used herein refers to a straight or branched alkynyl group attached to oxygen (alkynyl-O). Exemplary alkynyloxy groups include, but are not limited to, groups with an alkynyl group of 3-6 carbon atoms, referred to herein as C_3-6alkynyloxy.

Exemplary alkynyloxy groups include, but are not limited to, propynyloxy, butynyloxy, etc.

[0028] The term“alkyl” as used herein refers to a saturated straight or branched hydrocarbon. Exemplary alkyl groups include, but are not limited to, straight or branched hydrocarbons of 1-6, 1-4, or 1-3 carbon atoms, referred to herein as Ci_-6alkyl, Ci_-4alkyl, and Ci_-3alkyl, respectively. Exemplary alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, 2-methyl-l-butyl, 3 -methyl -2 -butyl, 2-methyl- 1 -pentyl, 3-methyl-l-pentyl, 4- methyl-l -pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2 -pentyl, 2,2-dimethyl- 1- butyl, 3, 3 -dimethyl -1 -butyl, 2-ethyl- 1 -butyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, hexyl, etc.

[0029] The term“alkylcarbonyl” as used herein refers to a straight or branched alkyl group attached to a carbonyl group (alkyl-C(O)-). Exemplary alkylcarbonyl groups include, but are not limited to, alkylcarbonyl groups of 1-6 atoms, referred to herein as Ci_-6alkylcarbonyl groups. Exemplary alkylcarbonyl groups include, but are not limited to, acetyl, propanoyl, isopropanoyl, butanoyl, etc.

[0030] The term“alkynyl” as used herein refers to an unsaturated straight or branched hydrocarbon having at least one carbon-carbon triple bond. Exemplary alkynyl groups include, but are not limited to, straight or branched groups of 2-6, or 3-6 carbon atoms, referred to herein as C_2-6alkynyl, and C_3-6alkynyl, respectively. Exemplary alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, methylpropynyl, etc.

[0031] The term“carbonyl” as used herein refers to the radical -C(O)-.

[0032] The term“cyano” as used herein refers to the radical -CN.

[0033] The term“cycloalkoxy” as used herein refers to a cycloalkyl group attached to oxygen (cycloalkyl-O-). Exemplary cycloalkoxy groups include, but are not limited to, cycloalkoxy groups of 3-6 carbon atoms, referred to herein as C_3-6cycloalkoxy groups. Exemplary cycloalkoxy groups include, but are not limited to, cyclopropoxy, cyclobutoxy, cyclohexyl oxy, etc

[0034] The terms“cycloalkyl” or a“carbocyclic group” as used herein refers to a saturated or partially unsaturated hydrocarbon group of, for example, 3-6, or 4-6 carbons, referred to herein as C3-6cycloalkyl or C4_-6cycloalkyl, respectively. Exemplary cycloalkyl groups include, but are not limited to, cyclohexyl, cyclopentyl, cyclopentenyl, cyclobutyl or cyclopropyl.

[0035] The terms“halo” or“halogen” as used herein refer to F, Cl, Br, or I.

[0036] The terms“heteroaryl” or“heteroaromatic group” as used herein refers to a monocyclic aromatic 5-6 membered ring system containing one or more heteroatoms, for example one to three heteroatoms, such as nitrogen, oxygen, and sulfur. Where possible, said heteroaryl ring may be linked to the adjacent radical though carbon or nitrogen. Examples of heteroaryl rings include but are not limited to furan, thiophene, pyrrole, thiazole, oxazole, isothiazole, isoxazole, imidazole, pyrazole, triazole, pyridine or pyrimidine etc.

[0037] The terms“heterocyclyl” or“heterocyclic group” are art-recognized and refer to saturated or partially unsaturated, 4-10 membered ring structures, including spirocyclic, bridged or fused rings, and whose ring structures include one to three heteroatoms, such as nitrogen, oxygen, and sulfur. Where possible, heterocyclyl rings may be linked to the adjacent radical through carbon or nitrogen. Examples of heterocyclyl groups include, but are not limited to, pyrrolidine, piperidine, morpholine, thiomorpholine, piperazine, oxetane, azetidine, tetrahydrofuran or dihydrofuran etc.

[0038] The term“heterocyclyloxy” as used herein refers to a heterocyclyl group attached to oxygen (heterocyclyl -0-).

[0039] The term“heteroaryloxy” as used herein refers to a heteroaryl group attached to oxygen (heteroaryl-O-).

[0040] The terms“hydroxy” and“hydroxyl” as used herein refers to the radical -OH.

[0041] The term“oxo” as used herein refers to the radical =0.

[0042] “Pharmaceutically or pharmacologically acceptable” include molecular entities and compositions that should not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate. For human administration, preparations should meet sterility, pyrogenicity, and general safety and purity standards as required by FDA Office of Biologies standards.

[0043] The term“pharmaceutically acceptable carrier” or“pharmaceutically acceptable excipient” as used herein refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical

administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions.

[0044] The term“pharmaceutical composition” as used herein refers to a composition comprising at least one compound as disclosed herein formulated together with one or more pharmaceutically acceptable carriers.

[0045] “ Individual,”“patient,” or“subject” are used interchangeably and include any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans. The compounds of the disclosure can be administered to a mammal, such as a human, but can also be administered to other mammals such as an animal in need of veterinary treatment, e.g., domestic animals (e.g, dogs, cats, and the like), farm animals (e.g, cows, sheep, pigs, horses, and the like) and laboratory animals (e.g, rats, mice, guinea pigs, and the like). “Modulation” includes antagonism (e.g, inhibition), agonism, partial antagonism and/or partial agonism.

[0046] In the present specification, the term“therapeutically effective amount” means the amount of the subject compound that will elicit the biological or medical response of a tissue, system or animal, (e.g. mammal or human) that is being sought by the researcher, veterinarian, medical doctor or other clinician. The compounds of the disclosure are administered in therapeutically effective amounts to treat a disease. Alternatively, a therapeutically effective amount of a compound is the quantity required to achieve a desired therapeutic and/or prophylactic effect, such as an amount that results in substantially mitigating a liver or kidney disease or disorder.

[0047] The term "pharmaceutically acceptable salt(s)" as used herein refers to salts of acidic or basic groups that may be present in compounds used in the compositions. Compounds included in the present compositions that are basic in nature are capable of forming a wide variety of salts with various inorganic and organic acids. The acids that may be used to prepare pharmaceutically acceptable acid addition salts of such basic compounds are those that form non-toxic acid addition salts, i.e., salts containing pharmacologically acceptable anions, including, but not limited to, malate, oxalate, chloride, bromide, iodide, nitrate, sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, / oluenesulfonate and pamoate (i.e., 1 , 1 '-methylene-/v.s-(2- hydroxy-3-naphthoate)) salts. Compounds included in the present compositions that are acidic in nature are capable of forming base salts with various pharmacologically acceptable cations. Examples of such salts include alkali metal or alkaline earth metal salts, particularly calcium, magnesium, sodium, lithium, zinc, potassium, and iron salts. Compounds included in the present compositions that include a basic or acidic moiety may also form pharmaceutically acceptable salts with various amino acids. The compounds of the disclosure may contain both acidic and basic groups; for example, one amino and one carboxylic acid group. In such a case, the compound can exist as an acid addition salt, a zwitterion, or a base salt.

[0048] The compounds of the disclosure may contain one or more chiral centers and, therefore, exist as stereoisomers. The term“stereoisomers” when used herein consist of all enantiomers or diastereomers. These compounds may be designated by the symbols“(+),”“(- ),”“R” or“S,” depending on the configuration of substituents around the stereogenic carbon atom, but the skilled artisan will recognize that a structure may denote a chiral center implicitly. The present invention encompasses various stereoisomers of these compounds and mixtures thereof. Mixtures of enantiomers or diastereomers may be designated“(±)” in nomenclature, but the skilled artisan will recognize that a structure may denote a chiral center implicitly.

[0049] The compounds of the disclosure may contain one or more double bonds and, therefore, exist as geometric isomers resulting from the arrangement of substituents around a carbon-carbon double bond. The symbol ^. denotes a bond that may be a single, double or triple bond as described herein. Substituents around a carbon-carbon double bond are designated as being in the“Z’ or“£” configuration wherein the terms“Z’ and“£” are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting double bonds encompass both the“ E” and“Z” isomers. Substituents around a carbon-carbon double bond alternatively can be referred to as“cis” or“trans,” where“cis” represents substituents on the same side of the double bond and“trans” represents substituents on opposite sides of the double bond.

[0050] Compounds of the disclosure may contain a carbocyclic or heterocyclic ring and therefore, exist as geometric isomers resulting from the arrangement of substituents around the ring. The arrangement of substituents around a carbocyclic or heterocyclic ring are designated as being in the“Z” or“E” configuration wherein the terms“Z” and“E” are used in accordance with IUPAC standards. Unless otherwise specified, structures depicting carbocyclic or heterocyclic rings encompass both“Z” and“E” isomers. Substituents around a carbocyclic or heterocyclic rings may also be referred to as“cis” or“trans”, where the term“cis” represents substituents on the same side of the plane of the ring and the term“trans” represents substituents on opposite sides of the plane of the ring. Mixtures of compounds wherein the substituents are disposed on both the same and opposite sides of plane of the ring are designated“cis/trans.”

[0051] Individual enantiomers and diasteriomers of compounds of the present invention can be prepared synthetically from commercially available starting materials that contain asymmetric or stereogenic centers, or by preparation of racemic mixtures followed by resolution methods well known to those of ordinary skill in the art. These methods of resolution are exemplified by (1) attachment of a mixture of enantiomers to a chiral auxiliary, separation of the resulting mixture of diastereomers by recrystallization or chromatography and liberation of the optically pure product from the auxiliary, (2) salt formation employing an optically active resolving agent, (3) direct separation of the mixture of optical enantiomers on chiral liquid chromatographic columns or (4) kinetic resolution using stereoselective chemical or enzymatic reagents. Racemic mixtures can also be resolved into their component enantiomers by well known methods, such as chiral-phase liquid chromatography or crystallizing the compound in a chiral solvent. Stereoselective syntheses, a chemical or enzymatic reaction in which a single reactant forms an unequal mixture of stereoisomers during the creation of a new stereocenter or during the transformation of a pre-existing one, are well known in the art. Stereoselective syntheses encompass both enantio- and diastereoselective transformations, and may involve the use of chiral auxiliaries. For examples, see Carreira and Kvaemo, Classics in Stereoselective Synthesis , Wiley-VCH: Weinheim, 2009

[0052] The compounds disclosed herein can exist in solvated as well as unsolvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like, and it is intended that the invention embrace both solvated and unsolvated forms. In one embodiment, the compound is amorphous. In one embodiment, the compound is a single polymorph. In another embodiment, the compound is a mixture of polymorphs. In another embodiment, the compound is in a crystalline form.

[0053] The invention also embraces isotopically labeled compounds of the invention which are identical to those recited herein, except that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as ¾ ¾ ¹³C, ¹⁴C, ¹⁵N, ¹⁸0, ¹⁷0, ³¹P, ³²P, ³⁵ S, ¹⁸F, and ³⁶Cl, respectively.

For example, a compound of the invention may have one or more H atom replaced with deuterium. [0054] Certain isotopically-labeled disclosed compounds ( e.g ., those labeled with ³H and ¹⁴C) are useful in compound and/or substrate tissue distribution assays. Tritiated (i.e., ³H) and carbon-l4 (i.e., ¹⁴C) isotopes are particularly preferred for their ease of preparation and detectability. Further, substitution with heavier isotopes such as deuterium (i.e., ²H) may afford certain therapeutic advantages resulting from greater metabolic stability (e.g., increased in vivo half-life or reduced dosage requirements) and hence may be preferred in some circumstances. Isotopically labeled compounds of the invention can generally be prepared by following procedures analogous to those disclosed in the examples herein by substituting an isotopically labeled reagent for a non-isotopically labeled reagent.

[0055] Also contemplated herein are prodrugs of disclosed compounds. The term“prodrug” refers to compounds that are transformed in vivo to yield a disclosed compound or a

pharmaceutically acceptable salt, hydrate or solvate of the compound. The transformation may occur by various mechanisms (such as by esterase, amidase, phosphatase, oxidative and or reductive metabolism) in various locations (such as in the intestinal lumen or upon transit of the intestine, blood or liver). Prodrugs are well known in the art (for example, see Rautio,

Kumpulainen, et al, Nature Reviews Drug Discovery 2008, 7, 255). For example, if a compound of the invention or a pharmaceutically acceptable salt, hydrate or solvate of the compound contains a carboxylic acid functional group, a prodrug can comprise an ester formed by the replacement of the hydrogen atom of the acid group with a group such as (Ci_-8)alkyl, (C_2-i2)alkylcarbonyloxymethyl, 1 -(alkyl carbonyl oxy)ethyl having from 4 to 9 carbon atoms, 1- methyl-l-(alkylcarbonyloxy)-ethyl having from 5 to 10 carbon atoms,

alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms, l-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms, 1 -methyl- l-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms,

l-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms, 3-phthalidyl,

4-crotonolactonyl, gamma-butyrol acton-4 -yl, di-N,N-(Ci_-2)alkylamino(C2.3)alkyl (such as b- dimethylaminoethyl), carbamoyl-(Ci_-2)alkyl, N,N-di(Ci_-2)alkylcarbamoyl-(Ci_-2)alkyl and piperidino-, pyrrolidino- or morpholino(C_2-3)alkyl.

[0056] Similarly, if a compound of the invention contains an alcohol functional group, a prodrug can be formed by the replacement of the hydrogen atom of the alcohol group with a group such as (Ci_-6)alkylcarbonyloxymethyl, l-((Ci_-6)alkylcarbonyloxy)ethyl, 1 -methyl- l-((Ci. ₆)alkylcarbonyloxy)ethyl (C_l-6)alkoxycarbonyloxym ethyl, N-(Ci.

₆)alkoxycarbonylaminomethyl, succinoyl, (Ci_-6)alkylcarbonyl, a-amino(Ci-4)alkylcarbonyl, arylalkylcarbonyl and a-aminoalkylcarbonyl, or a-aminoalkylcarbonyl-a-aminoalkylcarbonyl, where each a-aminoalkylcarbonyl group is independently selected from the naturally occurring L-amino acids, P(0)(0H)₂, -P(0)(0(Ci_-6)alkyl)₂ or glycosyl (the radical resulting from the removal of a hydroxyl group of the hemiacetal form of a carbohydrate).

[0057] If a compound of the invention incorporates an amine functional group, a prodrug can be formed, for example, by creation of an amide or carbamate, an N-alkylcarbonyloxyalkyl derivative, an (oxodioxolenyl)methyl derivative, an N-Mannich base, imine or enamine. In addition, a secondary amine can be metabolically cleaved to generate a bioactive primary amine, or a tertiary amine can metabolically cleaved to generate a bioactive primary or secondary amine. For examples, see Simplicio, et al, Molecules 2008, 13, 519 and references therein.

I. Compounds

[0058] In certain embodiments, the present disclosure provides a pharmaceutically acceptable base salt of a compound represented by:

wherein:

L¹ is -NR^XR²;

R¹ is H or Ci-3alkyl, and R² is selected from the group consisting of: a 4-9 membered monocyclic heterocyclic ring, heteroaryl, -Ci_-2alkylene-heteroaryl, phenyl, -Ci_-2alkylene- phenyl and -Ci_-6alkylene; wherein -Ci_-6alkylene may be straight or branched and may optionally be substituted with one or more substituents each independently selected from R^p; or

R¹ and R² taken together with the nitrogen to which they are attached form a 4-9 membered monocyclic, fused bicyclic, or spirocyclic heterocyclic ring; wherein the 4-9 membered monocyclic, fused bicyclic, or spirocyclic heterocyclic ring is optionally substituted with one or more substituents each independently selected from R^h;

L² is selected from the group consisting of: Ci_-4alkylene, -0-Ci_-6alkylene, -NR^a-Ci. ₄alkylene, -S-Ci_-4alkylene, - S(0)₂-Ci_-4alkylene, -S(0)-Ci_-4alkylene, =CR^a-, -Ci_-2alkylene- heteroaryl, a 4-9 membered monocyclic heterocyclic ring, and a bond; wherein Ci_-4alkylene and -0-Ci_-6alkylene may be straight or branched and may optionally be substituted with one or more substituents each independently selected from R^p; T is selected from the group consisting of: -C(0)OH, tetrazole, -SO3H, isoxazol-3-ol, isothiazol-3-ol, l-alkyl-lH-pyrazol-3-ol, l,2,4-oxadiazol-5-ol, l,2,4-thiadiazol-5-ol, 1H- imidazole-2,5-dione, oxazolidine-2,4-dione-5-yl, thiazolidine-2,4-dione-5-yl, 1,2,4- oxadiazolidine-3,5-dione-2-yl, 4-hydroxy-l,5-dihydro-2H-pyrrol-2-one-l-yl, 6-hydroxy-4H- l,3-dioxin-4-one-2-yl, -C(0)NHS(0)₂-Ci-3alkyl, -NH-S(0)₂-Ci-3alkyl, -S(0)₂-NH-Ci-3alkyl, and -P(0)(OH)(OR^b); wherein -C(0)NHS(0)₂-Ci_-3alkyl, -NH-S(0)₂-Ci_-3alkyl and -S(0)₂-NH- C_l-3alkyl may optionally be substituted with one or more substituents each independently selected from R^p;

R^p is independently selected, for each occurrence, from the group consisting of fluorine, hydroxyl, cyano, and -C(0)NR^aR^b; or two R^p are taken together to form =0;

R^h is independently selected, for each occurrence, from the group consisting of hydrogen, Ci_-3alkyl, fluoro and hydroxyl; or two R^h are taken together to form =0; and

R^a and R^b are independently selected, for each occurrence, from the group consisting of hydrogen and C 1.3 alkyl.

[0059] In some embodiments, L¹ is selected from the group consisting of:

wherein independently for each occurrence p is 1 or 2, q is 1 or 2, r is 0, 1 or 2, Z is CH₂ or O, and Y is N or CH.

[0060] In some embodiments, L¹ is selected from the group consisting of:

[0061] In some embodiments, L¹ is selected from the group consisting of:

wherein independently for each occurrence p is 1 or 2, and q is 1 or 2. [0062] In some embodiments, L¹ is selected from the group consisting of:

[0063] In some embodiments, L² is selected from the group consisting of: -CH₂-, -CH₂-CH₂-, -0-CH₂-, -0-CH₂-CH₂-, -NR^a-CH₂-, -CH₂-C(CH₃)(CH₃)-, -0-CH₂-C(CH₃)(CH₃)-,-0-

C(CH₃)(CH₃)-, -0-CH₂-CH₂-CHR^P-CH₂-CHR^P-CH₂-, -CH₂C(R^p)₂-, -CH₂-CHR^p-, -S-CH₂-, -

, and a bond; wherein independently for each occurrence X^a is CH or N. [0064] In some embodiments, the base salt is an alkali metal salt. For example, in some embodiments, the alkali metal is selected from the group consisting of, e.g., sodium, potassium, and lithium.

[0065] In some embodiments, T is selected from the group consisting of:

[0066] In some embodiments, T may be selected from a base salt (e.g., a lithium, sodium or potassium salt) of a moiety selected from the group consisting of:

[0067] Also provided herein are compounds that may be selected from the group consisting of: sodium 3-((S)-2-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)-3- methylbutyl)-5-oxo-l,2,4-oxadiazol-4-ide; sodium 2-(l-((((3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)azetidin-3-yl)acetate; sodium l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)piperidine-4-carboxylate; sodium 5-((l-((((3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)azetidin-3-yl)methyl)tetrazol-2-ide; sodium 2-((l-((((3R,4S,5S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)oxy)acetate; sodium 3-(l-((((3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)azetidin-3-yl)propanoate; sodium 5-(2-(l-((((3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)azetidin-3-yl)ethyl)tetrazol-2-ide; sodium (2-(l-((((3R,4S,5S,6R)-5-methoxy- 4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)acetyl)(methylsulfonyl)amide; sodium ethyl(2-(l- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)ethyl)phosphonate; sodium 2-((l- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)oxy)-2-methylpropanoate; sodium 3-(l- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-2,2-dimethylpropanoate; sodium (3-(l- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)propanoyl)(methylsulfonyl)amide; sodium 5-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2- yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)methyl)isoxazol-3-olate; sodium 5- (2-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2- yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)ethyl)isoxazol-3-olate; sodium 3-((l- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)oxy)propanoate; sodium 2-(((S)-l- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)pyrrolidin-3-yl)oxy)acetate; sodium 2-((l- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)amino)acetate; sodium 2-(((R)-l- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)pyrrolidin-3-yl)oxy)acetate; sodium 2-(3- (((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)azetidin-l-yl)acetate; sodium 4- ((((((3R,4S,5S,6R)-5-methoxy-4-((2S,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)methyl)benzoate; sodium 4-(((((3R,4S,5S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)benzoate; sodium 6-((((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)methyl)nicotinate; sodium l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)- 2 -methyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidine-3-carboxylate; sodium 2-((R)-3-(((((3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)amino)pyrrolidin-l-yl)acetate; sodium 2-((S)-3-(((((3R,4S,5S,6R)-5-methoxy- 4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)pyrrolidin-l-yl)acetate; sodium 2-(4-(((((3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)amino)piperidin-l-yl)acetate; sodium ethyl (l-((((3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)azetidin-3-yl)phosphonate; sodium 3-(3-(((((3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)amino)azetidin-l-yl)propanoate; sodium 2-(4-((((3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)piperazin-l-yl)acetate; sodium 2-(4-((((((3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)amino)methyl)-lH-l,2,3-triazol-l-yl)acetate; sodium 2-(4-((((((3R,4S,5S,6R)-

5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-

6-yl)oxy)carbonyl)(methyl)amino)methyl)-lH-l,2,3-triazol-l-yl)acetate; sodium (3R,5R)-3,5- dihydroxy-7-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)heptanoate; sodium 3-(3- (((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)azetidin-l-yl)-2,2-dimethylpropanoate; sodium (3R,5R)-3,5-dihydroxy-7-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)oxy)heptanoate; sodium 2-(6-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2,6- diazaspiro[3.3]heptan-2-yl)acetate; sodium 2-(4-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2- oxopiperazin-l-yl)acetate; sodium 2-((2-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2- azaspiro[3.3]heptan-6-yl)oxy)acetate; sodium 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-3- methylazetidin-3-yl)oxy)acetate; sodium 2-(4-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)piperazin-l-yl)acetate; sodium l-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)- 2 -methyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)-lH-pyrazole-4-carboxylate; sodium 5-((((((3R,4S,5S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)methyl)picolinate; sodium l-(l-((((3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)azetidin-3-yl)-lH-pyrazole-3-carboxylate; sodium 6-(l-((((3R,4S,5S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)nicotinate; sodium 5-(3-hydroxy-l-((((3R,4S,5S,6R)-5-methoxy- 4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)picolinate; sodium 6-(3-hydroxy-l-((((3R,4S,5S,6R)-5-methoxy- 4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)nicotinate; sodium 3-fluoro-l-((((3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)azetidine-3-carboxylate; sodium 6-(3-fluoro-l-((((3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)azetidin-3-yl)nicotinate; sodium 5-(l-((((3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)azeti din-3 -yl) picolinate; sodium l-[l-({[(3R,4S,5S,6R)-5-methoxy-4- [(2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6- yl]oxy}carbonyl)azetidin-3-yl]-lH-imidazole-4-carboxylate; sodium 5-(3-fluoro-l- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)picolinate; sodium 2-(l-((((3R,4S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-ylidene)acetate; sodium 2-((l-((((3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)azetidin-3-yl)thio)acetate; sodium 2-((l-((((3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)azetidin-3-yl)sulfonyl)acetate; sodium 2-((((3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)-2-azaspiro[3.3]heptane-6-carboxylate; sodium 2,2-difluoro-3-(l- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)propanoate; sodium 2-((l- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)sulfmyl)acetate; sodium l-(l- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-6-oxo-l,6-dihydropyridine-3-carboxylate; sodium l-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-2-oxo-l,2- dihydropyridine-4-carboxylate; sodium l-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azeti din-3 -yl)-2-oxo-l,2-dihydropyridine-3-carboxylate; sodium 4-[l- ({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl]-l- oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3-yl]-l,3-thiazole-2-carboxylate; sodium 3-(l- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-2-oxopropanoate; sodium l-(l- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-4-oxo-l,4-dihydropyridine-3-carboxylate; sodium 2-hydroxy-3-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2- en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)propanoate; sodium 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2- yl)-l-oxaspiiO[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)methyl)-2H-l, 2, 3-tri azole-4- carboxylate; sodium 6-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en- l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-6,7-dihydro-5H-pyrrolo[3,4- b]pyridine-3 -carboxylate; sodium 2-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2,3-dihydro-lH- pyrrolo[3,4-c]pyridine-6-carboxylate; sodium l-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)methyl)-lH-l,2,3-triazole-4-carboxylate; sodium 3-fluoro-l- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)pyrrolidine-3-carboxylate; sodium 2-((((3R,4S,5S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)-5-oxa-2-azaspiro[3.4]octane-6-carboxylate ; and sodium 2-(l- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)ethane-l-sulfonate; and a pharmaceutically acceptable salt or stereoisomer thereof.

II. Methods

[0068] In some embodiments, the disclosure provides a method of treating or mitigating a liver, intestine and/or kidney disorder in a patient in need thereof, comprising administering an effective amount of a disclosed compound to the patient. In some embodiments, for example, the liver disorder is one or more of, e.g.: non-alcoholic steatohepatitis, alcoholic

steatohepatitis, hepatocellular carcinoma, liver cirrhosis, and hepatitis B. In other

embodiments, the kidney disorder may be one or more of chronic kidney disease, glomerular disease such as iGA nephropathy, lupus nephritis, polycystic kidney disease

[0069] Contemplated patients include not only humans, but other animals such as companion animals (e.g., dogs, cats).

[0070] Methods of treating hepatocellular carcinoma in a patient in need thereof are also contemplated herein, comprising adminsetering an effective amount of a disclosed compound to the patient. In some embodiments, the patient suffers from type 2 diabetes, hepatitis B, cirrhosis, alpha-l antitrypsin deficiency, and/or is obese.

[0071] For example, provided herein are methods of treating, or preventing the development of, hepatic preneoplastic lesions in patient in need thereof, comprising administering an effective amount of a disclosed compound to the patient. In some embodiments, the patient is at risk for but not yet suffering from NASH. In some embodiments, the patient has at least one of, for example: minimal liver fibrosis, minimal hepatic steatosis, lobular inflammation, and/or balloon degeneration. For example, in some embodiments, the patient is diabetic (e.g. type 2 diabetes) or in pre-diabetic condition. The patient may or may not also suffer from obesity. In some embodiments, the patient has a risk of developing NASH (and/or alcoholic steatohepatitis or cirrhosis), but is not suffering from NASH and/or alcoholic steatohepatitis or cirrhosis, or in other embodiments, the patient has developed NASH, alcoholic steatohepatitis or cirrhosis, but has not suffered from hepacellular carcinoma. For example, a patient may have minimal liver fibrosis, minimal hepatic steatosis, lobular inflammation and/or balloon degeneration. Compounds may be administered in an amount insufficient to modulate or suppress angiogenesis in said patient. In some embodiments, the patient suffers from type 2 diabetes and/or is obese. Contemplated patients may optionally be currently administering or have been administered amiodarone, antiviral drugs such as nucleoside analogues, aspirin, NSAIDS, corticosteroids, methotrexate, tamoxifen, or tetracycline.

[0072] A patient suscepted of suffering from NASH or one or more of NASH symptoms may be scored using a NASH scoring. A NASH scored is the combined score of fibrosis (0-4), steatosis (0-3), lobular inflammation (0-3), and ballooning degeneration (0-2) as illustrated in table A (see Design and validation of a histological scoring system for nonalcoholic fatty liver disease, Kleiner et ak, Hepatology 41; 2005).

Table A

[0073] Without being bound to any particular theory, contemplated compounds disclosed herein may beneficially affect hepatic glucose uptake, insulin action, steatosis, inflammation and/or cell dysplasia.

[0074] Disclosed compounds may be administered to patients (animals and/or humans) in need of such treatment in dosages that will provide optimal pharmaceutical efficacy. It will be appreciated that the dose required for use in any particular application will vary from patient to patient, not only with the particular compound or composition selected, but also with the route of administration, the nature of the condition being treated, the age and condition of the patient, concurrent medication or special diets then being followed by the patient, and other factors which those skilled in the art will recognize, with the appropriate dosage ultimately being at the discretion of the attendant physician. For treating clinical conditions and diseases noted above, a compound of this invention may be administered orally, subcutaneously, topically, parenterally, by inhalation spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. Parenteral

administration may include subcutaneous injections, intravenous or intramuscular injections or infusion techniques.

[0075] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0076] In general, a suitable daily dose of a disclosed compound will be that amount of the compound which is the lowest dose effective to safely produce the maximal therapeutic effect. Such an effective dose will generally depend upon the factors described above. For example, a disclosed compound can be administered at about 0.01 mg/kg to about 200 mg/kg, at about 0.1 mg/kg to about 100 mg/kg, or at about 0.5 mg/kg to about 50 mg/kg. When the compounds described herein are co-administered with another agent (e.g., as sensitizing agents), the effective amount may be less than when the agent is used alone. Contemplated methods may include administration of a disclosed compounds, for example, hourly, twice hourly, every three to four hours, daily, twice daily, 1, 2, 3 or 4 times a week, every three to four days, every week, or once every two weeks depending on half-life and clearance rate of the particular compound.

[0077] Treatment can be continued for as long or as short a period as desired. The compositions may be administered on a regimen of, for example, one to four or more times per day. A suitable treatment period can be, for example, at least about one week, at least about two weeks, at least about one month, at least about six months, at least about 1 year, or indefinitely. A treatment period can terminate when a desired result disclosed herein, e.g., treating or mitigating a liver disorder disclosed herein, is achieved. A treatment regimen can include a corrective phase, during which a dose sufficient to treat or mitigate a liver disorder disclosed herein, and can be followed by a maintenance phase, during which a e.g. a lower dose sufficient to prevent recurrence of the liver disorder is administered. A suitable maintenance dose is likely to be found in the lower parts of the dose ranges provided herein, but corrective and maintenance doses can readily be established for individual subjects by those of skill in the art without undue experimentation, based on the disclosure herein.

[0078] Contemplated methods may also include administering a disclosed compound in combination with one or more other agents (and/or in combination with weight loss surgery and/or weight loss therapy). In some embodiments, other agents may be selected from an antidiabetic medication (e.g., metformin, a modulator of glucagon-like peptide -1 receptor such as exenatide, liraglutide, lixisenatide, albiglutide and dulaglutide; and/or a thiazolidinedione such as rosiglitazone, pioglitazone, troglitazone, and/or netoglitazone). In some embodiments, other agents may include a C-C chemokine receptor type 5/type 2 (CCR5/CCR2) modulator, such as but not limited to cenicriviroc. In some embodiments, the other agent may be fibroblast growth factor 21 (FGF21) modulator, such as but not limited to BMS-986036, an

investigational pegylated analogue of FGF21. In some embodiments, the other agent is a peroxisome proliferation-activated receptor (PPAR) agonist, and/or may include agents such as elafibranor, clofibrate, gemfibrozil, ciprofibrate, bezafibrate, and/or fenofibrate,

thiazolidinediones, NSAIDs, indoles, aleglitazar, muraglitazar and tesaglitazar. In some embodiments, the other agent is a obeticholic acid, aramchol, emricasan, simtuzumab, GS-4997 (Gilead), GR-MD-02 (Galectin Therapeutics), Tipelukast, ARI-3037MO (Arisaph

Pharmaceuticals), and/or volixibat. In some embodiments, a contemplated other agent is selected from an antioxidant (e.g., vitamin E, selenium or betaine), a blood pressure lowering medication (e.g., isinopril, losartan, metoprolol, amlodipine, aliskiren), and a cholesterol lowering medication (e.g., atorvastatin, rosuvastatin, or nicotinic acid). In other embodiments, a contemplated other agent is selected from amiodarone, antiviral drugs such as nucleoside analogues, aspirin, corticosteroids, methotrexate, tamoxifen, and tetracycline.

[0079] For example, a patient with NASH may also have other conditions, such as diabetes, high blood pressure or high cholesterol levels. Thus, contemplated herein are disclosed compounds in combination with at least one other agent that may be used to treat one or more of these conditions. For example, the second active agent can be, for example, an antidiabetic medication (e.g., metformin, and/or a thiazolidinedione such as rosiglitazone, pioglitazone, troglitazone, and/or netoglitazone), an antioxidant (e.g., vitamin E, selenium or betaine), a blood pressure lowering medication (e.g., lisinopril, losartan, metoprolol, amlodipine, aliskiren), or a cholesterol lowering medication (e.g., atorvastatin, rosuvastatin, or nicotinic acid), or another agent such as vitamin E. In other embodiments, contemplated methods further include administering to the patient an active agent such as one or more of: amiodarone, antiviral drugs such as nucleoside analogues, aspirin, NSAIDS, corticosteroids, methotrexate, tamoxifen, or tetracycline.

III. Pharmaceutical Compositions and Kits

[0080] The disclosure also provides pharmaceutical compositions comprising compounds as disclosed herein formulated together with a pharmaceutically acceptable carrier. In particular, the present disclosure provides pharmaceutical compositions comprising compounds as disclosed herein formulated together with one or more pharmaceutically acceptable carriers. These formulations include those suitable for oral, rectal, topical, buccal, parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous) rectal, vaginal, or aerosol

administration, although the most suitable form of administration in any given case will depend on the degree and severity of the condition being treated and on the nature of the particular compound being used. For example, disclosed compositions may be formulated as a unit dose, and/or may be formulated for oral or subcutaneous administration.

[0081] Exemplary pharmaceutical compositions may be used in the form of a pharmaceutical preparation, for example, in solid, semisolid or liquid form, which contains one or more of disclosed compounds, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for external, enteral or parenteral applications. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, and any other form suitable for use. The active object compound is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the process or condition of the disease. [0082] For preparing solid compositions such as tablets, the principal active ingredient may be mixed with a pharmaceutical carrier, e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g, water, to form a solid preformulation

composition containing a homogeneous mixture of a disclosed compound, or a non-toxic pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.

[0083] In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), the subject composition is mixed with one or more

pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4)

disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such a talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the compositions may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.

[0084] A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface- active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the subject composition moistened with an inert liquid diluent. Tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. [0085] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the subject composition, the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, cyclodextrins and mixtures thereof.

[0086] Suspensions, in addition to the subject composition, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0087] Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing a subject composition with one or more suitable non irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the body cavity and release the active agent.

[0088] Dosage forms for transdermal administration of a subject composition include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active component may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

[0089] The ointments, pastes, creams and gels may contain, in addition to a subject composition, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

[0090] Powders and sprays may contain, in addition to a subject composition, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. [0091] Disclosed compositions and compounds may alternatively be administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A non-aqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers may be used because they minimize exposing the agent to shear, which may result in degradation of the compounds contained in the subject compositions. Ordinarily, an aqueous aerosol is made by formulating an aqueous solution or suspension of a subject composition together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary with the requirements of the particular subject composition, but typically include non-ionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols. Aerosols generally are prepared from isotonic solutions.

[0092] Pharmaceutical compositions contemplated herein may be suitable for parenteral administration comprise a subject composition in combination with one or more

pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.

[0093] Examples of suitable aqueous and non-aqueous carriers which may be employed in contemplated pharmaceutical compositions include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate and cyclodextrins. Proper fluidity may be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

EXAMPLES

[0094] The compounds described herein can be prepared in a number of ways based on the teachings contained herein and synthetic procedures known in the art. In the description of the synthetic methods described below, it is to be understood that all proposed reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment and workup procedures, can be chosen to be the conditions standard for that reaction, unless otherwise indicated. It is understood by one skilled in the art of organic synthesis that the functionality present on various portions of the molecule should be compatible with the reagents and reactions proposed. Substituents not compatible with the reaction conditions will be apparent to one skilled in the art, and alternate methods are therefore indicated. The starting materials for the examples are either commercially available or are readily prepared by standard methods from known materials.

[0095] At least some of the compounds identified as“Intermediates” herein are contemplated as compounds of the invention.

[0096] All reagents were purchased from commercial suppliers (Sigma-Aldrich, AlfaAesar, Acros Organics, etc.) and used without further purification unless otherwise stated. THF was continuously refluxed and freshly distilled from sodium and benzophenone under nitrogen, dichloromethane was continuously refluxed and freshly distilled from CaH₂ under nitrogen.

[0097] Reactions were monitored by LCMS and/or TLC on silica gel 60 HSGF254 percolated plates (0.15-0.2 mm Si02) and visualized using UV light (254 nm or 365 nm) and/or staining with phosphomolybdic acid ethanol solution (10 g in 100 mL ethanol) and subsequent heating.

[0098] Analytical LCMS were typically performed on SHIMADZU LCMS-2010EV

(ChromolithSpeedROD, RP-l8e, 50x4.6 mm, mobile phase: Solvent A: CH₃CN/H₂0

/HCOOH=l 0/90/0.05, Solvent B: CH₃CN/H₂0 /HCOOH=90/l0/0.05, 0.8min@ 10% B, 2.7min gradient (10-95% B), then 0.8min@95%B, Flow rate: 3mL/min, temperature: 40°C).

[0099] Preparative HPLC were typically performed either by Method A: SHIMADZU LC- 8A (Column: YMC Pack ODS-A (l50*30mm, lOpm)) or Method B: LC-6AD (Column:

Shim=Pack PREP-ODS-H (250*20mm, lOpm)) with UV detection. Both methodswere controlled by LC solution Chemstation software using H₂0 (containing 0.1% HCOOH) and MeOH (or MeCN) as mobile phase at the indicated flow rate.

[0100] Analytical HPLC were typically performed on a SHIMADZU LC-2010A

(ChromolithSpeedROD, RP-l8e, 50x4.6 mm, mobile phase: Solvent A: CH₃CN/H₂0

/HCOOH=l 0/90/0.05, Solvent B: CH₃CN/H₂0 /HCOOH=90/l0/0.05, 0.8min@ 10% B, 2.7min gradient (10-95% B), then 0.8min@95%B, Flow rate: 3mL/min, temperature: 40°C).

[0101] Chiral HPLC were performed on a SHIMADZU LC-2010A (Chiral column, mobile phase: Solvent A: hexane (optionally containing 0.1% diethylamine), Solvent B: Ethanol or Isopropanol; Flow rate: 0.8 mL/min, temperature: 30°C).

[0102] 1H spectra were recorded on BrukerAvance II 400MHz NMR. Chemical shifts (d) were reported in ppm relative to tetramethylsilane (d = 0.000 ppm), and the spectra were calibrated to the residual solvent signal of chloroform (d = 7.26), Dimethyl sulfoxide (d = 2.50), or methanol (d = 3.30). Data for 1H NMR spectra were reported as follows: chemical shift (multiplicity, number of hydrogens). Abbreviations were described as follows: s (singlet), d (doublet), t (triplet), q (quartet), quint (quintet), m (multiple), br (broad).

Abbreviations

CDI = l,l '-carbonyl diimidazole

DBU = l,8-diazabicyclo(5.4.0)undec-7-ene

DCM = dichloromethane

DIPEA = diisopropylethylamine

DMAP = (4-dimethylamino)pyridine

DMSO = dimethyl sulfoxide

DMF = dimethyl formamide

EA = ethyl acetate

FA = formic acid

LDA = lithium diisopropylamine

PE = petroleum ether

r.t. = room temperature

TBTU = 2-(lH -Benzoin azole- 1 -yl)- 1,1, 3 ,3 -tetram ethyl aminium tetrailuoroborate

TEA = trithylamine

TFA = trifluoroacetic acid

THF = tetrahydrofuran

Example 1: Preparation of sodium 3-((S)-2-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)-3-methylbutyl)-5-oxo-l,2,4-oxadiazol-4-ide

Step 1. Preparation of (S)-tert-butyl (l-amino-4-methyl-l-oxopentan-3-yl)carbamate

1 . BOC₂0, Py

dioxane

[0103] To a mixture of (S)-3-((tert-butoxycarbonyl)amino)-4-methylpentanoic acid (2.02 g, 8.73 mmol) in l,4-dioxane (20 mL) was added di-tert-butyl dicarbonate (5.03 g, 23.04 mmol), followed by dropwise addition of pyridine (0.43 mL, 5.38 mmol) at 0°C. The mixture was stirred at 0°C for 10 min and ammonium bicarbonate (898 mg, 11.35 mmol) was added. After addition of the ammonium bicarbonate, the resulting mixture was stirred at room temperature overnight. The mixture was then diluted with ethyl acetate and washed with water and brine, dried over Na₂S0₄ and concentrated to give (S)-tert-butyl ( l-amino-4-m ethyl- 1 -ox opentan-3- yl)carbamate (2.02 g) as white solid. LC-MS (ESI) found: 175 [M+l-56]⁺.

Step 2. Preparation of (S)-tert-butyl (l-cyano-3-methylbutan-2-yl)carbamate

[0104] To a solution of (S)-tert-butyl (l-amino-4-methyl-l-oxopentan-3-yl)carbamate (1.72 g, 7.47 mmol) in DMF (15 mL) was added 2,4,6-trichloro-l,3,5-triazine (2.34 g, 12.68 mmol) at 0°C under N₂ atmosphere. The resulting mixture was stirred at room temperature for 3 hr. The mixture was then diluted with ethyl acetate and washed with aqueous lithium chloride solution, dried over Na₂S0₄ and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 2 to 5 % ethyl acetate in petroleum ether) to give (S)-tert- butyl (l-cyano-3-methylbutan-2-yl)carbamate (1.02 g) as white solid. LC-MS (ESI) found: 157 [M+l-56]⁺. 1HNMR (400 MHz, DMSO-i¾) S 7.04 (m, 1H), 3.57 - 3.42 (m, 1H), 2.66 (m, 1H), 2.55 - 2.51 (m, 2H), 1.69 (m, 1H), 1.40 (s, 9H), 0.83 (m, 6H).

Step 3. Preparation of (S)-tert-butyl (l-(hydroxyamino)-l-imino-4-methylpentan-3- yl)carbamate

[0105] To a mixture of (S)-tert-butyl (l-cyano-3-methylbutan-2-yl)carbamate (1.02 g, 4.80 mmol) in ethanol (6mL) was added aqueous hydroxylamine solution (1.5 mL, 24.02 mmol, 50% wt). The resulting mixture was stirred at 50°C overnight. The mixture was then

concentrated, diluted with ethyl acetate, and washed with water and brine. The organic layer was dried over Na₂S0₄ and concentrated to give a residue, which was purified by flash chromatography (silica gel, 1 to 2% methanol in dichloromethane) to give (S)-tert-butyl (1- (hydroxyamino)-l-imino-4-methylpentan-3-yl)carbamate (1.0 g) as white solid. LC-MS (ESI) found: 190 [M+l-56]⁺.

Step 4. Preparation of (S)-tert-butyl (3-methyl-l-(5-oxo-2,5-dihydro-l,2,4-oxadiazol-3- yl)butan-2-yl)carbamate

[0106] To a solution of (S)-tert-butyl (l-(hydroxyamino)-l-imino-4-methylpentan-3- yl)carbamate (434 mg, 1.77 mmol) in dioxane (8 mL) was added DBU (315 mg, 1.95 mmol) and CDI (296 mg, 1.94 mmol), the mixture was heated to 80°C for 2hrs. The mixture was diluted with water and extracted with EA, and the aqueous water was acidified to pH2 with 1 N

HC1 and extracted with EA. The organic layers were washed with brine and dried over Na₂S0₄, filtered and concentrated to provide the title compound (434 mg) as white solid. LC-MS (ESI) found: 216 [M+l-56]⁺.

Step 5. Preparation of (S)-3-(2-amino-3-methylbutyl)-l,2,4-oxadiazol-5(2H)-one trifluoroacetic acid salt

[0107] To a mixture of (S)-tert-butyl (3-methyl-l-(5-oxo-2,5-dihydro-l,2,4-oxadiazol-3- yl)butan-2-yl)carbamate (434 mg, 1.60 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid (2.5 mL) dropwise at 0°C. The reaction was stirred at room temperature for 1 hr and concentrated under reduced pressure to give (S)-3-(2-amino-3-methylbutyl)- 1,2,4- oxadiazol-5(2H)-onetrifluoroacetic acid salt (425 mg) as yellow syrup, which was directly used in the next step without purification. LC-MS (ESI) found: 172 [M+l]⁺.

Step 6. Preparation of (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2- en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl ((S)-3-methyl-l-(5-oxo-4,5-dihydro-l,2,4- oxadiazol-3-yl)butan-2-yl)carbamate

General Procedure for Coupling:

Intermediate 1

[0108] To a solution of (S)-3-(2-amino-3-methylbutyl)-l,2,4-oxadiazol-5(2H)-one (268 mg, 1.57 mmol) in acetonitrile (8 mL) was added N,N-Diisopropylethylamine (0.94 mL, 5.24 mmol) dropwise at 0°C. The mixture was stirred at 0°C for 10 min, and (3R,4S,5S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl (4-nitrophenyl) carbonate (Intermediate 1, 585 mg, 1.31 mmol) was added. After addition, the reaction was stirred at room temperature overnight under N₂ atmosphere. The mixture was then concentrated under vacuum to remove the solvent while keeping the temperature below 40°C. The residue was diluted with dichloromethane and washed with ammonium acetate buffer (pH 4.0, 20 mL x 2) and 5% sodium bicarbonate solution (20 mL). The organic layer was dried over Na₂S0₄, filtered and concentrated to give crude product. The crude product was purified by flash chromatography (silica gel, 1 to 2% MeOH in DCM) and preparative HPLC (Method A, H₂0 (0.1% FA)/ C¾CN) to give (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl- 3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6-yl((S)-3 -methyl- 1 -(5 -oxo-4, 5 - dihydro-l,2,4-oxadiazol-3-yl)butan-2-yl)carbamate (160 mg) as white solid. LC-MS (ESI) found: 480 [M+l]⁺. 1H NMR (400 MHz, CDCI₃) d 10.15 (m, 1H), 5.45 (m, 1H), 5.14 (m, 1H), 4.64 (m, 1H), 3.63 (s, 2H), 3.37 (s, 3H), 2.91 (s, 1H), 2.77 - 2.49 (m, 4H), 2.08 (m, 1H), 1.99 - 1.72 (m, 6H), 1.69 (s, 3H), 1.60 (s, 3H), 1.17 (d, J= 8.0 Hz, 3H), 1.03 (s, 1H), 0.91 (m, 6H).

Step 7. Preparation of sodium 3-((S)-2-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl- 3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)-3- methylbutyl)-5-oxo-l,2,4-oxadiazol-4-ide

[0109] To a solution of (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2- en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl (90 mg, 0.18 mmol) in MeCN was added dropwised 0.1 N NaOH (1.6 mL,0.16 mmol) solution at -60 °C with stirring. After

lypholization, 3-((S)-2-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2- en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)-3-methylbutyl)-5-oxo- l,2,4-oxadiazol-4-ide sodium salt (90 mg) was obtained as white solid. LC-MS (ESI) found: 480 [M+l]⁺. 1H NMR (400 MHz, DMSO- d) d 6.84 (d, J= 9.3 Hz, 1H), 5.18 (dd, J= 20.7, 13.3 Hz, 2H), 3.62 - 3.45 (m, 2H), 3.24 (s, 3H), 2.80 (d, J= 4.3 Hz, 1H), 2.60 - 2.50 (m, 2H), 2.24 (dd, J = 14.7, 6.2 Hz, 2H), 2.20 - 2.08 (m, 2H), 1.98 - 1.73 (m, 3H), 1.68 (s, 3H), 1.58 (s,

3H), 1.07 (s, 3H), 0.79 (t, J= 7.0 Hz, 6H).

Example 2: Preparation of sodium 2-(l-((((.?/?, S,5A,6/?)-5-methoxy-4-((2/?,.?/?)-2-methyl- 3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)acetate

Step 1 : Preparation of 2-(azetidin-3-yl)acetic acid(TFA salt)

[0110] To a solution of 2-(l-(tert-butoxycarbonyl)azetidin-3-yl)acetic acid (1.00 g, 4.64 mmol) in DCM (5 mL) was added TFA (2 mL) at 0°C. The reaction was then stirred at room temperature overnight. The reaction mixture was concentrated to give 2-(azeti din-3 -yl)acetic acid ( 1.35 g, TFA salt) as a yellow oil, which was directly used in the next step without further purification. LC -MS (ESI) found: 116 [M+l]⁺.

Step 2 : Preparation of 2-(l-((((J ?, A,5A,6 ?)-5-methoxy-4-((2 ?,J ?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)acetic acid

[0111] 2-(Azeti din-3 -yl)acetic acid (TFA salt, l.3g crude, 4.64 mmol) in CH₃CN (10 mL) was coupled with Intermediate 1 (1.63 g, 3.71 mmol) by following the General Procedure for Coupling. The solvent was removed under vacuum while maintaining the temperature below 40°C, then diluted with DCM (60 mL) and washed with ammonium acetate buffer (pH~4, 15 mL x 2) and 5% sodium bicarbonate solution (20 mL). The aqueous layer was extracted with DCM (10 mL x 2). The combined organic layers were dried over Na₂S0₄ and concentrated to give a yellow oil, which was purified by flash chromatography (silica gel, DCM:MeOH= 100:0 to 60: 1) to give the pure 2-(\-((((3Ii,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methy\-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)acetic acid (1.1 g) as light yellow syrup with HPLC purity 96%.LC-MS (ESI) found: 424 [M+l]⁺. 1H-NMR (400 MHz, DMSO- d₆ ) d 12.25 (br, 1 H), 5.29 (s, lH),5. l9(t, J= 7.6 Hz, 1H), 3.96- 4.00(m,2H), 3.32-3.58 (m, 3 H), 3.28 (s, 3H), 2.78-2.86 (m, 2 H), 2.50-2.59 (m, 4 H), 2.15-2.19 (m, 2 H), 1.91-1.98 (m, 1 H), 1.75-1.80 (m, 6 H), 1.62 (s, 3H), 1.13 (s, 3H), 0.99 (d, J= 13.6 Hz, 1H).

Step 3 : Preparation of sodium 2-(l-((((.?/?, S,5A,6/?)-5-methoxy-4-((2/?,.?/?)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)acetate

[0112] To a solution of 2-(l-((((3f?, S',5,S^,, d/?)-5-methoxy-4-((2f?,3f?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)acetic acid (100 mg, 0.236 mmol) in MeCN (5 mL) was added dropwise 0.1 N NaOH solution (2.1 mL, 0.9 eq) at -60°C with stirring. After lypholization, 120 mg of the title compound was obtained as white solid. LC-MS (ESI) found: 446 [M+l]⁺. 1H-NMR (400 MHz, DMSO-i¾) d 5.28 (s, 1H), 5.20(t, J= 7.6 Hz, 1H), 3.89-3.93(m,2H),3.45-3.53 (m, 3H), 3.28 (s, 3H), 2.84 (d, J= 4.4Hz, 1H), 2.67-2.73 (m, 1H), 2.54-2.58 (m, 2H), 2.11-2.19 (m, 4H), 1.91-1.96 (m, 1H), 1.75-1.80 (m, 6H), 1.62 (s, 3H), 1.14 (s, 3H), 0.98 (d, J= 13.6 Hz, 1H).

Example 3: Preparation of sodium l-((((.?/?, S,5A,6/?)-5-methoxy-4-((2/?,.?/?)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)piperidine-4- carboxylate

Step 1. Preparation of l-((((J ?, »V,5»V,6 ?)-5-methoxy-4-((2 ?,J ?)-2-methyl-3-(3-methylbut-

2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)piperidine-4-carboxylic acid

[0113] Piperidine-4-carboxylic acid (200 mg, 1.55 mmol) was coupled with Intermediate 1

(830 mg, 1.85 mmol) by following the General Procedure for Coupling. The mixture was concentrated under vacuum to remove the solvent while keeping the temperature below 40°C, then diluted with DCM (60 mL) and washed with ammonium acetate buffer (pH~4, 15 mL x 2) and 5% sodium bicarbonate solution (20 mL). The aqueous layer was extracted with DCM (10 mL x 2), dried over Na₂S0₄ and concentrated to give a yellow oil, the resided was purified by column chromatography (silica gel, 1—10 % MeOH in DCM) and prep. HPLC (C18, 0 to 90% acetonitrile in H₂0 with 0.1 % formic acid) to give l-((((3f?,4ri^,,5ri', df?)-5-methoxy-4-((2f?,3f?)- 2-m ethyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6- yl)oxy)carbonyl)piperidine-4-carboxylic acid (260 mg) as white solid. LC-MS (ESI) m/z (M+l): 438. 1H NMR (400 MHz, DMSO-76) d 12.28 (s, 1H), 5.36 (s, 1H), 5.19 (s, 1H), 3.87

(d, J= 13.0 Hz, 2H), 3.54 (dd, 7= 11.1, 2.6 Hz, 1H), 3.32 (s, 3H), 2.86 (d, J= 4.4 Hz, 3H),

2.58 (s, 2H), 2.42 (d, J= 10.8 Hz, 1H), 2.21 - 2.13 (m, 2H), 1.93 (dd, J= 13.5, 4.3 Hz, 1H), 1.79 (s, 4H), 1.71 (s, 4H), 1.61 (s, 3H), 1.41 (s, 2H), 1.08 (s, 3H), 1.02 (d, J= 13.5 Hz, 1H).

Step 2: Preparation of sodium l-((((3/?, V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)piperidine-4- carboxylate

[0114] To a solution of l-((((3f?, ,S^,,5S^,, 6/?)-5-methoxy-4-((2f?,3f?)-2-methyl-3-(3-methylbut- 2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)piperidine-4-carboxylic acid (122 mg, 0.27 mmol) in MeCN (10 mL) was added dropwise a 0.1 N NaOH solution (2.5 mL, 0.24 mmol) at -6°C with stirring, followed by lyophilization to give sodium 1 -((((3R,-fS,5S, 6R)- 5-methoxy-4-((2ri, 3f?)-2-methyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6- yl)oxy)carbonyl)piperidine-4-carboxylate (106 mg) as white solid. LC-MS (ESI) m/z (M+l): 438. 1H MR (400 MHz, DMSO-76) d 5.34 (s, 1H), 5.19 (t, J= 7.4 Hz, 1H), 3.77 (s, 2H), 3.55 - 3.50 (m, 1H), 3.32 (s, 3H), 2.85 (d, J= 4.4 Hz, 3H), 2.58 (d, J= 10.5, 5.2 Hz, 2H), 2.21 - 2.11 (m, 2H), 1.92 (d, 7= 6.3 Hz, 2H), 1.78 (d, 7= 11.0 Hz, 2H), 1.66 (d, 7= 38.7 Hz, 9H),

1.36 (s, 2H), 1.08 (s, 3H), 1.02 (d, J= 13.6 Hz, 1H).

Example 4: Preparation of sodium 5-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)methyl)tetrazol-2-ide

Step 1: Preparation of tert-butyl 3-(cyanomethylene)azetidine-l-carboxylate

B i oc -OEt B

O i oc

[0115] To a solution of NaH (0.8 g, 20 mmol) in THF (90 mL) at 0°C was added diethyl (cyanomethyl)phosphonate (3.45 g, 19.5 mmol). The reaction was stirred at 0°C for 45 min. Then asolution of tert-butyl 3-oxoazetidine-l-carboxylate (.9 g, 16.9 mmol) in THF (20 mL) was added at rt. After addition, the reaction was stirred at r.t. overnight. The reaction was then slowly poured into ice-water and extracted with EA twice. The organic layer was dried over Na₂S0₄ and concentrated to give a residue, which was purified by flash chromatography (silica gel, 10 to -20% EA in PE) to give tert-butyl 3-(cyanomethylene)azetidine-l-carboxylate as a white solid (2 g). LC-MS (ESI) found: 139 [M+l-56]⁺.

Step 2: Preparation of tert-butyl 3-(cyanomethyl)azetidine-l-carboxylate

Boc Boc

[0116] To a solution of /cvV-butyl 3-(cyanomethylene)azetidine-l-carboxylate (1.9 g, 9.79 mmol) in methanol (30 mL) was added Pd/C (0.2 g). The reaction mixture was de-gassed 3 times with N₂ then stirred under H₂ atmosphere at r.t. overnight. The reaction mixture was filtered, and the filtrate was concentrated to give /cvv-butyl 3-(cyanomethyl)azetidine-l- carboxylate as a white solid (1.5 g), which was directly used in the next step without purification. LC-MS (ESI) found: 141 [M+l-56]⁺.

Step 3: Preparation of tert-butyl 3-((2H-tetrazol-5-yl)methyl)azetidine-l-carboxylate

Boc

i

[0117] To a solution of tert- butyl 3 -(cyanomethyl)azetidine-l -carboxylate (1.2 g, 6.12 mmol ) in DMF (15 mL) was added NaN₃ (0.47 g, 7.34 mmol). The reaction mixture was stirred at l20°C overnight. The reaction was diluted by water and extracted with DCM for 3 times. The dichloromethane solution was dried over Na₂S0₄, filtered and concentrated to give crude product, which was purified by prep-HPLC (Method B: 0.l%NH₄OH) to give tert-butyl 3- ((2H-tetrazol-5-yl)methyl)azetidine-l -carboxylate as a yellow solid (280 mg). LC-MS (ESI) found: 184 [M+l-56]⁺.

Step 4: Preparation of 5-(azetidin-3-ylmethyl)-2H-tetrazole

[0118] To a solution of tert- butyl 3-((2H-tetrazol-5-yl)methyl)azetidine-l-carboxylate (280 mg, 1.1 mmol) in DCM (3 mL) was added TFA (400 mg, 3.51 mmol). The reaction was stirred at r.t. overnight. The reaction was concentrated to give 5-(azetidin-3-ylmethyl)-2H-tetrazole as a yellow oil (430 mg, TFA salt), which was directly used in the next step without any purification.

Step 5: Preparation of (3T?, V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-(3-methylbut-2- en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl lH-imidazole-l-carboxylate

Intermediate 2

[0119] To a solution of (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2- en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-ol (2 g, 7.08 mmol) in DCM (30 mL) was added CDI (3.4 g, 20.97 mmol) at 0 °C. After addition, the reaction mixture was stirred at 0°C for 2 to 3 h. The reaction was monitored by HPLC. Once the starting material was completely consumed, the mixture reaction was diluted with DCM (20 mL) and washed with water (30 mL x 3). The organic layers were combined and dried over Na₂S0₄. After filtration, the filtrate was concentrated under reduced pressure to afford the title compound (2.3 g) as oil, which was confirmed by 1HNMR. 1HNMR (400 MHz, CDCl₃) ό 8.12 (s, 1 H), 7.38 (s, 1 H), 7.05 (s, 1 H), 5.81 (s, 1 H), 5.18 (t, J = 6 Hz,l H), 3.74 (d, J = 16 Hz, 1 H), 3.49 (s, 3 H), 3.01 (d, J = 4 Hz, 1

H), 2.58-2.60 (m, 2 H), 2.26-2.35 (m, 1 H), 2.07-2.13 (m, 5 H), 1.90 (d, J = 12 Hz, 1 H), 1.73

(s, 3 H), 1.64 (s, 3 H), 1.10-1.21 (m, 4 H).

Step 6: Preparation of (3/?, V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-(3-methylbut-2- en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl 3-((2H-tetrazol-5-yl)methyl)azetidine-l- carboxylate

[0120] To a solution of 5-(azetidin-3-ylmethyl)-2H-tetrazole (430 mg, TFA salt) in DCM (5 mL) was added DIPEA (750 mg, 5.85 mmol) at 0°C. A solution of (3R, -iS,5S, ri/i)-5-methoxy-4- ((2i?,3i?)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl 1H- imidazole-l -carboxylate (350 mg, 0.936 mmol) in DCM (3 mL) was added to the reaction mixture. The reaction was stirred at r.t. overnight. The reaction mixture was washed with ammonium acetate buffer (pH 4.0, 5 mL x2). The dichloromethane layer was separated and dried over Na₂S0₄. After filtration, the filtrate was concentrated to give crude product, which was purified by prep. HPLC (Method B: 0. l%NH₄OH) to give (3R, -fS, 5S, ri/i)-5-methoxy-4- ((2f?,3f?)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl 3-((2H- tetrazol-5-yl)methyl)azetidine-l-carboxylate as a yellow oil (45 mg). LC-MS (ESI) found: 448 [M+l]⁺.

Step 7: Preparation of Sodium 5-((l-(((( /?, S,5A,6/?)-5-methoxy-4-((2/?, /?)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)methyl)tetrazol-2-ide

[0121] To a stirring solution of (3f?,4ri^,,5ri^,, 6/?)-5-methoxy-4-((2f?,3f?)-2-methyl-3-(3- methylbut-2-en-l -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl 3-((2H-tetrazol-5- yl)methyl)azetidine-l-carboxylate (45 mg, 0.10 mmol) in MeCN (0.5 mL) was added 0.1M NaOH (0.9 mL, 0.09 mmol) at -78°C. The reaction was lyophilized to give sodium 5-((l- ((((3R, 4S, 5S, 6R)-5-methoxy-4-((2R, 3f?)-2-methyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 - oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)methyl)tetrazol-2-ide as yellow oil (45 mg). LC-MS (ESI) found: 448 [M-23+l]⁺. 1H NMR (400 MHz, DMSO-i¾) d 5.26 (s, 1 H), 5.17 (s, 1 H), 3.90 (s, 2 H), 3.59 (s, 1 H), 3.49 (d, J= 9.6 Hz, 2 H), 3.26 (s, 3 H), 3.21 (s, 1 H), 3.10 (s, 1 H), 2.99 - 2.89 (m, 1 H), 2.88 - 2.76 (m, 2 H), 2.57 - 2.52 (m, 1 H), 2.40 (dd, J = 14.1, 7.0 Hz, 1 H), 2.11 (dd, = 14.9, 7.2 Hz, 2 H), 2.01 - 1.86 (m, 1 H), 1.78 - 1.66 (m, 4 H), 1.65 - 1.53 (m, 4 H), 1.35 - 1.15 (m, 5 H), 1.02 (d, J= 22.2 Hz, 2 H), 0.91 (dd, = 11.5, 5.6 Hz, 5 H), 0.82 (d, J= 6.8 Hz, 1 H).

Example 5: Preparation of sodium 2-((l-(((( /?, S,5A,6/?)-5-methoxy-4-((2/?, /?)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)oxy)acetate N

Step 1: Preparation of 2-(azetidin-3-yloxy)acetic acid

[0122] To a solution of 2-((l-(/er/-butoxycarbonyl)azeti din-3 -yl)oxy)acetic acid (500 mg, 2.16 mmol) in DCM (5 mL) was added TFA (2 mL). The mixture was stirred at r.t. for 2hr. The reaction mixture was concentrated and the residue was used in the next step without further purification.

Step 2: Preparation of 2-((l-((((J/?, V,5,V,6/?)-5-methoxy-4-((2/?,J/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)oxy)acetic acid

[0123] 2-(Azeti din-3 -yloxy)acetic acid (200 mg, 0.87 mmol) was coupled with Intermediate 1 (469 mg, 1.05 mmol) by following the General Procedure for Coupling. The mixture was concentrated under vacuum to remove the solvent while keeping the temperature below 40°C. Then diluted with DCM (60 mL) and washed with ammonium acetate buffer (pH~4, 15 mL x 2) and 5% sodium bicarbonate solution (20 mL). The aqueous layer was extracted with DCM (10 mL x 2). The combined organic layers were washed with aq. NaHC0₃ solution (15 mL x 2, 5% w/w), dried over Na₂S0₄ and concentrated to give yellow oil. The residue was purified by column chromatography (silica gel, 1 % ~ 10 % MeOH in DCM) and prep. HPLC (Cl 8, 0 to ~90 % acetonitrile in H₂0 with 0.1 % formic acid) to give 2-((l-((((3i?,47,57, d ?)-5-methoxy-4- ((2i?,3i?)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)oxy)acetic acid (180 mg) as white solid. LC-MS (ESI) m/z (M+l): 440.1H NMR (400 MHz, DMSO) d 5.30 (s, 1H), 5.19 (s, 1H), 4.36 (d, J= 4.0 Hz, 1H), 4.05 (d, J= 8.9 Hz, 2H), 4.00 (s, 2H), 3.81 - 3.70 (m, 2H), 3.52 (dd, 7= 11.0, 2.4 Hz, 1H), 2.85 (d, 7= 4.3 Hz, 1H), 2.59 - 2.52 (m, 2H), 2. l7 (s, 2H), 1.92 (dd, 7 = 13.2, 4.4 Hz, 1H), 1.75 (d, J = 11.0 Hz, 3H), 1.71 (s, 4H), 1.61 (s, 3H), 1.07 (s, 3H), 1.01 (d, 7= 13.6 Hz, 1H).

Step 3: Preparation of sodium 2-((l-((((3/?,7V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)oxy)acetate

[0124] To a solution of 2-(( l -{{{{3R,4S,5S,6R)- -mei\\oxy- -{{2R, 3//)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)oxy)acetic acid (120 mg, 0.27 mmol) in MeCN (10 ml) was added dropwise 0.1 N NaOH solution (2.5 ml, 0.24 mmol) at -60°C with stirring. Lyophilization gave 2-(( 1 -((((3R, -fS,5S, 6R)- 5-methoxy-4-((2i?,5i?)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azeti din-3 -yl)oxy)acetate sodium salt (94 mg) as white solid. LC-MS (ESI) m/z (M+l): 440. 1H NMR (400 MHz, DMSO-76) d 5.29 (s, 1H), 5.19 (s, 1H), 4.36 (s, 1H),

4.03 - 3.92 (m, 2H), 3.74 (s, 2H), 3.54 - 3.43 (m, 3H), 2.85 (d, J = 4.3 Hz, 1H), 2.56 (d, J = 4.1 Hz, 2H), 2.17 (d, J = 7.3 Hz, 2H), 1.93 (s, 1H), 1.75 (d, J = 10.9 Hz, 3H), 1.71 (s, 3H), 1.61 (s, 3H), 1.07 (s, 3H), 1.01 (d, J = 13.3 Hz, 1H).

Example 6: Preparation of sodium 3-(l-((((J ?, »V,5»V,6 ?)-5-methoxy-4-((2 ?,J ?)-2-methyl-

3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)propanoate

Step 1: Preparation of 3-(l-(terf-butoxycarbonyl)azetidin-3-yl)propanoic acid

Boc

[0125] To a solution of /cvV-butyl 3-(2-cyanoethyl)azetidine-l-carboxylate (1.0 g, 4.75 mmol) in THF (10 mL) was added aqueous lithium hydroxide solution (7.1 mL, 7.1 mmol) at 0°C. After addition, the reaction was stirred at room temperature for 16 hr. The mixture was washed with ether and the aqueous layer was acidified to pH = 3~4 by adding 1M hydrochloride acid. The mixture was extracted with chloroform and isopropyl alcohol (3 / 1) three times. The combined organic layers were dried over Na₂S0₄, filtered and concentrated to give 3-(l-(tert- butoxycarbonyl)azetidin-3-yl)propanoic acid (0.9 g) as white solid.

Step 2 : Preparation of 3-(azetidin-3-yl)propanoic acid (TFA salt)

[0126] To a solution of 3-(l-(/er/-butoxycarbonyl)azetidin-3-yl)propanoic acid (0.9 g, 3.92 mmol) in DCM (5 mL) was added TFA (2 mL) at 0°C. The reaction was then stirred at room temperature overnight. The reaction mixture was concentrated to give 3-(azetidin-3- yl)propanoic acid (1.3 g, TFA salt) as yellow oil, which was used for the next step reaction directly without any purification. Step 3: Preparation of 3-(l-((((J ?, A,5A,6 ?)-5-methoxy-4-((2 ?,J ?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)propanoic acid

[0127] 3-(Azetidin-3-yl)propanoic acid (TFA salt, 260 mg crude, 0.78mmol) was coupled with Intermediate 1 (0.28 g, 0.62 mmol) by following the General Procedure for Coupling. The solvent was removed under vacuum below 40°C, then diluted with DCM (10 mL) and washed with ammonium acetate buffer (pH~4, 5 mL x 2) and 5% sodium bicarbonate solution (20 mL). The aqueous layers were extracted with DCM (10 mL x 2). The combined organic layers was dried over Na₂S0₄ and concentrated to give a yellow oil, which was purified by flash chromatography (silica gel, 0 to -1.5% MeOH in DCM) to give the pure 3-(l- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)propanoic acid (l30mg) as light yellow syrup. Step 4: Preparation of sodium 3-(l-((((J ?, A,5A,6/?)-5-methoxy-4-((2 ?,J ?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)propanoate

[0128] To a solution of 3-(l-((((3i?,4S^,,5_<S^,,d/?)-5-methoxy-4-((2i?,3i?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)propanoic acid (60 mg, 0.137 mmol) in MeCN (3 mL) was added dropwise of the 0.1 N NaOH solution (1.2 mL, 0.9 eq) at -60°C with stirring. After lypholization, 62 mg of the title compound was obtained as yellowish solid. LC-MS (ESI) found: 438,460,461 [M+l]⁺. 1H- NMR (400 MHz, DMSO- d₆) d 5.28 (s, 1H), 5.20(t, J= 7.6 Hz, 1H), 3.89-3.93(m,2H), 3.45- 3.53 (m, 3H), 3.28 (s, 3H), 2.84 (d, J= 4.4Hz, 1H), 2.67-2.73 (m, 1H), 2.54-2.58 (m, 2H), 2.11- 2.19 (m, 4H), 1.91-1.96 (m, 1H), 1.75-1.80 (m, 6H), 1.62 (s, 3H), 1.14 (s, 3H), 0.98 (d, J= 13.6 Hz, 1H).

Example 7: Preparation of sodium 5-(2-(l-((((3/?, S,5»S,6/?)-5-methoxy-4-((2/?,.?/?)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)ethyl)tetrazol-2-ide

Step 1: Preparation of terf-butyl 3-(2-hydroxyethyl)azetidine-l-carboxylate Boc

[0129] To a solution of 2-(l-(tert-butoxycarbonyl)azetidin-3-yl)acetic acid (2.5 g, l l .63mmol ) in THF (anhydrous, 20 mL) was added a solution of BH₃-THF (31.3 mL, 3 l .3mmol, 1M in THF) dropwise at -20°C for 30 min. The resulting mixture was stirred at r.t. overnight. The reaction mixture was cooled to 0-5°C. MeOH (lOmL) was added dropwise to the mixture while controlling the temperature below l0°C. The mixture was stirred at r.t. for 20 min and then concentrated under vacuum to give a residue, which was purified by flash chromatography (silica gel, 0 to ~2% MeOH in DCM) to give tert- butyl 3 -(2-hydroxy ethyl)azeti dine- 1- carboxylate (2.15 g) as colorless oil. LC-MS: m/z=l46 (M+H-56)⁺.

Step 2: Preparation of tert- butyl 3-(2-(tosyloxy)ethyl)azetidine-l-carboxylate Boc

0 °C to r.t.

[0130] To a solution of /cvV-butyl 3-(2-hydroxyethyl)azetidine-l-carboxylate (1.8 g, 8.95 mmol) in DCM (15 mL) was added triethylamine (3.71 mL, 26.8 mmol) at 0°C, followed by dropwise addition of a solution of 4-methylbenzene-l-sulfonyl chloride (2.2 g, 11.61 mmol) in DCM (10 mL) over 40 min. The resulting mixture was stirred at 25°C overnight. The mixture was washed with water (10 mL), brine (10 mL), dried over anhydrous Na₂S0₄ and

concentrated. The residue was purified by flash chromatography (silica gel, 0 to -1.5% MeOH in DCM) to provide /cvV-butyl 3-(2-(tosyloxy)ethyl)azetidine-l-carboxylate (1.7 g) as pale yellow oil. LC-MS:m/z=300 (M+H-56)⁺.

Step 3: Preparation of tert- butyl 3-(2-cyanoethyl)azetidine-l-carboxylate Boc

[0131] To a solution of /cvV-butyl 3-(2-(tosyloxyethyl)azetidine-l-carboxylate (1.7 g, 4.78 mmol) in DMSO (10 mL) was added NaCN (0.65 g, 7. l7mmol). The resulting mixture was stirred at l00°C overnight under N₂ atmosphere. The mixture was diluted with water (20 mL) and extracted with ethyl acetate (30 mL x 2). The organic layers were combined, dried over anhydrous Na₂S0₄ and concentrated to give crude product, which was purified by flash chromatography (silica gel, 0 to ~2% MeOH in DCM) to give tert- butyl 3-(2- cyanoethyl)azetidine-l-carboxylate (0.85 g) as yellow oil.LC-MS: m/z=2l l (M+H)⁺.

Step 4: Preparation of tert- butyl 3-(2-(2H-tetrazol-5-yl)ethyl)azetidine-l-carboxylate

[0132] To a solution of /cvV-butyl 3-(2-cyanoethyl)azetidine-l-carboxylate (0.5 g, 2.38mmol) in DMF (5 mL) was added tri -//-butylti n azide (2.37 g, 7.l4mmol). The resulting mixture was stirred at l00°C overnight under N₂ atmosphere. The mixture was diluted with water (10 mL) and extracted with ethyl acetate (30 mL x 2). The organic layers were combined, dried over anhydrous Na₂S0₄ and concentrated to give crude product, which was purified by prep. HPLC to give /ert-butyl 3-(2-(2H-tetrazol-5-yl)ethyl)azetidine-l-carboxylate (200 mg) as oil. LC-MS: m/z=254 (M+H)⁺.

Step 5: Preparation of 5-(2-(azetidin-3-yl)ethyl)-2H-tetrazole (TFA salt)

[0133] To a solution of /cvV-butyl 3-(2-(2H-tetrazol-5-yl)ethyl)azetidine-l-carboxylate (200 mg, 0.79 mmol) in DCM (2 mL) was added TFA (1 mL) at 0°C. The reaction was then stirred at room temperature overnight. The reaction mixture was concentrated to give 5-(2-(azetidin-3- yl)ethyl)-2H-tetrazole (0.32 g, TFA salt) as yellow oil, which was directly used in the next step without any purification. LC-MS (ESI) found: 154 [M+l]⁺. Step 6: (J/?, V,5,V,6/?)-5-methoxy-4-((2/?,J/?)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2- yl)-l-oxaspiro[2.5]octan-6-yl 3-(2-(2H-tetrazol-5-yl)ethyl)azetidine-l-carboxylate

[0134] 5-(2-(Azetidin-3-yl)ethyl)-2H-tetrazole (TFA salt, 0.32g crude, 0.79mmol) was coupled with Intermediate 1 (0.28 g, 0.63 mmol) by following the General Procedure for Coupling. The solvent was removed under vacuum while keeping the temperature below 40°C, then diluted with DCM (10 mL) and washed with ammonium acetate buffer (pH~4, 5 mL x 2) and 5% sodium bicarbonate solution (20 mL). The aqueous layers were extracted with DCM (10 mL x 2). The combined organic layers were washed with aq. NaHC0₃ solution (15 mL x 2, 5% w/w), dried over Na₂S0₄ and concentrated to give a yellow oil, which was purified by prep. HPLC (Method B: 0.l%NH₄OH) to give (3R, -fS,5S, 6R)-5-methoxy-4-((2R, 3/f)-2-methyl-3-(3- methylbut-2-en-l -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl 3-(2-(2H-tetrazol-5- yl)ethyl)azetidine-l-carboxylate (50 mg) as light yellow syrup. LC-MS (ESI) found: 462

[M+l]⁺. Step 7: Preparation of sodium 5-(2-(l-((((3/?, S,5A,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)ethyl)tetrazol-2-ide

[0135] To a stirring solution of (3f?, ri',5ri', 6/?)-5-methoxy-4-((2f?,3f?)-2-methyl-3-(3- methylbut-2-en-l -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl 3-(2-(2H-tetrazol-5- yl)ethyl)azetidine-l-carboxylate (50 mg, 0.1 lmmol) in MeCN (2mL) was added dropwise a solution of 0.1 M NaOH (1 mL, 0.9 eq) at -70°C. After lypholization, 55 mg of the title compound was obtained as yellowish solid. LC-MS (ESI) found: 484 [M+Na],462 [M+l]⁺. 1H- NMR (400 MHz, DMSO-i¾) d 5.47 (s, 1H), 5. l2(m, 1H), 3.97(m,2H), 3.60-3.62 (m, 3 H), 3.37 (s, 3 H), 2.91-2.95 (m,3H), 2.51-2.55 (m, 3H), 2.11-2.14 (m, 1H), 1.78-1.88 (m, 7 H), 1.67 (s, 3H), 1.59 (s, 3H), 1.18 (s, 3H), 1.00 (d, 7= 12.4Hz, 1H).

Example 8: Preparation of sodium ( 2-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2 - methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)acetyl)(methylsulfonyl)amide

Step 1: Preparation of tert- butyl 3-formylazetidine-l-carboxylate

[0136] To a solution of 2-(l-(/er/-butoxycarbonyl)azetidin-3-yl)acetic acid (2.0 g, 9.3 mmol) in DCM (20 mL) was added methanesulfonamide (1.06 g, 11.1 mmol), TBTU (4.47 g, 13.95 mmol) and DIPEA (2.4 g, 18.6 mmol). The mixture reaction was stirred at r.t. overnight. The mixture was extracted with 0.1N HC1, then the acidic water was extracted with DCM and the dichloromethane solution was dried over Na₂S0₄, filtered and concentratedto give crude product, which was purified by flash chromatography (silica gel, 0 to ~2% MeOH in DCM) to give /c/7-butyl 3-formylazetidine-l-carboxylate as yellow oil (1.3 g). LC-MS (ESI) found: 237 [M+l-56]⁺.

Step 2: Preparation of 2-(azetidin-3-yl)-N-(methylsulfonyl)acetamide

[0137] To a solution of /c/7-butyl 3-formylazetidine-l-carboxylate (1.3 g, 4.5 mmol) in DCM

(10 mL) was added TFA (2.53 g, 22.2 mmol). The reaction was stirred at r.t. overnight. The reaction was concentrated to give 2-(azetidin-3-yl)-N-(methylsulfonyl)acetamide (2.1 g, TFA salt), which was directly used in the next step without any purification.

Step 3: Preparation of (J/?, V,5,V,6/?)-5-methoxy-4-((2/?,J/?)-2-methyl-3-(3-methylbut-2- en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl 3-(2-(methylsulfonamido)-2- oxoethyl)azetidine-l-carboxylate

[0138] To a solution of 2-(azeti din-3 -yl)-N-(methylsulfonyl)acetamide (2.1 g, TFA salt ) in DCM (20 mL) was added DIPEA (4.6 g, 36 mmol) at 0°C. A solution of (3R,4S,5S,6R)-5- methoxy-4-((2i?,3i?)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl lH-imidazole-l-carboxylate (1.3 g, 3.6 mmol) in DCM (5 mL) was added dropwise to the above reaction. The reaction mixture was stirred at r.t. overnight. The reaction mixture was washed with ammonium acetate buffer (pH 4.0, 5 mL x 2). The dichloromethane layer was separated, dried over Na₂S0₄, filtered and concentrated to give crude product, which was purified by prep. HPLC (Method B: 0.l%NH₄OH) to give (3R, 4S, 5S, ri/i)-5-methoxy-4- ((2i?,3i?)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl 3-(2-

(methylsulfonamido)-2-oxoethyl)azetidine-l-carboxylate as yellow oil (310 mg). LC-MS (ESI) found: 501 [M+l]⁺.

Step 4: Preparation of sodium(2-(l-((((3/?, V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)acetyl)(methylsulfonyl)amide

[0139] To a solution of (3R,-(S,5S, 6R)-5-methoxy-4-((2R, J/i)-2-methyl-3-(3-methylbut-2-en- l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl 3-(2-(methylsulfonamido)-2-oxoethyl)azetidine-l- carboxylate (310 mg, 0.62 mmol) in MeCN (5 mL) was added 0.1M NaOH (5.5 mL, 0.55 mmol) at -78°C. The reaction was lyophilized to give sodium ( 2-(l-((((3R4S,5S,6R)-5 - methoxy-4-((2i?,3i?)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)acetyl)(methylsulfonyl)amide (300 mg). LC-MS (ESI) found: 501 [M-23+2]⁺. 1H NMR (400 MHz, DMSO-76) d 5.26 (s, 1 H), 5.17 (t, J= 7.4 Hz, 1 H), 3.90 (t, j= 8.4 Hz, 2 H), 3.49 (dd, j= 10.8, 2.4 Hz, 3 H), 3.26 (s, 3H), 2.82 (d, j= 4.2 Hz, 1 H), 2.78 - 2.68 (m, 1 H), 2.66 (s, 3 H), 2.55 (dd, J= 12.6, 5.4 Hz, 2 H), 2.23 (d, J= 7.8 Hz, 2 H), 2.20 - 2.05 (m, 2 H), 1.91 (td, J= 13.2, 4.2 Hz, 1 H), 1.74 (t, 7= 9.6 Hz, 2 H), 1.68 (s, 4 H),

1.59 (s, 3 H), 1.05 (s, 3 H), 0.98 (d, J= 13.4 Hz, 1 H).

Example 9: Preparation of sodium ethyl(2-(l-((((J ?, »V,5»V,6 ?)-5-methoxy-4-((2 ?,J ?)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)ethyl)phosphonate

Step 1: Preparation of tert-butyl 3-formylazetidine-l-carboxylate

Boc

[0140] To a solution of (COCl)₂ (4.0 g, 31.5 mmol) in DCM (30 mL) was added DMSO (2.5 g, 31.4 mmol) at -78°C. The mixture was stirred at -78°C for 30min. A solution of tert- butyl 3- (hydroxymethyl)azetidine-l-carboxylate (3.0 g, 15.8 mmol) in DCM (30 mL) was added dropwise to the reaction at -78°C over 10 min. The reaction was stirred at -78°C for lh. TEA (8.1 g, 31.4 mmol) was added dropwise to the mixture at -78°C slowly. After addition, the reaction was stirred at -78°C for 30min then warmed to 0°C. The reaction was quenched by sat.NELCl (100 mL) and then extracted with DCM for 3 times. The dichloromethane layer was separated and dried over Na₂S0_4. After filtration, the filtrate was concentrated to give tert- butyl 3-formylazetidine-l-carboxylate (4.2 g crude), which was directly used in the next step without any purification.

Step 2: Preparation of (E)-tert-but \ 3-(2-(diethoxyphosphoryl)vinyl)azetidine-l- carboxylate

[0141] To a solution of NaH (0.63 g, 17.38 mmol, 60%) in THF (20 mL) was added tetraethyl methylenebis(phosphonate) (4.72 g, 17.38 mmol) at 0°C. The reaction was stirred at 0°C for lh. A solution of /c/T-butyl 3 -for yl azeti di ne- 1 -carboxyl ate (4.2 g crude) in THF (20 mL) was added dropwise to the mixture at 0°C. After addition, the reaction was stirred at r.t. overnight. The reaction was quenched with sat.MLCl (100 mL) and then extracted with DCM for 3 times. The dichloromethane layer was separated and dried over Na₂S0_4. After filtration, the filtrate was concentratedto give a residue, which was purified by flash chromatography (silica gel, 0 to ~2% MeOH in DCM) to give (E)-tert- butyl 3-(2-

(diethoxyphosphoryl)vinyl)azetidine-l-carboxylate as a yellow oil (1.2 g). LC-MS (ESI) found: 264 [M+l-56]⁺.

Step 3: Preparation of tert- butyl 3-(2-(ethoxy(hydroxy)phosphoryl)ethyl)azetidine-l- carboxylate

[0142] To a solution of (E)-tert- butyl 3-(2-(diethoxyphosphoryl)vinyl)azetidine-l- carboxylate (1.2 g, 3.76 mmol) in ethanol (30 mL) was added Pd/C (0.12 g). The hydrogenation was performed with 1 atm and r.t. overnight. The reaction mixture was filtered and the filtrate was concentrated to give /c/V-butyl 3-(2-(ethoxy(hydroxy)phosphoryl)ethyl)azetidine-l- carboxylate as a yellow oil (1.2 g), which was used directly in the next reaction without purification. LC-MS (ESI) found: 266 [M+l-56]⁺. Step 4: Preparation of ethyl hydrogen (2-(azetidin-3-yl)ethyl)phosphonate

[0143] To a solution of /cvV-butyl 3-(2-(ethoxy(hydroxy)phosphoryl)ethyl)azetidine-l- carboxylate (1.2 g, 3.74 mmol) in DCM (10 mL) was added TMSBr (1.14 g, 7.47 mmol). The mixture was stirred under N₂ atmosphere at r.t. overnight. The reaction solvent was removed under vacuum to give crude residue, which was purified by prep. HPLC (Method B: 0.1%

NH4OH) to give ethyl hydrogen (2-(azetidin-3-yl)ethyl)phosphonateas a yellow oil (108 mg). LC-MS (ESI) found: 194 [M+l]⁺.

Step 5: Preparation of (3/?, V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-(3-methylbut-2- en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl 3-(2- (ethoxy(hydroxy)phosphoryl)ethyl)azetidine-l-carboxylate

[0144] To a solution of ethyl hydrogen (2-(azetidin-3-yl)ethyl)phosphonate (108 mg, 0.56 mmol) in DCM (2 mL) was added DIPEA (72 mg, 0.56 mmol) at 0°C. A solution of

( 3R , 4S, 5S, 6/?)-5-methoxy-4-((2A, 3f?)-2-methyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 - oxaspiro[2.5]octan-6-yl lH-imidazole-l-carboxylate (186 mg, 0.488 mmol) in DCM (2 mL) was added to the above mixture. The reaction was stirred at r.t. overnight. The reaction mixture was monitored by TLC and then washed with ammonium acetate buffer (pH 4.0, 5 mL x 2). The dichloromethane layer was separated, dried over Na₂S0₄, filtered and concentratedto give crude product, which was purified by prep. HPLC (Method B: 0.l%NH₄OH) to give

( 3R , 4S, 5S, 6/?)-5-methoxy-4-((2A, 3f?)-2-methyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 - oxaspiro[2.5]octan-6-yl 3-(2-(ethoxy(hydroxy)phosphoryl)ethyl)azetidine-l-carboxylate as a yellow oil (53 mg). LC-MS (ESI) found: 502 [M+l]⁺.

Step 6: Preparation of sodium ethyl(2-(l-((((3/?, V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)ethyl)phosphonate

[0145] To a stirring solution of (3f?, ri',5ri', 6/?)-5-methoxy-4-((2f?,3f?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl 3-(2- (ethoxy(hydroxy)phosphoryl)ethyl)azetidine-l-carboxylate (53 mg, 0.1 mmol) in MeCN (0.5 mL) was added 0.1M NaOH (0.9 mL, 0.09 mmol) at -78°C. The reaction mixture was lyophilized to give sodium ethyl(2-(l-((((3i?,¥ri',5ri', 6/?)-5-methoxy-4-((2i?,3i?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)ethyl)phosphonate as a solid (57 mg). LC-MS (ESI) found: 502 [M-23+l]⁺. ¹HNMR (400 MHz, DMSO-£¾) d 5.28 (s, 1 H), 5.19 (t, J= 7.4 Hz, 1 H), 3.90 (t, J= 7.9 Hz, 2 H), 3.66 - 3.56 (m, 2 H), 3.51 (dd, j= 11.0, 2.5 Hz, 1 H), 3.43 (s, 2 H), 3.28 (s, 3 H), 2.84 (d, j= 4.3 Hz, 1 H), 2.55 (t, J= 5.4 Hz, 3 H), 2.26 - 2.08 (m, 2 H), 1.97 (dt, J= 26.2, 8.2 Hz, 1 H), 1.80 - 1.74 (m, 2 H), 1.70 (s, 4 H), 1.63 (d, = 14.9 Hz, 5 H), 1.23 (s, 2 H), 1.16 - 1.05 (m, 7 H), 1.01 (d, 7 = 13.7 Hz, 1 H).

Example 10: Preparation of sodium 2-((l-((((3/?, S,5»S,6/?)-5-methoxy-4-((2/?,3/?)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)oxy)-2-methylpropanoate

Step 1: Preparation of ferf-Z>Hiy/ 3-((l-methoxy-l-oxopropan-2-yl)oxy)azetidine-l- carboxylate

[0146] To a solution of tert-butyl 3-hydroxyazetidine-l-carboxylate (2 g, 11.5 mmol) in THF

(20 mL) was added NaH (692 mg, 17.3 mmol) while cooling in an ice bath. After 30min, methyl 2-bromopropanoate (2.3 g, 13.8 mmol) was added. The resulting mixture was stirred at r.t. for 1 hr. The mixture was diluted with water and extracted with EtOAc (100 mL x2). The organic layer was washed with H₂0 (100 ml), dried over Na₂S0₄, filtered and concentrated. The reside was purified by column chromatography (silica gel, 1 to -10 % ethyl acetate in petroleum ether) to give tert- butyl 3-((l-methoxy-l-oxopropan-2-yl)oxy)azetidine-l- carboxylate (2.3 g) as yellow oil. LC-MS (ESI) m/z (M+23): 282. Step 2: Preparation of tert-butyl 3-((l-methoxy-2-methyl-l-oxopropan-2- yl)oxy)azetidine-l-carboxylate

[0147] To a solution of tert-butyl 3-((l-methoxy-l-oxopropan-2-yl)oxy)azetidine-l- carboxylate (2.3 g, 8.8 mmol) in THF (20 mL) was added LDA (6.6 mL, 13.3 mmol) at -78°C. After 30min, CH₃I (1.5 g, 10.5 mmol) was added. The resulting mixture was stirred at r.t. for 2 hrs. The mixture was diluted with water and extracted with EtOAc (100 mL x2). The organic layer was washed with H₂0 (100 mL), dried over Na₂S0₄, filtered and concentrated. The reside was purified by flash chromatography (silica gel, 5 to -10 % ethyl acetate in petroleum ether) to give tert-butyl 3-((l-methoxy-2-methyl-l-oxopropan-2-yl)oxy)azetidine-l-carboxylate (1.8 g) as yellow oil. LC-MS (ESI) m/z (M+l): 274.

Step 3: Preparation of 2-((l-(tert-butox carbonyl)azetidin-3-yl)oxy)-2-methylpropanoic acid

Boc

[0148] To a solution of tert-butyl 3-((l-methoxy-2-methyl-l-oxopropan-2-yl)oxy)azetidine- l-carboxylate (1.8 g, 6.5 mmol) in THF/H₂0 (15 ml/5 ml) was added LiOH (2.7 g, 65 mmol). The mixture was stirred at r.t. overnight. The reaction mixture was concentrated and diluted with H₂0, the pH adjusted to~3 with cone. HC1, and extracted with EtOAc (30 mL x2). the ethyl acetate extracts were washed with water and brine, dried over Na₂S0₄ and concentrated to give 2-((l-(/er/-butoxycarbonyl)azeti din-3 -yl)oxy)-2-methylpropanoic acid (1.3 g) as a colorless oil. LC-MS (ESI) m/z (M+l): 260.

Step 4: Preparation of 2-(azetidin-3-yloxy)-2-methylpropanoic acid

[0149] To a solution of 2-((l -(tert-butoxycarbonyl)azeti din-3 -yl)oxy)-2-methylpropanoic acid (300 mg, 1.15 mmol) in DCM (3 mL) was added TFA (1 mL). The mixture was stirred at r.t. for 1 h. The reaction mixture was concentrated and the residue was used in next step without further purification. LC-MS (ESI) m/z (M+l): 160. Step 5: Preparation of 2-((l-((((J ?, A,5»V,6 ?)-5-methoxy-4-((2 ?,J ?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)oxy)-2-methylpropanoic acid

[0150] 2-(Azetidin-3-yloxy)-2-methylpropanoic acid (200 mg, 0.78 mmol) in CH₃CN (20 ml) was coupled with Intermediate 1 (418 mg, 0.93 mmol) by following the General Procedure for Coupling. The mixture was concentrated under vacuum to remove the solvent while keeping the temperature below 40°C. The reaction mixture was washed with ammonium acetate buffer (pH 4.0, 5 mL x 2) and 5% sodium bicarbonate solution (20 mL). The mixture was extracted with DCM. The DCM layer was dried over Na₂S0₄, concentrated to give crude product for prep. HPLC (C18, 0 to ~90 % acetonitrile in H₂0 with 0.1 % formic acid) to give 2-((l- ((((3A, 4S, 5S, 6R)-5-methoxy-4-((2R, 3f?)-2-methyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 - oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)oxy)-2-methylpropanoic acid (82mg) as a white solid. LC -MS (ESI) m/z (M+l): 468.

Step 6: Preparation of sodium 2-(( l-((((3/?, V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)oxy)-2-methylpropanoate

[0151] To a solution of 2-((l-((((3f?, S^,,5ri^,,6/?)-5-methoxy-4-((2f?,3f?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)oxy)-2- methylpropanoic acid (83 mg, 0.17 mmol) in MeCN (5 mL) was added dropwise 0.1 N NaOH solution (1.6 mL, 0.15 mmol) at -60°C. After lyophilization, 2-(( l -((((3R,-fS,5S,6R)-5- ethoxy- 4 -{{2R, 3i?)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)azeti din-3 -yl)oxy)-2-methylpropanoatesodium salt (75 mg) was obtained as a white solid. LC-MS (ESI) m/z (M+l): 468. 1H NMR (400 MHz, DMSO- d) d 5.29 (d, J= 2.3 Hz, 1H), 5.19 (t, J= 7.4 Hz, 1H), 4.52 - 4.45 (m, 1H), 4.05 - 3.95 (m, 2H), 3.66 (s, 2H), 3.51 (dd, j= 11.0, 2.6 Hz, 1H), 3.28 (s, 3H), 2.84 (d, j= 4.3 Hz, 1H), 2.55 (t, J= 5.3 Hz, 2H), 2.22 - 2.12 (m, 2H), 1.93 (td, 7= 13.4, 4.5 Hz, 1H), 1.75 (d, = 11.0 Hz, 2H), 1.70 (s, 3H), 1.61 (s, 3H), 1.15 (s, 6H), 1.07 (s, 3H), 1.00 (d, 7= 13.2 Hz, 1H), 0.94 (d, J= 6.6 Hz, 1H).

Example 11: Preparation of sodium 3-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)-2,2-dimethylpropanoate

Step 1: Preparation of (E)-tert-butyl 3-(3-ethoxy-3-oxoprop-l-en-l-yl)azetidine-l- carboxylate

[0152] To a solution of tert-butyl 3-formylazetidine-l-carboxylate (7 g, 0.037 mol) in anhydrous DCM (30 mL) was added ethyl 2-(triphenylphosphoranylidene)acetate (14.4 g, 0.042 mol) in portions at 0°C. The reaction mixture was stirred at room temperature overnight. The resulting mixture was concentrated under vacuum. The residue was purified by flash chromatography (silica gel, 1 to -20% ethyl acetate in petroleum ether) to give (E)-tert-butyl 3- (3 -ethoxy-3 -oxoprop-l-en-l-yl)azeti dine- l-carboxylate (7.0 g) as a pale-yellow oil.

Step 2: Preparation of tert-butyl 3-(3-ethoxy-3-oxopropyl)azetidine-l-carboxylate

[0153] A solution of (E)-tert-butyl 3-(3-ethoxy-3-oxoprop-l-en-l-yl)azetidine-l-carboxylate (5 g, 0.019 mol) in MeOH (80 mL) was degassed under N₂ atmosphere three times, then Pd/C (1.5 g, 10% wt) was added in one portion. The mixture was stirred under an H₂ balloon at room temperature overnight. The resulting mixture was filtered through a pad of celite and the filtrate was concentrated under vacuum. The residue was purified by flash chromatography (silica gel,

1 to -3% MeOH in DCM) to give tert-butyl 3-(3-ethoxy-3-oxopropyl)azetidine-l-carboxylate (4.5 g) as a colorless oil.

Step 3: Preparation of tert-butyl 3-(3-ethoxy-2,2-dimethyl-3-oxopropyl)azetidine-l- carboxylate

[0154] To a solution of tert-butyl 3 -(3 -ethoxy-3 -oxopropyl)azeti dine- l-carboxylate (1 g, 3.87 mmol ) in anhydrous THF (20 mL) was added a solution of LDA (11.6 mL, 11.6 mmol, 1M in THF) dropwise at -78°C over 30 min. After the addition, the mixture was stirred at -78°C for 1 hr. Then Mel (2.2 g, 15.5 mmol) was added dropwise. The resulting mixture was quenched by sat.NH₄Cl at 0 °C and extracted with ethyl acetate (20 mL x 2). The combined organic layers were dried over Na₂S0₄, filter and concentrated to give crude product. The crude product was purified by flash chromatography (silica gel, 1 to -20% ethyl acetate in petroleum ether) to give tert-butyl 3-(3-ethoxy-2,2-dimethyl-3-oxopropyl)azetidine-l-carboxylate (380 mg).

Step 4: Preparation of 3-(l-(tert-butoxycarbonyl)azetidin-3-yl)-2,2-dimethylpropanoic acid

[0155] To a mixture of tert-butyl 3-(3-ethoxy-2,2-dimethyl-3-oxopropyl)azetidine-l- carboxylate (380 mg, 1.32 mmol) in THF (4 mL) was added 4M aq.NaOH solution (3.3 mL, 13.2 mmol). The mixture was stirred at 50°C for 4 hrs. The volatiles were removed under reduced pressure, and the residue was diluted with water (7 mL) and washed with EA (10 mL x 2). The aqueous layer was adjusted pH to 3 with 1N hydrochloric acid and extracted with EA (10 mL x 2). The combined organic layers were washed with brine, dried over Na₂S0₄ and concentrated under reduced pressure to give 3-(l-(tert-butoxycarbonyl)azetidin-3-yl)-2,2- dimethylpropanoic acid (320 mg) as a pale-yellow oil.

Step 5: Preparation of 3-(azetidin-3-yl)-2,2-dimethylpropanoic acid

CF₃C0₂H

[0156] To a solution of 3-(l-(tert-butoxycarbonyl)azetidin-3-yl)-2,2-dimethylpropanoic acid (320 mg, 1.24 mmol) in DCM (2 mL) was added TFA (1 mL, l2.4mmol) dropwise at 0°C. The mixture was stirred at room temperature for lhr. The resulting mixture was concentrated under vacuum to give a residue, which was co-evaporated with toluene (5 mL x 2) to give 3-(azetidin- 3-yl)-2,2-dimethylpropanoic acid (295 mg, contained TFA) as brown oil, which was directly used in the next step without purification. LC-MS: m/z 158 (M+H)⁺

Step 6: Preparation of 3-(3-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)cyclobutyl)-2,2- dimethylpropanoic acid

[0157] 3-(Azetidin-3-yl)-2,2-dimethylpropanoic acid (295 mg, -1.24 mmol) was coupled to Intermediate 1 (500 mg, 1.12 mmol) by following the General Procedure for Coupling. The solvent was removed under vacuum below 40°C, then diluted with DCM (20 mL) and washed with ammonium acetate buffer (pH~4, 5 mL x 2) and 5% sodium bicarbonate solution (20 mL). The aqeous layers were extracted with DCM (5 mL x 2). The combined organic layers were dried over Na₂S0₄ and concentrated to give a yellow oil, which was purified by flash chromatography (silica gel, 1 to -3% MeOH in DCM) to give pure 3-(3-((((3R,4S,5S,6R)-5- methoxy-4-((2f?,3f?)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)cyclobutyl)-2,2-dimethylpropanoic acid (l60mg)as a light yellow syrup.

Step 7: Preparation of sodium 3-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-

2,2-dimethylpropanoate

[0158] To a solution of 3-(3-((((3f?, ,S^,,5S^,,6/?)-5-methoxy-4-((2f?,3f?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)cyclobutyl)-2,2- dimethylpropanoic acid (160 mg, 0.34 mmol) in CH₃CN (0.5 mL) was added O. lM NaOH (3 mL, 0.31 mmol) dropwise at 0~5°C. The mixture was freeze dried to give the 3-(3-

((((37?, 4S, 5S, 6/ )- 5 -m ethoxy-4 -((2/?, 37?)-2-methyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 - oxaspiro[2.5]octan-6-yl)oxy)carbonyl)cyclobutyl)-2,2-dimethylpropanoate as a white solid (170 mg). LC-MS (ESI) found: 465 [M+l]⁺.1H NMR (400 MHz, DMSO) d 5.28 (s, 1H), 5.19 (t, J= 7.4 Hz, 1H), 3.89 (t, J= 8.2 Hz, 2H), 3.51 (dd, J= 10.9, 2.5 Hz, 3H), 3.27 (s, 3H), 2.84 (d, J= 4.3 Hz, 1H), 2.69 - 2.61 (m, 1H), 2.56 (dd, J= 8.2, 4.2 Hz, 2H), 2.17 (dd, J= 12.5, 6.3

Hz, 2H), 1.90 (dd, J= 13.2, 4.5 Hz, 1H), 1.74 - 1.59 (m, 11H), 1.05 (d, 7= 19.9 Hz, 4H), 0.92 (d, J= 10.7 Hz, 6H).

Example 12: Preparation of sodium (3-(l-((((3/?, S,5»S,6/?)-5-methoxy-4-((2/?,3/?)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)propanoyl)(methylsulfonyl)amide

Step 1: Preparation of terf-butyl3-(3-(methylsulfonamido)-3-oxopropyl)azetidine-l- carboxylate Boc

DCM

[0159] To a solution of 3-(l-(/er/-butoxycarbonyl)azetidin-3-yl)propanoic acid (2.0 g, 8.7 mmol) in DCM (20 mL) was added methanesulfonamide (0.99 g, 10.4 mmol), TBTU (4.18 g, 13.05 mmol) and DIPEA (2.20 g, 17.4 mmol). The mixture reaction was stirred at r.t.

overnight. The reaction was washed with water 3 times and the dichloromethane solution was dried over Na₂S0₄, filtered and concentrated to give crude product, which was purified by flash chromatography (silica gel, 0 to ~3% MeOH in DCM) to give ter/-butyl3-(3- (methylsulfonamido)-3-oxopropyl)azetidine-l-carboxylate as a yellow oil (0.80 g). LC-MS (ESI) found: 251 [M+l-56]⁺.

Step 2: Preparation of 3-(azetidin-3-yl)-N-(methylsulfonyl)propenamide

[0160] To a solution of /c/V-butyl 3-(3-(methylsulfonamido)-3-oxopropyl)azetidine-l- carboxylate (0.8 g, 2.6 mmol) in DCM (8 mL) was added TFA (1.5 g, 13.1 mmol). The reaction was stirred at r.t. overnight. The reaction was concentrated to give 3-(azetidin-3-yl)-N- (methylsulfonyl)propanamide (1.4 g, TFA salt), which was directly used in the next step without any purification.

Step 3: Preparation of (3T?, V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-(3-methylbut-2- en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl 3-(3-(methylsulfonamido)-3- oxopropyl)azetidine-l-carboxylate

[0161] To a solution of 3-(azetidin-3-yl)-N-(methylsulfonyl)propanamide (1.4 g, TFA salt) in DCM (20 mL) was added DIPEA (3.46 g, 24 mmol) at 0°C. Then a solution of ( 3R,4S,5S,6R)- 5-methoxy-4-((2f?,3f?)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl lH-imidazole-l-carboxylate (0.97 g, 2.4 mmol) in DCM (5 mL) was added to the above reaction. The reaction mixture was stirred at r.t. overnight. The reaction mixture was extracted with ammonium acetate buffer (pH 4.0, 5 mL x 2) and stirred for 15 min. The dichloromethane solution was separated, dried over Na₂S0₄, filtered and concentrated to give crude product, which was purified by Prep. HPLC (Method B: 0.1% NH₄OH) to give ( 3R,4S,5S, 6R)-5 - methoxy-4-((2i?,3i?)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl 3-(3-(methylsulfonamido)-3-oxopropyl)azetidine-l-carboxylate as a yellow oil (220 mg). LC- MS (ESI) found: 515 [M+l]⁺.

Step 4: Preparation of Sodium (3-( l-((((3/?, V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)propanoyl)(methylsulfonyl)amide

[0162] To a solution of (3R,-(S,5S, 6R)-5-methoxy-4-((2R, J/i)-2-methyl-3-(3-methylbut-2-en- l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl3-(3-(methylsulfonamido)-3-oxopropyl)azetidine-l- carboxylate (220 mg, 0.43 mmol) in MeCN (5 mL) was added 0.1M NaOH (3.8 mL, 0.38 mmol) at -78°C. The mixture was lyophilized to give sodium ( 3-(l-((((3R,4S,5S, 6R)-5 - methoxy-4-((2i?,3i?)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)propanoyl)(methylsulfonyl)amide as white solid. (200 mg). LC- MS (ESI) found:5 l5 [M-23+2]⁺. 1H NMR (400 MHz, CDCl₃) d 5.52 (s, 1 H), 5.19 (d, J= 7.2 Hz, 1 H), 4.04 (dd, J= 17.1, 8.8 Hz, 2 H), 3.82 - 3.56 (m, 3 H), 3.46 (d, J= 8.8 Hz, 3 H), 3.09 (s, 3 H), 2.95 (d, j= 3.9 Hz, 1 H), 2.75 - 2.49 (m, 4 H), 2.28 (s, 2 H), 2.16 (dd, j= 8.9, 6.4 Hz, 2 H), 2.06 (dd, j= 14.7, 10.3 Hz, 2 H), 1.93 (s, 1 H), 1.91 (d, j= 13.2 Hz, 2 H), 1.85 (d, j= 5.7 Hz, 1 H), 1.74 (s, 3 H), 1.66 (s, 3 H), 1.25 (s, 1 H), 1.21 (s, 3 H), 1.05 (d, j= 12.2 Hz, 1 H). Example 13: Preparation of sodium 5-((l-((((J ?, A,5A,6 ?)-5-methoxy-4-((2 ?,J ?)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)methyl)isoxazol-3-olate

1 ,1 '-carbonyl- diimidazole,

Step 1: Preparation of tert-butyl 3-(4-ethoxy-2,4-dioxobutyl)azetidine-l-carboxylate

1 ,1'-carbonyl- diimidazole,

[0163] To a stirred solution of 2-(l -(tert-butoxycarbonyl)azeti din-3 -yl)acetic acid (2.0 g, 9.3 mmol) in THF (20 mL) was added 1,1’ -carbonyl diimidazole (2.2 g, 13.9 mmol). The mixture was stirred at r.t. for lh, then magnesium chloride (883 mg, 9.3 mmol) and ethyl potassium malonate (2.44 g, 9.3mmol) were added successively. The mixture was then heated to 50°C for 15 hr. The mixture was filtered and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, 5 % MeOH in DCM) to afford tert-butyl 3-(4- ethoxy-2,4-dioxobutyl)azetidine-l-carboxylate (1.5 g) as a yellow oil. LC-MS (ESI) m/z (M+l): 286.

Step 2: Preparation of tert-butyl 3-((3-hydroxyisoxazol-5-yl)methyl)azetidine-l- carboxylate

[0164] To a solution of tert-butyl 3-(4-ethoxy-2,4-dioxobutyl)azetidine-l-carboxylate (500 mg, 1.75 mmol) in H₂0 (10 ml) was added NH₂OH/HCl (945 mg, l3.5mmol) at 0°C. After 30 min, MeOH/THF (5 mL/5 mL) was added. The resulting mixture was stirred at 0°C for 20 min and r.t. for 2 hr. The reaction mixture was concentrated and diluted withH₂0, and the pH was adjusted to ~4 with 2NHC1. The acidic water mixture was extracted with EtOAc (30 mL x2), dried over Na₂S0₄ and concentrated. The residue was purified by flash chromatography (silica gel, 5 % MeOH in DCM) to give tert-butyl 3-((3-hydroxyisoxazol-5-yl)methyl)azetidine-l- carboxylate (300 mg) as a colorless oil. LC-MS (ESI) m/z (M+l): 255.

Step 3: Preparation of 5-(azetidin-3-ylmethyl)isoxazol-3-ol

Boc

[0165] To a solution of tert-butyl 3-((3-hydroxyisoxazol-5-yl)methyl)azetidine-l-carboxylate (300 mg, 1.18 mmol) in DCM(3 mL) was added TFA (1 mL). The mixture was stirred at r.t. for

1 h and concentrated under vacuum. The crude product was used in next step without further purification.

Step 4: Preparation of (3T?, V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-(3-methylbut-2- en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl 3-((3-hydroxyisoxazol-5- yl)methyl)azetidine-l-carboxylate

[0166] 5-(Azetidin-3-ylmethyl)isoxazol-3-ol (260 mg, 1.03 mmol) was coupled with

Intermediate 1 (505 mg, 1.13 mmol) by following the General Procedure for Coupling. The reaction mixture was concentrated under vacuum to remove the solvent while keeping the temperature below 40°C. The residue was purified by column chromatography (silica gel, 1 to~l0 % MeOH in DCM) and prep. HPLC (C18, 0 ~90 % acetonitrile in H₂0 with 0.1 % formic acid) to give (3i?,4ri',5ri',6/?)-5-methoxy-4-((2i?,3i?)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl 3-((3-hydroxyisoxazol-5-yl)methyl)azetidine-l- carboxylate (160 mg) as a white solid.LC-MS (ESI) m/z (M+l): 463.

Step 5: Preparation of sodium 5-((l-((((3/?, ,V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)methyl)isoxazol-3-olate

[0167] To a solution of (3R,-(S,5S, 6R)-5-methoxy-4-((2R, J/i)-2-methyl-3-(3-methylbut-2-en- l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl 3-((3-hydroxyisoxazol-5-yl)methyl)azetidine-l- carboxylate (160 mg, 0.34 mmol) in MeCN (5 mL) was added dropwise 0.1 N NaOH solution (3.1 mL, 0.31 mmol) at -60°C with stirring. Lyophilization provided 5 -( ( 1 - (( (( 3/( -fS, 5S, 6R)-5- methoxy-4-((2i?,3i?)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)methyl)isoxazol-3-olate sodium salt (139 mg) as a white solid. LC-MS (ESI) m/z (M+l): 463. 1H NMR (400 MHz, DMSO-76) d 5.28 (s, 1H), 5.19 (t, J= 7.4 Hz, 1H), 3.91 (t, J= 8.3 Hz, 2H), 3.66 (s, 1H), 3.59 - 3.46 (m, 3H), 3.28 (s, 3H), 2.84 (d, J = 4.3 Hz, 1H), 2.77 - 2.66 (m, 1H), 2.56 (dd, 7= 11.5, 5.3 Hz, 2H), 2.42 (d, J= 7.3 Hz, 2H), 2.24

- 2.07 (m, 2H), 1.92 (tt, 7= 19.9, 9.9 Hz, 1H), 1.76 (t, J= 8.8 Hz, 2H), 1.70 (s, 4H), 1.61 (s, 3H), 1.07 (s, 3H), 1.00 (d, J= 13.5 Hz, 1H).

Example 14: Preparation of sodium 5-(2-(l-((((3/?, £,5»£,<i/?)-5-inethoxy-4-((2/?,3/?)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)ethyl)isoxazol-3-olate

Step 1: Preparation of 3-(l-(terf-butoxycarbonyl)azetidin-3-yl)propanoic acid

[0168] To a solution of /cvV-butyl 3 -(3 -ethoxy-3 -oxopropyl)azeti dine- l-carboxylate (2.0 g, 7.78 mmol) in MeOH (30 mL) was added aqueous LiOH (10 mL, 23.35 mmol,l M) at 0°C. The reaction was stirred at 25°C for 6 hrs. The mixture was diluted with THF and extracted with diethyl ether (20 mL x 2). The aqueous layer was adjusted to ~pH3-4 withHCl (1M) and extracted with dichloromethane (30 mL x 3). The dichloromethane layers were dried over anhydrous Na₂S0₄ and concentrated under reduced pressure to give 3-(l -{tert- butoxycarbonyl)azetidin-3-yl)propanoic acid (1.78 g) as a white solid. LC-MS (ESI) found:

230 [M+H] ⁺

Step 2: Preparation of tert- butyl 3-(5-ethoxy-3,5-dioxopentyl)azetidine-l-carboxylate

[0169] To a suspension of MgCl₂ (998 mg, 10.48 mmol) and TEA (1.88 mL, 13.54 mmol) in MeCN (90 mL) was added potassium 3 -ethoxy-3 -oxopropanoate (1.56 g, 9.17 mmol) at 25°C. The reaction was stirred at 25°C for 2hrs, to prepare mixture 1. 3-( 1 -(tert- butoxycarbonyl)azetidin-3-yl)propanoic acid (1.0 g, 4.36 mmol) was added to DMF (30 mL), followed by adding CDI (778 mg, 4.80 mmol), the resulting mixture was stirred at 25°C for 2 hrs, to prepare mixture 2. The mixture 1 was added into the mixture 2, and then the resulting reaction mixture was stirred overnight at room temperature, the reaction mixture was extratcted with EA and the EA layer was washed with water several times. The organic layers were dried over anhydrous Na₂S0₄ and concentrated under reduced pressure to give /er/-butyl 3-(5- ethoxy-3,5-dioxopentyl)azetidine-l-carboxylate (1.05 g) as a colorless oil. LC-MS (ESI) found: 300 [M+H] ⁺

Step 3: Preparation of tert-butyl 3-(2-(3-hydroxyisoxazol-5-yl)ethyl)azetidine-l- carboxylate

[0170] To a cold slurry of /er/-butyl 3-(5-ethoxy-3,5-dioxopentyl)azetidine-l-carboxylate (220 mg, 0.76 mmol) in water (3 mL) was added hydroxylamine hydrochloride (330 mg, 4.78 mmol) at 0°C, followed by NaOH (380 mg, 9.5 mmol). After 5min, THF (1.5 mL) and MeOH (1.5 mL) were added, the resulting cloudy light yellow solution was stirred at 0°C for 20 min, then warmed up to room temperature for 6hr. The reaction was acidified with cone. HC1, diluted by water and extracted with diethyl ether (10 mL x 4). The combined organic layers were dried over anhydrous Na₂S0₄, concentrated under reduced pressure and purified by flash chromatography to give /cvV-butyl 3-(2-(3-hydroxyisoxazol-5-yl)ethyl)azetidine-l-carboxylate (l80mg) as a light yellow oil. LC-MS (ESI) found: 269 [M+H] ⁺

Step 4: Preparation of 5-(2-(azetidin-3-yl)ethyl)isoxazol-3-ol

[0171] To a mixture of /er/-butyl 3-(2-(3-hydroxyisoxazol-5-yl)ethyl)azetidine-l-carboxylate (180 mg, 0.67 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid (2.5 mL) dropwise at 0°C. The reaction was stirred at room temperaturefor 1 hr. The mixture was concentrated under reduced pressure to give 5-(2-(azetidin-3-yl)ethyl)isoxazol-3-ol trifluoroacetic acid salt (200 mg) as yellow resin, which was directly used in the next step without purification. LC-MS (ESI) found: 169 [M+l] ⁺.

Step 5: Preparation of sodium 5-(2-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-

3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)ethyl)isoxazol-3-olate

[0172] Under N₂ atmosphere, a mixture of 5-(2-(azetidin-3-yl)ethyl)isoxazol-3- oltrifluoroacetic acid salt (200 mg, 0.839 mmol) in acetonitrile (5 mL) was added DIPEA (0.3 mL, 1.69 mmol) dropwise at 0°C. The mixture was stirred at 0°C for 10 min, then

Intermediate 1 (140 mg, 0.335 mmol) and 4-dimethylaminopyridine (5 mg) were added. After addition, the reaction was stirred at room temperature overnight under N₂ atmosphere. The mixture was then concentrated under vacuum to remove the solvent while keeping the temperature below 40°C. The residue was diluted with dichloromethane and washed twice with ammonium acetate buffer (pH 4.0, 5 mL x 2). The dichloromethane solution was dried over Na₂S0₄, filtered and concentrated. The crude product was purified by preparative HPLC (Method A, H₂0 (0.1% FA)/ CH₃CN) to give (3/(-/A5k, 6//)-5-rnethoxy-4-((2/(3//)-2-rnethyl- 3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl3-(2-(3-hydroxyisoxazol-5- yl)ethyl)azetidine-l-carboxylate (130 mg) as a colorless oil. LC-MS (ESI) found:477 [M+l] ⁺.

[0173] To a solution of (3R, S,5S, 6R)-5-methoxy-4-((2R, 3//)-2-methyl-3-(3-methylbut-2-en- l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl 3-(2-(3-hydroxyisoxazol-5-yl)ethyl)azetidine-l- carboxylate (130 mg, 0.273 mmol) in acetonitrile/H₂0 (0.5 mL:2 mL) was added NaOH (2 mL, 0.1 N in water). Lyophilization give 5-(2-( l -((((3//,-/ri',5A6//)-5-methoxy-4-((2//,3//)-2-methyl- 3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)ethyl)isoxazol-3-olate sodium salt (130 mg) as a colorless foam. LC-MS (ESI) found:477 [M+l] ⁺. 1H MR (400 MHz, DMSO-i/d) d 5.28 (s, 1H), 5.19 (t, J= 7.6 Hz, 1H), 3.99 - 3.82 (m, 2H), 3.67 (s, 1H), 3.51 (dd, j= 10.9, 2.6 Hz, 3H), 3.28 (s, 3H), 2.84 (d, j= 4.4 Hz, 1H), 2.61 - 2.51 (m, 4H), 2.17 (h, J= 7.7 Hz, 2H), 2.08 (t, J= 7.5 Hz, 2H), 1.99 - 1.88 (m, 1H),

1.77 - l .72(m, 2H),l .72- 1.68 (m, 5H), 1.61 (s, 3H), 1.07 (s, 3H), 1.01 (d, J= 13.7 Hz, 1H). Example 15: Preparation of sodium 3-((l-((((J ?, »V,5»V,6 ?)-5-methoxy-4-((2 ?,J ?)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)oxy)propanoate

Boc H

Step 1: Preparation of tert- butyl 3-(3-(/er/-butoxy)-3-oxopropoxy)azetidine- l-carboxylate

[0174] /er/-Butyl 3-hydroxyazetidine-l-carboxylate (500 mg 2.89 mmol) was combined with acrylic acid tert. -butyl ester (1.80 g, 14.45 mmol) and tetra-n- butylammoniumhydrogensulphate (197 mg, 0.578 mmol) in dichloromethane (10 mL) at 0 °C. Aqueous 50% sodium hydroxide (2.67 mL, 3.44 mmol) was added and the mixture was stirred vigorously at 0° C for 1 h. The reaction was warmed up to RT, and the stirring was continued overnight at RT. The reaction was diluted with dichloromethane and water, acidifed with 10% citric acid solution and the phases were separated. The aqueous phase was extracted once again with dichloromethane. The organic layers were combined, washed with aqueous sodium chloride, dried over magnesium sulphate and concentrated by evaporation under vacuum. Flash chromatography gave /c/T-butyl 3-(3-(/c/V-butoxy)-3-oxopropoxy)azetidine- l -carboxyl ate (600 mg) as a colorless oil. LC-MS (ESI) found: 302 [M+H] ⁺ Step 2: Preparation of 3-(azetidin-3-yloxy)propanoic acid

[0175] To a mixture of /er/-butyl 3-(3-(tert-butoxy)-3-oxopropoxy)azetidine-l-carboxylate (840 mg, 2.79 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid (2.5 mL) dropwise at 0°C. The reaction was stirred at room temperature for 16 hr. The mixture was concentrated under reduced pressure to give 3-(azetidin-3-yloxy)propanoic acid (1.0 g) as yellow syrup, which was directly used in the next step without purification. LC-MS (ESI) found: 146 [M+l]⁺.

Step 3: Preparation of 3-((l-((((.?/?, S,5A,6/?)-5-methoxy-4-((2/?,.?/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)oxy)propanoic acid

[0176] DIPEA (0.35mL, 2.0 mmol) was added dropwise to a solution of 3-(azetidin-3- yloxy)propanoic acid (290 mg, 2.0 mmol) in acetonitrile (5 mL) at 0°C under N_2. The mixture was stirred at 0°C for 10 min and Intermediate 1 (150 mg, 0.33 mmol) and DMAP (5 mg) were added. After addition, the reaction was stirred at room temperature overnight under N₂ atmosphere. The mixture was then concentrated under vacuum to remove the solvent while keeping the temperature below 40°C. The residue was acidified by ammonium acetate buffer and 5% sodium bicarbonate solution (20 mL), then extratcted with dichloromethane. The dichloromethane solution was dried over Na₂S0₄, filtered and concentrated. The crude product was purified by preparative HPLC (Method A, H₂0 (0.1% FA)/ CH₃CN) to give 3-((l- ((((3R, 4S, 5S, 6// )- 5 -m ethoxy-4 -((2//, 3i?)-2-methyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 - oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)oxy)propanoic acid (140 mg) as a colorless oil. LC-MS (ESI) found:454 [M+l] ⁺. Step 4: Preparation of sodium 3-(( l-((((3/?, V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)oxy)propanoate

[0177] To a solution of S-^l-^^df^^S'.J^d/^-S-methoxy-d-^i.R.df^^Z-inethyl-S-p- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)oxy)propanoic acid (140 mg, 0.3 lmmol) in acetonitrile/H₂0 (0.5 mL/2 mL) was added NaOH (3 mL, 0.1 N in water). Lyophilization gave 3 -(( 1 -((((3/( -(S, 5S, 6/i)-5-methoxy-4- ((2i?,3i?)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)oxy)propanoate sodium salt (142 mg) as a colorless foam. LC- MS (ESI) found: 454 [M+l]⁺. 1H MR (400 MHz, DMSO- d) d 5.34 - 5.26 (m, 1H), 5.19 (ddt, J= 7.4, 5.9, 1.5 Hz, 1H), 4.23 (tt, J= 6.4, 4.2 Hz, 1H), 4.09(m, 2H), 3.77- 3.61 (m, 2H), 3.54 - 3.44 (m, 3H), 3.28 (s, 3H), 2.85 (d, J= 4.4 Hz, 1H), 2.59 - 2.53 (m, 2H), 2.16 (p, J = 7.6, 7.1 Hz, 2H), 2.08 (t, J= 7.0 Hz, 2H), 1.93 (dt, J= 13.3, 6.7 Hz, 1H), 1.81 - 1.64 (m, 6H), 1.61 (s, 3H), 1.07 (s, 3H), 1.01 (dt, j= 13.6, 3.7 Hz, 1H).

Example 16: Preparation of sodium 2-(((S)-l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)pyrrolidin-3-yl)oxy)acetate

Step 1: Preparation of (S)-tert-butyl 3-(2-(tert-butoxy)-2-oxoethoxy)pyrrolidine-l- carboxylate

[0178] A solution of (S)-tert-butyl 3-hydroxypyrrolidine-l-carboxylate (500 mg, 2.70 mmol) in anhydrous THF (5.0 mL) was added dropwise to a slurry of sodium hydride (60% dispersion in oil, 128 mg, 3.21 mmol) in THF (10.0 mL) at 0°C under a nitrogen atmosphere, and the mixture stirred for 15 mins at 0 °C. A solution of tert-butyl 2-bromoacetate (576 mg, 3.0mmol) in THF (5.0 mL) was added and the mixture warmed to room temperature and stirred for 18 hours. Water (20.0 mL) was added, and the mixture was extracted with ethyl acetate (50.0 mL). The organic extract was washed with brine, dried over anhydrous Na₂S0₄, and evaporated in vacuo. The residue was purified by flash chromatography (silica gel, 1 to ~50 % ethyl acetate in petroleum ether) to give (S)-tert-butyl 3-(2-(tert-butoxy)-2-oxoethoxy)pyrrolidine-l- carboxylate (620 mg) as a white solid. LC-MS (ESI) found: 302 [M+l]⁺.

Step 2: Preparation of (S)-2-(pyrrolidin-3-yloxy)acetic acid trifluoroacetic acid salt

[0179] To a mixture of (S)-tert-butyl 3-(2-(tert-butoxy)-2-oxoethoxy)pyrrolidine-l- carboxylate (620 mg, 2.1 mmol) in dichloromethane (10 mL) was added trifluoroacetic acid (4 mL) dropwise at 0°C. The reaction was stirred at room temperature for 1 hr. The mixture was concentrated under reduced pressure to give (S)-2-(pyrrolidin-3-yloxy)acetic acid

trifluoroacetic acid salt (480 mg) as yellow syrup, which was directly used in the next reaction without purification. LC-MS (ESI) found: 146[M+1]⁺.

Step 3: Preparation of 2-(((S)-l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)pyrrolidin-3- yl)oxy)acetic acid

[0180] (S)-2-(Pyrrolidin-3-yloxy)acetic acid trifluoroacetic acid salt (480 mg, 2.0 mmol) was coupled with Intermediate 1 (550 mg, 1.2 mmol) by following the General procedure for Coupling. The mixture was then concentrated under vacuum to remove the solvent while keeping the temperature below 40°C. The residue was diluted with dichloromethane and washed with ammonium acetate buffer (pH 4.0, 20 mL x 2) followed by 5% sodium

bicarbonate solution (20 mL). The dichloromethane solution was dried over Na₂S0₄, filtered and concentrated to give crude product. The crude product was purified by silica gel chromatography (eluting with 1 to~2 % MeOH in DCM) and preparative HPLC (Method A, ¾0 (0.1% FA)/ CHsCN) to give 2-(((S)-l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl- 3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)pyrrolidin-3- yl)oxy)acetic acid (375 mg) as a white solid. LC-MS (ESI) found: 454 [M+l]⁺.

Step 4: Preparation of sodium 2-(((S)-l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl- 3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)pyrrolidin- 3-yl)oxy)acetate

[0181] To a solution of 2-(((S)-l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)pyrrolidin-3- yl)oxy)acetic acid (135 mg, 0.3 mmol) in acetonitrile (2 mL) was added aq.NaOH (2.7 mL,

0.27 mmol) dropwise at -20°C. The mixture was freeze drying to give sodium 2-(((S)-l- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)pyrrolidin-3-yl)oxy)acetate (150 mg, 0.29 mmol) as a white solid. LC-MS (ESI) found: 454 [M+l]⁺. 1H NMR (400 MHz, DMSO- d) d 5.36 (d, J = 5.5 Hz, 1H), 5.19 (s, 1H), 4.20 (d, J= 22.1 Hz, 1H), 3.62 - 3.41 (m, 3H), 3.29 (d, J= 5.0 Hz, 8H), 2.91 - 2.80 (m, 1H), 2.72 - 2.53 (m, 2H), 2.30 - 2.08 (m, 2H), 1.98 (dd, = 15.9, 11.1 Hz, 2H), 1.81 (dd, 11.1, 2.9 Hz, 3H), 1.71 (s, 3H), 1.61 (s, 3H), 1.08 (d, J= 3.5 Hz, 3H), 1.01 (d, 7= 13.5 Hz, 1H).

Example 17: Preparation of (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl3-(2- (((isopropoxycarbonyl)oxy)methoxy)-2-oxoethoxy) azetidine-l-carboxylate

[0182] To a solution of 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)oxy)acetic acid (235 mg, 0.54 mmol) in dry DMF (20 mL) was added K₂C0₃ (221 mg, 1.60 mmol) at r.t. The mixture was cooled to 0°C, and chloromethyl isopropyl carbonate (160 mg, 1.07 mmol) and Nal (79.70 mg, 0.54 mmol) were added to the reaction solution at 0°C under N₂ atmosphere. After the completion of addition, the reaction mixture stirred at r.t. overnight. The mixture was diluted with EA and washed with water. The organic layer was then washed with brine, dried over Na₂S0₄, and concentrated. The residue was purified by prep-HPLC (Method A, H₂0 (0.1% FA)/ C¾CN) to give (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl- 3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6-yl3 - (2(isopropoxycarbonyl)oxy)methoxy)-2-oxoethoxy)azetidine-l-carboxylate (68.6 mg) as a yellow solid. LC-MS (ESI) found: 556 [M+l]⁺. 1H NMR (400 MHz, CDCl₃) d 5.80 (s, 2H), 5.51 (s, 1H), 5.21 (t, 7= 7.5 Hz, 1H), 4.92 (dt, 7= 9.9, 6.3 Hz, 1H), 4.37 (s, 1H), 4.16 - 4.09 (m, 3H), 4.01 - 3.90 (m, 2H), 3.61 (dd, 7= 11.1, 2.7 Hz, 1H), 3.45 (d, 7= 2.4 Hz, 3H), 2.99 (t, 7= 8.5 Hz, 1H), 2.65 - 2.52 (m, 2H), 2.39 - 1.83 (m, 7H), 1.74 (s, 3H), 1.64 (d, 7= 8.6 Hz, 3H), 1.33 (s, 3H), 1.32 (s, 3H), 1.19 (d, 7= 6.3 Hz, 3H), 1.07 (d, 7= 12.7 Hz, 1H)

Example 18: Preparation of (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl3-(2-oxo-2-

((pivaloyloxy)methoxy)ethoxy)azetidine-l-carboxylate

[0183] To a solution of 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)oxy)acetic acid (235 mg, 0.54 mmol) in dry DMF (20 mL) was added K₂C0₃ (221 mg, 1.60 mmol) at r.t. The mixture was cooled to 0°C, chloromethylpivalate (163 mg, l .08mmol) and Nal (79.70 mg, 0.54 mmol) was added to the reaction solution at 0°C under N₂ atmosphere. After the completion of addition, the reaction mixture was stirred at r.t. overnight. The mixture was diluted with EA and washed with water. The combined organic layers were washed with brine and dried over Na₂S0₄, concentrated. The residue was purified by prep-HPLC (Method A, H₂0 (0.1% FA)/ CH₃CN) to give (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl3-(2-oxo-2- ((pivaloyloxy)methoxy)ethoxy)azetidine-l-carboxylate (98.3 mg) as a yellow solid. LC-MS (ESI) found: 554 [M+l]⁺. 1H NMR (400 MHz, CDCl₃) d 5.80 (s, 2H), 5.51 (s, 1H), 5.20 (t, 7 = 7.5 Hz, 1H), 4.36 (s, 1H), 4.11 (dd, 7= 11.8, 8.5 Hz, 3H), 4.02 - 3.90 (m, 2H), 3.61 (dd, 7 = 11.1, 2.7 Hz, 1H), 3.44 (d, 7= 2.5 Hz, 3H), 2.99 (t, 7= 8.8 Hz, 1H), 2.65 - 2.49 (m, 2H), 2.44 - 2.30 (m, 1H), 2.21 - 1.77 (m, 6H), 1.74 (s, 3H), 1.65 (s, 3H), 1.22 (s, 9H), 1.19 (d, 7= 6.3 Hz, 3H), 1.07 (d, 7= 10.3 Hz, 1H). Example 19: Preparation of sodium 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)amino)acetate

OF ₃C0₂

ate 1

Step 1: Preparation of tert-butyl 3-((2-(tert-butoxy)-2-oxoethyl)amino)azetidine-l- carboxylate

[0184] To a solution of tert-butyl 3-aminoazetidine-l-carboxylate (500 mg, 2.90mmol) in THF (20 ml) was added K₂C0₃ (802 mg, 5.8mmol) and tert-butyl 2-bromoacetate (678 mg, 3.48mmol). The resulting mixture was stirred at r.t. overnight. The mixture was diluted with water and extracted with EtOAc (100 mL x 2). The organic layers were washed with H₂0 (100 mL), dried over Na₂S0₄, filtered and concentrated. The residue was purified by flash chromatography (silica gel, 0 to ~2 % EtOAc in PE) to give tert-butyl 3-((2-(tert-butoxy)-2- oxoethyl)amino)azetidine-l-carboxylate (650 mg) as a colorless oil. LC-MS (ESI) m/z (M+l): 287.

Step 2: Preparation of 2-(azetidin-3-ylamino)acetic acid

Boc

N. TFA

DCM

HN

[0185] To a solution of tert-butyl 3-((2-(/er/-butoxy)-2-oxoethyl)amino)azetidine-l- carboxylate (650 mg, 2.27 mmol) in DCM (1 mL) was added TFA (3 mL). The mixture was stirred at r.t. overnight. The reaction mixture was concentrated, and the crude product was used in the next step without further purification. LC-MS (ESI) m/z (M+l): 131.

Step 3: Preparation of 2-((l-((((J ?, A,5A,6 ?)-5-methoxy-4-((2 ?,J ?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)amino)acetic acid

[0186] A solution of 2-(azetidin-3-ylamino)acetic acid (310 mg, l.36mmol) was coupled with Intermiate 1 (731 mg, 1.63 mmol) by following the General Procedure for Coupling. The mixture was concentrated under vacuum to remove the solvent while keeping the temperature below 40°C. The residue was purified by flash chromatography (silica gel, 1 % ~ 5 % MeOH in DCM) and prep. HPLC (Cl 8, 0 to ~90 % acetonitrile in H₂0 with 0.1 % ammonium hydroxide) to give, upon lyophilization, 2-(( l -((((3R,-(S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azeti din-3 -yl)amino)acetic acid (160 mg) as a white solid. LC-MS (ESI) m/z (M+l): 439.

Step 4: Preparation of sodium 2-(( l-((((3/?, V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)amino)acetate

[0187] To a solution of 2-(( l -{{{{3R,4S,5S,6R)- -mei\\oxy- -{{2R, 3//)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)amino)acetic acid (100 mg, 0.22 mmol) in MeCN (5 mL) was added dropwise 0.1 N NaOH solution (2 mL, 0.2 mmol) at -60 °C with stirring. After lyophilization, 2-(( l -((((3R,4S,5S,6R)- 5-methoxy-4-((2f?,3f?)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azeti din-3 -yl)amino)acetate sodium salt (84mg) was obtained as a white solid. LC-MS (ESI) m/z (M+l): 439. 1H NMR (400 MHz, DMSO) d 5.28 (s, 1H), 5.19 (t, J= 7.4 Hz, 1H), 3.91 (t, J= 7.5 Hz, 2H), 3.61 - 3.48 (m, 4H), 3.28 (s, 3H), 2.84 (d, J= 4.3 Hz, 1H), 2.74 (s, 2H), 2.59 - 2.53 (m, 2H), 2.17 (dd, j= 14.0, 7.1 Hz, 2H), 1.93 (t, 7= 11.5 Hz, 1H), 1.76 (s, 2H), 1.70 (s, 3H), 1.65 (s, 1H), 1.61 (s, 3H), 1.07 (s, 3H), 1.01 (d, 7= 13.5 Hz, 1H). Example 20: Preparation of sodium 2-(((/?)-l-(((( /?, S,5A,6/?)-5-methoxy-4-((.2/?, /?)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)pyrrolidin-3-yl)oxy)acetate

Step 1: Preparation of ( ?)-tert-butyl 3-(2-(tert-butoxy)-2-oxoethoxy)pyrrolidine-l- carboxylate

Boc

Boc

i

,N,

OH

[0188] To a solution of (R)-tert-butyl 3-hydroxypyrrolidine-l-carboxylate (500 mg, 2.67 mmol) in THF (20 mL) was added NaH (160 mg, 4.0 mmol, 60% in mineral oil) while cooling with an ice-bath. Thirty mins later, tert-butyl 2-bromoacetate (624 mg, 3.20 mmol) was added. The resulting mixture was stirred at r.t. overnight. TLC showed the reaction was complete. The mixture was diluted with water and extracted with EtOAc (100 ml x 2). The organic layer was washed with H₂0 (100 mL), dried over anhydrous Na₂S0₄, filtered and concentrated. The residue was purified by silica gel column chromatography (silica gel, 10 % EtOAc in PE) to give (A)-tert-butyl 3-(2-(tert-butoxy)-2-oxoethoxy)pyrrolidine-l-carboxylate (450 mg) as a colorless oil. LC-MS (ESI) m/z (M+l): 302.

Step 2: Preparation of (R)-2-(pyrrolidin-3-yloxy)acetic acid

[0189] To a solution of (R)-tert- butyl 3-(2-(/er/-butoxy)-2-oxoethoxy)pyrrolidine-l- carboxylate (450 mg, 1.49 mmol) in DCM (1 mL) was added TFA (3 mL). The mixture was stirred at r.t. overnight. TLC showed the reaction was complete. The reaction mixture was concentrated, and the residue was used in the next step without further purification. LC-MS (ESI) m/z (M+l): 146.

Step 3: Preparation of 2-((( ?)-l-((((J ?, A,5A,6 ?)-5-methoxy-4-((2 ?,J ?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)pyrrolidin-3- yl)oxy)acetic acid

[0190] To a solution of (f?)-2-(pyrrolidin-3-yloxy)acetic acid (280 mg, 1.15 mmol) in CH₃CN (20 mL) was added DIPEA (0.8 mL, 4.6 mmol) drop-wise at 0 °C. The mixture was stirred at 0 °C for 10 min, and Intermediate 1 (620 mg, 1.38 mmol) was added. The reaction mixture was stirred at r.t. overnight under N₂ atmosphere. TLC showed the reaction was complete. The mixture was concentrated under vacuum to remove the solvent while keeping the temperature below 40 °C. The residue was purified by silica gel column chromatography (silica gel, 1 % ~ 5 % MeOH in DCM) and prep-HPLC (Cl 8, 0 ~ 90 % acetonitrile in H₂0 with 0.1 % ammonium hydroxide) to give 2-(((R)- l -((((3//,-/N5A6//)-5-methoxy-4-((2//,3//)-2-methyl-3-(3-methylbut-

2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)pyrrolidin-3 -yl)oxy)acetic acid (240 mg) as a white solid. LC-MS (ESI) m/z (M+l): 454.

Step 4: Preparation of sodium 2-(((R)-l-((((3/?, S,5A,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl- 3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)pyrrolidin-

3-yl)oxy)acetate

[0191] To a solution of 2-(((R)-l-((((3i?, S^,,5,S^,, d ?)-5-methoxy-4-((2i?,3i?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)pyrrolidin-3- yl)oxy)acetic acid (130 mg, 0.28 mmol) in MeCN (5 mL) was added dropwise a 0.1 N NaOH solution (2.5 mL, 0.25 mmol) at -60 °C with stirring. After lyophilization, sodium 5-((l- ((((3R, 4S, 5S, 6// )- 5 -m ethoxy-4 -((2//, 3f?)-2-methyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 - oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)methyl)isoxazol-3-olate (104 mg) was obtained as a white solid. LC-MS (ESI) m/z (M+l): 454. ¹H NMR (400 MHz, DMSO-d6 ) d 5.35 (s, 1H), 5.19 (t, J= 7.5 Hz, 1H), 4.20 (s, 1H), 3.53 (s, 3H), 3.32 - 3.25 (m, 7H), 2.89 - 2.81 (m, 1H), 2.63 - 2.52 (m, 2H), 2.17 (d, J= 5.3 Hz, 2H), 2.02 - 1.89 (m, 2H), 1.87 - 1.75 (m, 3H), 1.69 (d, j= 13.3 Hz, 4H), 1.61 (s, 3H), 1.08 (d, j= 2.2 Hz, 3H), 1.01 (d, j= 13.8 Hz, 1H). Example 21: Preparation of sodium 2-(3-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)azetidin-l-yl)acetate

Step 1: Preparation of ethyl 2-(3-((tert-butoxycarbonyl)amino)azetidin-l-yl)acetate

[0192] To a solution of tert-butyl azeti din-3 -ylcarbamate (344 mg, 2.0mmol) in

dichloromethane (10 mL) was added tert-butyl 2-oxoacetate (1.04 g, 4.0 mmol, 50% in toluene) dropwise at 0°C. The mixture was stirred at 0°C for 10 min, and sodium triacetoxyborohydride (848 mg, 4.0 mmol) was added at 0°C. The reaction was stirred at room temperature overnight under N₂ atmosphere. The reaction mixture was then washed with water. The dichloromethane solution was dried over Na₂S0₄, filtered and concentrated to give crude product. The crude product was purified by chromatography (silica gel, 1 to ~50 % ethyl acetate in petroleum ether) to give ethyl 2-(3-((tert-butoxycarbonyl)amino)azetidin-l-yl)acetate (410 mg) as a white solid. LC-MS (ESI) found: 259 [M+l]⁺.

Step 2: Preparation of 2-(3-((tert-butoxycarbonyl)amino)azetidin-l-yl)acetic acid

[0193] A mixture of ethyl 2-(3-((tert-butoxycarbonyl)amino)azetidin-l-yl)acetate (400 mg, l.55mmol), lithium hydroxide (74 mg, 3.lmmol) in THF (5mL) and water (2 mL) was stirred at room temperature overnight. The mixture was concentrated, and the aq. layer was washed with ethyl acetate (50 mL X 2). The aq. layer was then acidified to ~pH4 with 1M HC1 and extracted with DCM. The DCM layer was dried over Na₂S0₄, filtered and concentrated under reduced pressure to give 2-(3-((tert-butoxycarbonyl)amino)azetidin-l-yl)acetic acid (310 mg) as a colorless oil, which was directly used in the next reaction without purification. LC-MS (ESI) found: 231 [M+l]⁺. Step 3: Preparation of 2-(3-aminoazetidin-l-yl)acetic acid trifluoroacetic acid salt

[0194] To a mixture of 2-(3-((tert-butoxycarbonyl)amino)azetidin-l-yl)acetic acid (310 mg, 1.35 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid (3 mL) dropwise at 0°C. The reaction was stirred at room temperature for 1 hr. The mixture was concentrated under reduced pressure to give 2-(3-aminoazetidin-l-yl)acetic acid trifluoroacetic acid salt (260 mg) as yellow syrup, which was directly used in the next step without purification. LC-MS (ESI) found: 13 l[M+l]⁺.

Step 4: Preparation of 2-(3-(((((.?/?, S,5»S,6/?)-5-methoxy-4-((2/?,.?/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)azetidin- l-yl)acetic acid

[0195] 2-(3-Aminoazetidin-l-yl)acetic acid trifluoroacetic acid salt (297mg, 1.2 mmol) was coupled with Intermediate 1 (440 mg, l.Ommol) by following the General Procedure for Coupling. The reaction was concentrated under vacuum to remove the solvent while keeping the temperature below 40°C. The residue was diluted with dichloromethane and washed with ammonium acetate buffer (pH 4.0, 20 mL x 2) followed by 5% sodium bicarbonate solution (20 mL). The organic layer was dried with Na₂S0₄, filtered and concentrated to give crude product. The residue was purified by prep-HPLC (Method B: 0.1% NH₄OH) to give 2-(3- (((((3R, 4S, 5S, 6R)-5 -m ethoxy-4 -((2 A 3f?)-2-methyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 - oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)azetidin-l-yl)acetic acid (115 mg) as a white solid. LC-MS (ESI) found: 439 [M+l]⁺.

Step 5: Preparation of sodium 2-(3-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)azetidin-l-yl)acetate

[0196] To a solution of 2-{2-{{{{{3R,4S,5S,6R)-S-mei\\oxy-4-{{2R, J/i)-2-methyl-3-(3- methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)azetidin- 1 - yl)acetic acid (115 mg, 0.26 mmol) in acetonitrile (2 mL) was added aq. NaOH (2.4 mL, 0.24 mmol) dropwise at -20°C. The mixture was freeze dried to give sodium 2-(((S)-l- ((((37?, 4S, 5S, 6/ )- 5 -m ethoxy-4 -((2/?, 37?)-2-methyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 - oxaspiro[2.5]octan-6-yl)oxy)carbonyl)pyrrolidin-3-yl)oxy)acetate (110 mg) as a white solid. LC-MS (ESI) found: 439 [M+l]⁺. 1H MR (400 MHz, DMSO- d) d 7.59 (d, J= 7.8 Hz, 1H), 5.30 (m, 1H), 5.19 (t, J= 7.2 Hz, 1H), 4.09 (m, 1H), 3.61 - 3.42 (m, 5H), 3.25 (m, 3H), 2.86 (m, 5H), 2.57 (d, J= 4.3 Hz, 1H), 2.26 - 2.06 (m, 2H), 2.02 - 1.73 (m, 3H), 1.69 (s, 3H), 1.61 (s, 3H), 1.20 - 0.83 (m, 4H).

Example 22: Preparation of Preparation of sodium 4-(((((( /?, S,5»S,6/?)-5-methoxy-4- ((2»V,J ?)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)methyl)benzoate

Step 1: Preparation of 4-(((tert-butoxycarbonyl)amino)methyl)benzoic acid

[0197] 4-(Aminomethyl)benzoic acid (2.0 g, 13.2 mmol) was dissolved in a mixture of dioxane (20 mL) and 1 M NaOH (20 mL). The resulting solution was cooled to 0 °C in an ice bath and di-tert-butyl dicarbonate (2.9 g, 13.6 mmol) was added. The reaction mixture was stirred at r.t. for lh. TLC showed the reaction was complete. The solvent was concentrated under reduced pressure, and a 1 M NaHS0₄ solution was added to adjust the pH value to 2. The mixture was filtered to give 4-(((/c/7-butoxycarbonyl)a ino) ethyl jbenzoic acid (2.3 g) as a white solid. LC-MS (ESI) m/z (M+l): 252.

Step 2: Preparation of allyl 4-(((tert-butox carbonyl)amino)methyl)benzoate

Boc Boc

[0198] To a mixture of 4-(((/c/7-butoxycarbonyl)a ino) ethyl jbenzoic acid (2.3 g, 9.1 mmol) in MeOH (20 mL) and H₂0 (10 mL) was added KOH (510 mg, 9.1 mmol). The mixture was stirred at r.t. for 30 min. The mixture was concentrated, the residue was dissolved in DMF (10 mL) and allyl bromide (1.2 g, 9.56 mmol) was added. After the completion of the addition of allyl bromide, the reaction mixture was stirred at r.t. overnight. TLC showed the reaction was complete. The mixture was concentrated, and the residue was purified by flash

chromatography (silica gel, 5 % ~ 10 % ethyl acetate in petroleum ether) to give allyl 4-(((tert- butoxycarbonyl)amino)methyl)benzoate (2.1 g) as a yellow oil. LC-MS (ESI) m/z (M+l): 292. Step 3: Preparation of allyl 4-(aminomethyl)benzoate

[0199] To a solution of allyl 4-(((tert-butoxycarbonyl)amino)methyl)benzoate (300 mg, 1.03 mmol) in DCM (3 mL) was added TFA (1 mL) at r.t. After the completion of the addition, the mixture was stirred at r.t. for additional 1 h. The reaction mixture was concentrated, and the residue was used in next step without further purification. LC-MS (ESI) m/z (M+l): 191. Step 4: Preparation of allyl 4-((((((3/?, V,5,V,6/?)-5-methoxy-4-((2V,3/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)methyl)benzoate

[0200] To a solution of allyl 4-(aminomethyl)benzoate (350 mg, 1.83 mmol) in CH₃CN (20 mL) was added DIPEA (1.3 mL, 7.32 mmol) drop-wise at 0 °C. After the completion of addition, the mixture was stirred at 0°C for another 10 min and Intermediate 1 (818 mg, 1.83 mmol) was added. The reaction mixture was stirred at r.t. overnight under N₂ atmosphere. TLC showed the reaction was complete. The mixture was concentrated under vacuum while keeping the temperature below 40°C. The residue was purified by flash chromatography (silica gel, 1 % ~ 10 % MeOH in DCM) to give allyl 4-((((((3/(-/A5k,d//)-5-methoxy-4-((2A3//)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)methyl)benzoate (350 mg) as a yellow oil. LC-MS (ESI) m/z (M+l):

500.

Step 5: Preparation of 4-((((((3/?, V,5,V,6/?)-5-methoxy-4-((2V,3/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)methyl)benzoic acid

[0201] To a solution of allyl 4-((((((3A, 4S,5S, df?)- 5 -m ethoxy-4 -((2 , 3f?)-2-methyl-3-3- methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)methyl)benzoate (350 mg, 0.7 mmol) in CEE CN/ Acetone was added Pd(PPh₃)₄ (87 mg, 0.7 mmol) and tetrahydro pyrrole (198 mg, 2.8 mmol ). The mixture was stirred at r.t. overnight. The reaction mixture was concentrated and the residue was purified by prep-ffPLC (Cl 8, 0 ~ 90 % acetoniltrile in H₂0 with 0.1 % NH₃ H₂0) to give 4- ((((((.3R, 4S, 5S, d/^)-5-methoxy-4-((25', 3f?)-2-methyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 - oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)methyl)benzoic acid (120 mg) as a yellow oil. LC-MS (ESI) m/z (M+l): 460.

Step 6: Preparation of sodium 4-((((((J/?, V,5,V,6/?)-5-methoxy-4-((2V,J/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)methyl)benzoate

[0202] To a solution of 4-((((((3f?, S',5,S^,, df?)-5-methoxy-4-((2S^,,3f?)-2-methyl-3-(3-methylbut- 2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)methyl) benzoic acid (120 mg, 0.26 mmol) in MeCN (3 mL) was added a 0.1 N NaOH solution (2.3 mL, 0.23 mmol) dropwise at -60°C. After lyophilization, sodium A-((((((3R,4S,5S, 6R)-5-me\\\oxy-A-((2S,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)methyl)benzoate (94 mg) was obtained as a white solid. LC-MS (ESI) m/z (M+l): 460. 1H NMR (400 MHz, DMSO-d6 ) d 7.81 (d, J= 7.5 Hz, 2H), 7.66 (s, 1H), 7.21 (d, J = 7.7 Hz, 2H), 5.32 (s, 1H), 5.19 (t, J= 7.3 Hz, 1H), 4.21 (d, J= 6.1 Hz, 1H), 3.57 - 3.53 (m, 1H), 3.29 (s, 3H), 2.84 (d, j= 4.3 Hz, 1H), 2.57 (d, j= 4.3 Hz, 1H), 2.18 (dd, j= 13.2, 6.6 Hz, 2H), 1.86 (m, 4H), 1.70 (s, 3H), 1.62 (s, 1H), 1.61 (s, 3H), 1.10 (s, 3H), 1.06 (s, 1H).

Example 23: Preparation of sodium 4-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-

3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)benzoate

Step 1: Preparation of 4-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)benzoic acid

[0203] To a solution of (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2- en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl lH-imidazole-l-carboxylate (250 mg, 0.66 mmol) in dry THF (5 mL) was added 4-aminobenzoic acid (109 mg, 0.80 mmol) and TFA (0.07 mL, 0.95 mmol) at 0°C. The reaction was then stirred at room temperature overnight. TLC showed the reaction was complete. The resulting mixture was diluted with DCM and washed with water. The combined organic layers were dried over anhydrous Na₂S0₄ and concentrated. The crude product was purified by flash chromatography (silica gel, l~ 3 % MeOH in DCM) and pre-HPLC (Cl 8, 0 ~ 90 % acetoniltrile in H₂0 with 0.1 % NH₃ H₂0) to give 4-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2- yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)benzoic acid (141.4 mg) as a white solid. LC-MS (ESI) found: 446 [M+l]⁺.

Step 2: Preparation of sodium 4-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)benzoate

[0204] To a solution of 4-(3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2- en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)benzoic acid (68 mg, 0.15 mmol) in MeCN (1.4 mL) was added a 0.1 M NaOH (1.4 mL, 0.14 mmol) solution dropwise at 0°C with stirring. After lyophilization, sodium 4-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)benzoate (60 mg) was obtained as a white solid. LC-MS (ESI) found: 446 [M+l]⁺. 1H NMR (400 MHz, DMSO-d6 ) d 9.51 (s, 1H), 7.70 (d, J= 8.5 Hz, 2H), 7.30 (d, J = 8.2 Hz, 2H), 5.39 (s, 1H), 5.16 (m, J= 7.4 Hz, 1H), 3.55 (m, J= 10.8, 2.6 Hz, 1H), 3.28 (s, 3H), 2.82 (d, J= 4.4 Hz, 1H), 2.55 (d, 7= 4.4 Hz, 1H), 2.51 - 2.47 (m, 1H), 2.22 - 2.07 (m, 2H), 1.91 (m, 7= 29.4, 16.6, 5.2 Hz, 3H), 1.73 (m, 7= 28.0, 14.1 Hz, 1H), 1.67 (s, 3H), 1.57 (s,

3H), 1.06 (s, 3H), 1.02 (d, J= 4.3 Hz, 1H).

Example 24: Preparation of sodium 6-((((((J ?, »V,5»V,6 ?)-5-methoxy-4-((2 ?,J ?)-2-methyl- 3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)methyl)nicotinate

Step 1: Preparation of 6-(aminomethyl)nicotinic acid

[0205] To a solution of 6-Cyanonicotinic acid (1 g, 6.7 mmol) in MeOH (20 mL) was added Pd(OH)₂ (10% Pd in carbon, 0.1 g). The mixture was stirred at r.t. under a hydrogen atmosphere overnight. Then the mixture was diluted with H₂0 (10 mL), the catalyst was filtered, and the filtrate was concentrated in vacuo to give 6-(aminomethyl)nicotinic acid (750 mg), which was used in the next step without further purification.

Step 2: Preparation of 6-(((tert-butox carbonyl)amino)methyl)nicotinic acid

[0206] To a solution of 6-(aminomethyl)nicotinic acid (750 mg, 4.92 mmol) in THF (50 mL) was added 50 mL of 1N aq NaOH and Boc₂0 (1.2 g, 5.66 mmol) at 25 °C. After the completion of addition, the mixture was stirred overnight. Then the mixture was diluted with water (25 mL) and brine (25 mL), acidified slowly to pH = 3 by 1N aq HC1, and extracted with EtOAc (3 x 20 mL). The combined organic extracts were dried over anhydrous Na₂S0₄ and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel, 5 % ~ 10 % ethyl acetate in petroleum ether) to give 6-(((tert- butoxycarbonyl)amino)methyl)nicotinic acid (720 mg) as a colorless oil. LC-MS (ESI) m/z (M+l): 253.

Step 3: Preparation of allyl 6-(((tert-butox carbonyl)amino)methyl)nicotinate

[0207] To a solution of 6-(((tert-butoxycarbonyl)amino)methyl)nicotinic acid (720 mg, 2.85 mmol) in DMF (8 mL) was added Cs₂C0₃ (1.4 g, 4.28 mmol) at rt. After completion of addition, the reaction mixture was stirred for another 10 min, and then allylbromide (518 mg, 4.28 mmol) and a catalytic amount of KI were added. After completion of addition, the reaction mixture was heated to l00°C for 4h. Upon cooling of the reaction, water (50 mL) was added, and the aqueous layer was extracted with EtOAc (3 x 20 mL). The combined organic extracts were dried over anhydrous Na₂S0₄ and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (silica gel, 5 % ~ 10 % ethyl acetate in petroleum ether) to give allyl 6-(((tert-butoxycarbonyl)amino)methyl)nicotinate (660 mg) as a white solid. LC-MS (ESI) m/z (M+l): 293.

Step 4: Preparation of allyl 6-(aminomethyl)nicotinate

[0208] To a solution of allyl 6-(((tert-butoxycarbonyl)amino)methyl)nicotinate (660 mg, 2.26 mmol) in DCM (4 mL) was added TFA (2 mL). The mixture was stirred at r.t. for 1 h. TLC showed the reaction was complete. The reaction mixture was concentrated and the residue was used in next step without further purification. LC-MS (ESI) m/z (M+l): 193.

Step 5: Preparation of allyl 6-((((((3/?, V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)methyl)nicotinate

[0209] To a solution of allyl 6-(aminomethyl)nicotinate (660 mg, 3.43 mmol) in CH₃CN (20 mL) was added DIPEA (2.4 mL, 13.6 mmol) drop-wise at 0°C. The mixture was stirred at 0 °C for another 10 min and Intermediate 1 (1.3 g, 3.1 mmol) was added. The reaction mixture was stirred at r.t. overnight under N₂ atmosphere. The mixture was concentrated under vacuum to remove the solvent while keeping the temperature below 40 °C. The residue was purified by flash chromatography (silica gel, 1 % ~ 10 % MeOH in DCM) to give allyl 6- (((((( 3R , 4S, 5S, 6/^)-5-methoxy-4-((2k, 3f?)-2-methyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 - oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)methyl)nicotinate (650 mg) as a yellow oil. LC- MS (ESI) m/z (M+l): 501.

Step 6: Preparation of 6-((((((3/?, V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)methyl)nicotinic acid

[0210] To a solution of allyl 6-((((((3f?,4ri^,,5ri^,,6/?)-5-methoxy-4-((2f?,3f?)-2-methyl-3-(3- methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)methyl)nicotinate (650 mg, 1.29 mmol) in CH₃CN/acetone (10 mL) was added Pd(PPh₃)₄ (161 mg, 0.13 mmol) and tetrahydropyrrole (366 mg, 5.16 mmol). The mixture was stirred at r.t. overnight. The reaction mixture was concentrated, and the residue was purified by prep-HPLC (C18, 0 ~ 90 % acetoniltrile in H₂0 with 0.1 % NH₃TT₂0) to give 6-((((((3/(-/N5.5', 6//)-5-methoxy-4-((2A3//)-2-methyl-3-(3-methylbut-2-en- l -yl)oxiran-2-yl)- l - oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)methyl)nicotinic acid (102 mg) as a white solid. LC-MS (ESI) m/z (M+l): 461.

Step 7: Preparation of sodium 6-((((((3/?, S,5A,6/?)-4-((2/?,3/?)-3-ethyl-2-methyloxiran-2- yl)-5-methoxy-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)methyl)nicotinate

[0211] To a solution of 6-((((((3f?, ,S^,,5S^,, 6/?)-5-methoxy-4-((2f?,3f?)-2-methyl-3-(3- methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)methyl)nicotinic acid (102 mg, 0.22 mmol) in MeCN (3 mL) was added 0.1 N NaOH solution (2.0 mL, 0.2 mmol) dropwise at -60°C. After lyophilization, sodium 6-((((((3A, 4S, 5S, 6R)-4-((2S, 3R)~ 3 -ethyl-2-methyloxiran-2-yl)-5-methoxy- 1 - oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)methyl)nicotinate (73 mg) was obtained as a white solid. LC-MS (ESI) m/z (M+l): 461. 1H NMR (400 MHz, DMSO-d6 ) d 8.84 (s, 1H),

8.04 (dd, 7= 7.9, 1.9 Hz, 1H), 7.65 (t, J= 5.9 Hz, 1H), 7.17 (d, J= 7.9 Hz, 1H), 5.32 (s, 1H), 5.20 (t, J= 7.5 Hz, 1H), 4.27 (d, J= 6.0 Hz, 2H), 3.55 (dd, J= 10.6, 2.3 Hz, 1H), 3.30 (s, 3H),

2.85 (d, J= 4.3 Hz, 1H), 2.62 - 2.54 (m, 1H), 2.19 (m, 2H), 1.99 - 1.77 (m, 3H), 1.71 (s, 3H), 1.61 (s, 3H), 1.10 (s, 3H), 1.05 (s, 1H).

Example 25: Preparation of sodium l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl- 3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidine-3- carboxylate

Step 1 : l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidine-3-carboxylic acid

[0212] To a mixture of azetidine-3 -carboxylic acid (202 mg, 2.0 mmol) in CH₃CN (5 mL) was added DIPEA (650 mg, 5 mmol) drop-wise at 0~5°C. The mixture was stirred at 0~5°C for 10 min, then (J/i,-/V,5,.y, ri/i)-5-methoxy-4-((2/i, J/i)-2-methyl-3-(3-methylbut-2-en- 1 -yl)oxiran- 2-yl)-l-oxaspiro[2.5]octan-6-yl (4-nitrophenyl) carbonate (Intermediate 1, 900 mg, 2.0 mmol) was added to the mixture in portions at 0°C under N₂ atmosphere. The reaction mixture was stirred at 25°C for another 16 hrs. TLC showed the reaction was complete. The solvent was removed under vacuum below 40°C, then diluted with DCM (60 mL) and washed with ammonium acetate buffer (pH~4, 15 mL x 2). The aqueous layer was extracted with DCM (10 mL x 2), dried over anhydrous Na₂S0₄ and concentrated to give a yellow oil, which was purified by prep-HPLC (Method B: 0.1 % NH OH) to give (385 mg) l-((((3R,4S,5S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidine-3-carboxylic acid as a white solid. LC-MS (ESI) found: 424 [M+l]⁺. Step 2: Preparation of sodium l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidine-3- carboxylate

[0213] To a solution of l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut- 2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidine-3 -carboxylic acid (123 mg, 0.3 mmol) in MeCN (2 mL) was added a 0.1 N NaOH solution (2.7 mL, 0.9 eq) dropwise at -60°C with stirring. After lyophilization, (140 mg) of the title compound was obtained as a white solid. LC-MS (ESI) found: 410 [M+l]⁺. 1H NMR (400 MHz, DMSO-d6 ) d 5.36 (d, J= 5.5 Hz, 1H), 5.19 (s, 1H), 4.20 (d, J= 22.1 Hz, 1H), 3.62 - 3.41 (m, 3H), 3.29 (d, J = 5.0 Hz, 4H), 2.91 - 2.80 (m, 1H), 2.72 - 2.53 (m, 2H), 2.30 - 2.08 (m, 2H), 1.98 (dd, J = 15.9, 11.1 Hz, 2H), 1.81 (dd, J= 11.1, 2.9 Hz, 3H), 1.71 (s, 3H), 1.61 (s, 3H), 1.08 (d, J= 3.5 Hz, 3H), 1.01 (d, J= 13.5 Hz, 1H).

Example 26: Preparation of sodium 2-((R)-3-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)pyrrolidin-l-yl)acetate

Step 1: Preparation of tert-butyl (R)-2-(3-((tert-butoxycarbonyl)amino)pyrrolidin-l- yl)acetate

[0214] The mixture of tert-butyl (R)-pyrrolidin-3-ylcarbamate (400 mg, 2.15 mmol), tert- butyl 2-bromoacetate (430 mg, 2.26 mmol) and K₂C0₃ (593 mg, 4.30 mmol) in DMF (10 mL) was stirred at 50°C for l6h. The slurry was diluted by EA, and washed by water and brine. The combined organic layers were dried over anhydrous Na₂S0₄, concentrated under reduced pressure and purified by flash chromatography to give tert-butyl (R)-2-(3-((tert- butoxycarbonyl)amino)pyrrolidin-l-yl)acetate (600 mg) as a white solid. LC-MS (ESI) found: 301 [M+H] ⁺

Step 2: Preparation of(R)-2-(3-aminopyrrolidin-l-yl)acetic acid

[0215] To a solution of tert-butyl (R)-2-(3-((tert-butoxycarbonyl)amino)pyrrolidin-l- yl)acetate (600mg, 2.00 mmol) in DCM (2 mL) was added TFA (1 mL). The reaction mixture was then stirred at room temperature for 3 hours. Concentration gave crude (R)-2-(3- aminopyrrolidin-l-yl)acetic acid TFA salt (560 mg) as a brown oil. LC-MS (ESI) found:306 [M+l] ⁺.

Step 3: Preparation of 2-((R)-3-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)pyrrolidin-l-yl)acetic acid

Intermediate 1 [0216] Under a N₂ atmosphere, to a solution of (R)-2-(3-aminopyrrolidin-l-yl)acetic acid

2,2,2-trifluoroacetate salt (crude, 560 mg, 1.0 mmol) and diisopropylethylamine (259 mg, 2.10 mmol) in CH₃CN (5 mL) was added a solution of (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl (4-nitrophenyl) carbonate (Intermediate 1, 300 mg, 0.67 mmol) in CH₃CN (lmL) drop-wise at 0°C, followed by addition of DMAP (5 mg, 0.067 mmol). Then the mixture was stirred at r.t. for 16 h. The reaction mixture was concentrated and purified by prep-HPLC (Method A, H₂0 (0.1% FA)/ CH₃CN) to give 2-((R)-3-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut- 2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)pyrrolidin- 1 -yl)acetic acid (50 mg) as a colorless oil. LC-MS (ESI) found: 453 [M+l] ⁺.

Step 4: Preparation of sodium 2-((R)-3-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)pyrrolidin-l-yl)acetate

[0217] To a solution of 2-((R)-3-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)pyrrolidin- 1 - yl)acetic acid (50 mg, 0.11 mmol) in CH₃CN (1 mL) was added a 0.1 N NaOH (1.6 mL, 0.099 mmol) solution at -60°C with stirring. After lypholization, sodium 2-((R)-3-(((((3R,4S,5S,6R)- 5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan- 6-yl)oxy)carbonyl)amino)pyrrolidin-l-yl)acetate salt (51 mg) was obtained as white solid. 1HNMR (400MHz, DMSO-76) 57.86 (d, J= 7.3 Hz, 1H), 5.27 (s, 1H), 5.18 (d, J= 7.3 Hz,

1H), 3.91 (s, 1H), 3.52 (d, j= 10.4 Hz, 1H), 3.28 (s, 3H), 2.85 - 2.80 (m, 2H), 2.76 (s, 2H),

2.70 (d, J= 7.8 Hz, 1H), 2.57 - 2.53 (m, 2H), 2.46 (d, 7= 9.1 Hz, 2H), 2.20 - 2.14 (m, 2H), 1.97 - 1.74 (m, 5H), 1.71 (s, 3H), 1.61 (s, 3H), 1.57 - 1.51 (m, 1H), 1.09 (s, 3H), 1.04 (d, J =

14.1HZ, 1H).

Example 27: Preparation of sodium 2-((S)-3-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)pyrrolidin-l-yl)acetate

Step 1: Preparation of (S)-tert-butyl 2-(3-((tert-butoxycarbonyl)amino)pyrrolidin-l- yl)acetate

⁰

H 1 — ( I _Br^ C_oJ I

N <

HN

V-NH

Boc DIPEA, MeCN B RoncnrO ^{^}J ^vU O<

[0218] To a solution of (S)-tert-butyl pyrrolidin-3-ylcarbamate (400 mg, 2.15 mmol) in

MeCN (8 mL) was added DIPEA (0.53 mL, 3.22 mmol) and a solution of tert-butyl 2- bromoacetate (0.31 mL, 2.15 mmol) in CH₃CN (2mL) dropwise. The reaction mixture was stirred at r.t. overnight. The mixture was diluted with water and extracted with EtOAc (15 mL x2). The combined organic layers were dried over Na₂S0₄, filtered and concentrated. The residue was purified by column chromatography (silica gel, 0 to 2 % MeOH in DCM) to give (S)-tert-butyl 2-(3-((tert-butoxycarbonyl)amino)pyrrolidin-l-yl)acetate (600 mg) as yellow oil. LC-MS (ESI) found: 454 [M+l]⁺: 301.

Step 2: Preparation of (S)-2-(3-aminopyrrolidin-l-yl)acetic acid

[0219] To a solution of (S)-tert-butyl 2-(3-((tert-butoxycarbonyl)amino)pyrrolidin-l- yl)acetate (600 mg, 2.0 mmol) in DCM (3 mL) was added TFA (1.5 mL) at 0°C. The mixture was stirred at r.t. for 1 h. The reaction mixture was concentrated and the residue was used in the next step without further purification. LC-MS (ESI) m/z (M+l): 145.

Step 3: Preparation of sodium 2-((S)-3-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-

3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)pyrrolidin-l-yl)acetate

[0220] (S)-2-(3-aminopyrrolidin-l-yl)acetic acid (192 mg, 1.33 mmol) in CH₃CN (5 mL) and water (lmL) was coupled with Intermediate 1 (500 mg, 1.11 mmol) by following the General Procedure for Coupling. The mixture was concentrated under vacuum to remove the solvent while keeping the temperature below 40°C. The residue was purified by prep-HPLC (Method B: 0.1% MEOH) to give 2-((S)-3-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)pyrrolidin- 1 - yl)acetic acid(60 mg, 10%). The acid was dissolved in MeCN (1 mL) and aq. NaOH (1.2 mL, 0.1M) was added at 0°C. After lyophilization, sodium 2-((S)-3-(((((3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)amino)pyrrolidin-l-yl)acetate (60 mg) was obtained as a white solid. LC-MS (ESI) m/z (M+l): 453. 1H NMR (400 MHz, DMSO- d) d 7.85 (d, J= 7.5 Hz, 1H), 5.27 (s,

1H), 5.20 (t, j= 7.4 Hz, 1H), 3.89 (s, 1H), 3.52 (dd, j= 10.7, 2.6 Hz, 1H), 3.29 (s, 3H), 2.88 - 2.64 (m, 5H), 2.55 (dd, J= 7.8, 5.4 Hz, 2H), 2.44 (dd, J= 13.3, 8.2 Hz, 2H), 2.25 - 2.11 (m, 2H), 2.04 - 1.65 (m, 8H), 1.64 - 1.47 (m, 4H), 1.14 - 0.97 (m, 4H). Example 28: Preparation of sodium 2-(4-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)piperidin-l-yl)acetate

Step 1: Preparation of ethyl 2-(4-((tert-butoxycarbonyl)amino)piperidin-l-yl)acetate

[0221] To a mixture of tert-butyl piperidin-4-yl carbamate (500 mg, 2.49 mmol) in DMF (20 mL) were added ethyl 2-bromoacetate (500 mg, 2.99 mmol) and K₂C0₃ (687 mg, 4.98 mmol). The mixture was stirred at r.t. overnight. Water (50 mL) was added, and the mixture was extracted with ethyl acetate (50 mL). The organic extract was washed with brine, dried over anhydrous Na₂S0₄, and evaporated under vacuum. The residue was purified by flash chromatography (silica gel, 1 % ~ 20 % ethyl acetate in petroleum ether) to give ethyl 2-(4- ((tert-butoxycarbonyl)amino)piperidin-l-yl)acetate (490 mg) as a white solid. LC-MS (ESI) found: 287 [M+l]⁺. Step 2: Preparation of ethyl 2-(4-aminopiperidin-l-yl)acetate

Bo

[0222] To a solution of ethyl 2-(4-((tert-butoxycarbonyl)amino)piperidin-l-yl)acetate (490 mg, 1.471 mmol) in DCM (2 mL) was added TFA (3 mL). The mixture was stirred at r.t. for 2 h. The reaction mixture was concentrated, and the crude product was used in the next step without further purification. LC-MS (ESI) m/z (M+l): 187

Step 3: Preparation of ethyl 2-(4-(((((J/?, V,5,V,6/?)-5-methoxy-4-((2/?,J/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)piperidin-l-yl)acetate

[0223] To a solution of ethyl 2-(4-aminopiperidin-l-yl)acetate (320 mg, 1.71 mmol) in CH₃CN (20 mL) was added DIPEA (1.2 mL, 6.8 mmol) dropwise at 0°C. The mixture was stirred at 0°C for 10 min and Intermediate 1 (764 mg, 1.71 mmol) was added. The reaction mixture was stirred at r.t. overnight under N₂ atmosphere. After the reaction complete, the mixture was concentrated under vacuum to remove the solvent while keeping the temperature below 40°C. The residue was purified by flash chromatography (silica gel, 1 % ~ 5 % MeOH in DCM) to give ethyl 2-(4-(((((3A, S^,,5S^,,di?)-5-methoxy-4-((2i?,3i?)-2-methyl-3-(3-methylbut-2- en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)piperidin- 1 -yl)acetate (510 mg) as a colorless oil. LC-MS (ESI) m/z (M+l): 495.

Step 4: Preparation of 2-(4-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)piperidin-l-yl)acetic acid

[0224] To a solution of ethyl 2-(4-(((((3f?, ,S^,,5S^,, 6/?)-5-methoxy-4-((2f?,3f?)-2-methyl-3-(3- methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)piperidin- 1 - yl)acetate (510 mg, 1.05 mmol) in THF/H₂0 (3 mL/l mL) was added LiOH (433 mg, 10.3 mmol). The mixture was stirred at r.t. overnight. The reaction mixture was concentrated and the residue was purified by prep-HPLC (Method B: 0.1% NH₄OH) to 2-(4-(((((3R,4S,5S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)piperidin-l-yl)acetic acid (60 mg) as a white solid. LC-MS (ESI) m/z (M+l): 467.

Step 5: Preparation of sodium 2-(4-(((((3/?, ,V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)piperidin-l-yl)acetate

[0225] To a solution of 2-(4-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)piperidin- 1 - yl)acetic acid (60 mg, 0.12 mmol) in MeCN (1 mL) was added dropwise a 0.1 N NaOH solution (1.1 mL, 0.12 mmol) at -60°C with stirring. After lyophilization, sodium 2-(4- (((((3R, 4S, 5S, 6R)-5 -m ethoxy-4 -((2 A 3i?)-2-methyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 - oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)piperidin-l-yl)acetate (40 mg) was obtained as a white solid. LC-MS (ESI) m/z (M+l): 467. 1H NMR (400 MHz, DMSO- d) d 7.00 (s, 1H), 5.23 (d, J= 29.6 Hz, 2H), 3.52 (dd, J= 10.7, 2.7 Hz, 1H), 3.28 (s, 3H), 3.22 (d, J= 8.3 Hz, 2H), 2.83 (d, J= 4.1 Hz, 3H), 2.66 (s, 2H), 2.56 (d, J= 4.3 Hz, 1H), 2.18 (d, J= 7.0 Hz, 2H), 2.10 - 1.73 (m, 6H), 1.71 (s, 3H), 1.66 (s, 2H), 1.61 (s, 3H), 1.40 (s, 2H), 1.09 (s, 2H), 1.06 (s, 1H). Example 29: Preparation of sodium ethyl (l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)phosphonate

Step 1: Preparation of l-benzhydrylazetidin-3-yl methanesulfonate

[0226] To a mixture of l-benzhydrylazeti din-3 -ol (10.0 g, 41.79 mmol) in DCM (100 mL) was added TEA (12.7 g, 125.37 mmol) and MsCl (8.6 g, 75.22 mmol) at 0°C. Then the reaction mixture was warmed to r.t. and stirred for 3 hours. After the reaction was complete, the mixture was washed with water and the water layer was back-extracted with DCM. The combined organic layers were dried over Na₂S0₄, and concentrated. The residue was purified by flash chromatography (silica gel, 2 % - 5 % MeOH in DCM) to give l-benzhydrylazetidin- 3-yl methanesulfonate (11.0 g) as a yellow solid. LC-MS (ESI) found: 318 [M+l]⁺.

Step 2: Preparation of diethyl (l-benzhydrylazetidin-3-yl)phosphonate

[0227] A solution of l-benzhydrylazeti din-3 -yl methanesulfonate (10 g, 31.54 mmol) in P(OEt)₃ (52 g, 315.4 mmol) was heated to l50°C and stirred at this temperature for 8 hours. After the reaction complete, it was cooled to r.t. and purified by flash chromatography (silica gel, 2 % -5 % MeOH in DCM) to give diethyl (l-benzhydrylazetidin-3-yl)phosphonate (17.0 g) as a yellow oil. LC-MS (ESI) found: 360 [M+l]⁺.

Step 3: Preparation of ethyl hydrogen (l-benzhydrylazetidin-3-yl)phosphonate

[0228] To a mixture of diethyl (l-benzhydrylazeti din-3 -yl)phosphonate crude (10 g, 27.86 mmol) in EtOH (80 mL) was added 2.0M NaOH (80 mL, 167.13 mmol) at r.t. Then the reaction mixture was heated to 80°C and stirred for 8 hours. After the reaction was complete, the mixture was adjusted to pH = 2~3 using 2M HC1 and extracted with DCM 3 times. The DCM extracts were dried over Na₂S0₄ and concentrated. The residue was purified by prep- HPLC (Method B: 0.1% NH₄OH) to give ethyl hydrogen (l-benzhydrylazeti din-3 - yl)phosphonate (1.8 g) as a white solid. LC-MS (ESI) found: 332 [M+l]⁺.

Step 4: Preparation of ethyl hydrogen azetidin-3-ylphosphonate

[0229] To a mixture of ethyl hydrogen (l-benzhydrylazetidin-3-yl)phosphonate (800 mg, 2.41 mmol) in EtOH (16 mL) was added Pd/C (10%, 400 mg) at r.t. Then the reaction mixture was stirred at r.t. for 2 hours under H_2. After the reaction complete, it was filtered and washed with EtOH. The filtrate was concentrated to give crude ethyl hydrogen azeti din-3 - ylphosphonate (700 mg) as an oil. LC-MS (ESI) found: 166 [M+l]⁺. Step 5: Preparation of ammonium ethyl (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl- 3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl 3-

(ethyl(hydroxy)phosphonyl)azetidine-l-carboxylate

[0230] To a solution of crude ethyl hydrogen azeti din-3 -ylphosphonate (700 mg) in MeCN (6 mL) was added DIPEA (267 mg) at 0°C. Then (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-

3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl (4-nitrophenyl) carbonate (617 mg) was added at 0°C. The reaction mixture was then stirred for 2 hours at r.t. After the reaction complete, the mixture was concentrated under reduced pressure to remove MeCN and diluted by water. The water mixture was extracted with DCM. The combined organic layers was dried over Na₂S0₄, concentrated to give the crude DIPEA salt (3R,4S,5S,6R)-5-methoxy-

4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl 3- (ethyl(hydroxy)phosphonyl)azetidine-l-carboxylate (1.3 g) as colorless oil. Then, 360 mg of the aforementioned crude material was purified by prep-HPLC (Method B: 0.1% NH₄OH) ammonium ethyl (l-((((3i?,4ri',5ri', 6/?)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)phosphonate (80 mg) as an oil. LC-MS (ESI) found: 474 [M+l]⁺. 1H MR (400 MHz, DMSO) d 7.38 (s, 4H), 5.28 (s, 1H), 5.19 (s, 1H), 3.85 (s, 4H), 3.72 - 3.64 (m, 2H), 3.50 (s, 1H), 3.28 (s, 3H), 2.84 (d, J= 4.3 Hz, 1H), 2.57 (d, J= 4.3 Hz, 2H), 2.17 (d, J= 6.6 Hz, 2H), 1.99 - 1.88 (m, 1H), 1.81 - 1.66 (m, 6H), 1.61 (s, 3H), 1.12 - 1.04 (m, 6H), 1.04 - 0.98 (m, 1H).

Step 6: Preparation of sodium ethyl (l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-

3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)phosphonate

[0231] To a mixture of (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en- l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl 3 -(ethyl (hydroxy)phosphonyl)azeti dine- 1- carboxylate (40 mg, 0.084 mmol) in MeCN (0.8 mL) was added 0.1M NaOH (0.76 mL, 0.076 mmol) at r.t. Then the mixture was freeze-dried to give sodium ethyl (l-((((3R,4S,5S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)phosphonate (41 mg) as a white solid. LC-MS (ESI) found: 496 [M+l]⁺. 1H NMR (400 MHz, DMSO- d) d 5.31 - 5.23 (m, 1H), 5.22 - 5.16 (m, 1H), 3.95 - 3.72 (m, 4H), 3.71 - 3.58 (m, 2H), 3.54 - 3.46 (m, 1H), 3.27 (s, 3H), 2.87 - 2.81 (m, 1H), 2.56 (s, 2H), 2.46 - 2.38 (m, 1H), 2.24 - 2.10 (m, 2H), 1.99 - 1.90 (m, 1H), 1.70 (s, 6H), 1.61 (s, 3H), 1.10 - 1.03 (m, 6H), 1.03 - 0.97 (m, 1H).

Example 30: Preparation of sodium 3-(3-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)azetidin-l-yl)propanoate

Step 1: Preparation of methyl 3-(3-((tert-butoxycarbonyl)amino)azetidin-l-yl)propanoate

[0232] To a solution of tert-butyl azeti din-3 -ylcarbamate (416 mg, 2.0 mmol) and K₂C0₃ (560 mg, 4.0 mmol) in DMF (10 mL) was added methyl 3-bromopropanoate (330 mg, 2.0 mmol) dropwise at 0°C. The mixture was stirred for 30 min at 0°C. Then the reaction was stirred at room temperature overnight under a N₂ atmosphere. After the reaction was complete water was added and the water later was extracted with DCM. The dichloromethane layer was dried over anhydrous Na₂S0₄, filtered and concentrated to give crude product. The crude product was purified by chromatography (silica gel, 1 to -10 % MeOH in DCM) to give methyl 3-(3-((tert-butoxycarbonyl)amino)azetidin-l-yl)propanoate (350 mg) as a white solid. LC-MS (ESI) found: 259 [M+H]⁺.

Step 2: Preparation of 3-(3-((tert-butoxycarbonyl)amino)azetidin-l-yl)propanoic acid

[0233] A mixture of methyl 3-(3-((tert-butoxycarbonyl)amino)azetidin-l-yl)propanoate (350 mg, 1.35 mmol) and lithium hydroxide (74 mg, 3.1 mmol) in THF (5mL) and water (2 mL) was stirred at room temperature overnight. TLC showed the reaction was complete, and the mixture was concentrated to remove THF. Then the water solution was washed with ethyl acetate (50 mL x 2), acidified to -pH = 4 with 1M HC1 and extracted with DCM. The DCM layer was dried over anhydrous Na₂S0₄, filtered and concentrated under reduced pressure to give 3-(3- ((tert-butoxycarbonyl)amino)azetidin-l-yl)propanoic acid (280 mg) as a colorless oil, which was used directly in the next reaction without further purification. LC-MS (ESI) found: 245 [M+H]⁺.

Step 3: Preparation of 3-(3-aminoazetidin-l-yl)propanoic acid trifluoroacetic acid salt

[0234] To a mixture of 3-(3-((tert-butoxycarbonyl)amino)azetidin-l-yl)propanoic acid (280 mg, 1.2 mmol) in dichloromethane (5 mL) was added trifluoroacetic acid (3 mL) dropwise at 0°C. The reaction was stirred at room temperature for 1 h. TLC showed the reaction was complete, and the mixture was concentrated under reduced pressure to give 3-(3-aminoazetidin- l-yl)propanoic acid trifluoroacetic acid salt (280 mg) as yellow syrup, which was used directly in the next step without further purification. LC-MS (ESI) found: 145[M+H]⁺.

Step 4: Preparation of 2-(3-(((((3/?, S,5A,6/?)-5-methoxy-4-((2/?,.?/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)azetidin- l-yl)acetic acid

[0235] 3-(3-aminoazetidin-l-yl)propanoic acid trifluoroacetic acid salt (280 mg, 1.0 mmol) was coupled with Intermediate 1 (440 mg, 1.0 mmol) by following the General Procedure for Coupling. The reaction was concentrated under vacuum to remove the solvent while keeping the temperature below 40°C. The residue was diluted with dichloromethane and washed with ammonium acetate buffer (pH 4.0, 20 mL x 2) . The organic layer was dried with anhydrous Na₂S0₄, filtered and concentrated to give crude product. The residue was purified by prep- HPLC (Method B: 0.1% NH₄OH) to give 3-(3-(M3R,JS,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)azetidin-l-yl)propanoic acid (105 mg) as a white solid. LC-MS (ESI) found: 453 [M+H]⁺.

Step 5: Preparation of sodium 2-(3-(((((3/?, V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)azetidin-l-yl)acetate

[0236] To a solution of 3-(3-(((((3f?,4,S^,,5S^,,d/?)-5-methoxy-4-((2f?,3f?)-2-methyl-3-(3- methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)azetidin- 1 - yl)propanoic acid (105 mg, 0.23 mmol) in acetonitrile (1 mL) was added aq. NaOH (2.4 mL, 0.24 mmol, 1M) dropwise at -20°C. After completion of the reaction, the mixture was freeze dried to give sodium 3-(3-(((((3f?,4ri^,,5ri^,,6/?)-5-methoxy-4-((2f?,3f?)-2-methyl-3-(3-methylbut-2- en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)azetidin-l-yl)propanoic acid (115 mg) as a white solid. LC-MS (ESI) found: 453 [M+H]⁺. 1H NMR (400 MHz, DMSO- d) d 7.64 (d, J= 7.8 Hz, 1H), 5.37 (d, J= 26.7 Hz, 1H), 5.26 (t, J= 7.2 Hz, 1H), 4.08 (dd, J= 14.3, 7.0 Hz, 1H), 3.59 (dd, J= 10.8, 2.6 Hz, 2H), 3.47 (s, 3H), 3.34 (s, 3H), 2.91 (d, J = 4.2 Hz, 1H), 2.77 (dt, J= 13.6, 6.9 Hz, 2H), 2.64 (d, J= 4.3 Hz, 1H), 2.55 - 2.47 (m, 2H), 2.32 - 2.16 (m, 2H), 2.13 - 1.81 (m, 5H), 1.78 (s, 3H), 1.73 (d, J= 7.4 Hz, 1H), 1.68 (s, 3H), 1.31 - 0.99 (m, 4H).

Example 31: Preparation of sodium 2-(4-((((J ?, V,5A,6 ?)-5-methoxy-4-((2 ?,J ?)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)piperazin-l-yl)acetate

Step 1: Preparation of tert-butyl 4-(2-(tert-butoxy)-2-oxoethyl)piperazine-l-carboxylate

[0237] To a solution of tert-butyl piperazine- l-carboxylate (1.5 g, 8.1 mmol) in THF (50 mL) were added tert-butyl 2-bromoacetate (1.4 mL, 9.6 mmol) and TEA (1.1 mL, 8.1 mmol). The mixture was stirred at r.t. for 2 hours. TLC showed the reaction was complete. Water (20 mL) was added and the mixture was extracted with ethyl acetate (50 mL). The organic extract was washed with brine, dried over anhydrous Na₂S0₄, and concentrated under vacuum. The residue was purified by flash chromatography (silica gel, 1 % ~ 20 %ethyl acetate in petroleum ether) to give tert-butyl 4-[2-(tert-butoxy)-2-oxoethyl]piperazine- l-carboxylate (2.1 g) as a white solid. LC-MS (ESI) m/z (M+l): 301. Step 2: Preparation of 2-(piperazin-l-yl)acetic acid

[0238] To a mixture of tert-butyl 4-(2-(tert-butoxy)-2-oxoethyl)piperazine-l-carboxylate (500 mg, 1.6 mmol) in DCM (1 mL) was added TFA (3 mL). The reaction mixture was stirred at room temperature for 16 hrs. TLC showed the reaction was complete. The resulting mixture was concentrated to give crude product, which was used in the next step without further purification. LC-MS (ESI) m/z (M+l): 145.

Step 3: Preparation of 2-(4-((((J/?, V,5,V,6/?)-5-methoxy-4-((2/?,J/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)piperazin-l- yl)acetic acid

[0239] To a solution of 2-(piperazin-l-yl)acetic acid, TFA salt (260 mg, 1.8 mmol) in CH₃CN (20 mL) was added DIPEA (1.2 mL, 7.2 mmol) drop-wise at 0°C. The mixture was stirred at 0°C for 10 min and Intermediate 1 (806 mg, 1.8 mmol) was added. The reaction mixture was stirred at r.t. overnight under N₂ atmosphere. TLC showed the reaction was complete. The mixture was concentrated under vacuum to remove the solvent while keeping the temperature below 40°C. The residue was purified by prep-HPLC (Cl 8,0 ~ 90 %

acetonitrile in H₂0 with 0.1 % formic acid) to give 2-(A-((((3R,4S,5S,6R)-S-me\\\oxy-4- ((2i?,3i?)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)piperazin-l-yl)acetic acid (320 mg) as a white solid. LC-MS (ESI) m/z (M+l): 453.

Step 4: Preparation of sodium 2-(4-((((3/?, V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)piperazin-l- yl)acetate

[0240] To a solution of 2-(4-((((3R, 4S,5S, 6/?)-5-methoxy-4-((2f?, 3f?)-2-methyl-3-(3- methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)piperazin- 1 -yl)acetic acid (300 mg, 0.67 mmol) in MeCN (2 mL) was added 0.1 N NaOH solution (5.9 mL, 0.0.59 mmol) dropwise at -60°C with stirring. The mixture was lyophilized to give sodium 2-(4- ((((37?, 4S, 5S, 6R)-5-methoxy-4-((2R, 3i?)-2-methyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 - oxaspiro[2.5]octan-6-yl)oxy)carbonyl)piperazin-l-yl)acetate (260 mg) as a white solid. LC-MS (ESI) m/z (M+l): 453. 1H NMR (400 MHz, DMSO-d6 ) d 5.36 (s, 1H), 5.19 (s, 1H), 3.53 (d, J = 10.8 Hz, 1H), 3.28 (s, 4H), 2.85 (s, 1H), 2.68 (s, 2H), 2.57 (s, 2H), 2.39 (d, J = 24.7 Hz, 4H), 2.17 (s, 2H), 1.94 (s, 1H), 1.78 (d, J = 11.7 Hz, 2H), 1.70 (s, 4H), 1.61 (s, 3H), 1.07 (s, 3H),

1.01 (d, J = 13.3 Hz, 1H).

Example 32: Preparation of sodium 2-(4-((((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)methyl)-lH-l,2,3-triazol-l-yl)acetate

Step 1: Preparation of ethyl 2-(4-(((tert-butoxycarbonyl)amino)methyl)-lH-l,2,3-triazol- l-yl)acetate

Boc

CU(OAC)₂, Sodium ascorbate

t-BuOH/H₂0

[0241] A mixture of tert-butyl prop-2-yn-l-ylcarbamate (600 mg, 3.86 mmol), ethyl 2- azidoacetate (648 mg, 5.02 mmol), Cu(OAc)₂ (230 mg, 1.15 mmol) and sodium ascorbate (384 mg, 1.93 mmol) in t-BuOH/H₂0 (10 mL, 1/1, v/v) was stirred at 30°C overnight. TLC showed the reaction was complete. The t-BuOH was removed under vacuum. The aqueous layer was extracted with ethyl acetate (5 mL x 2). The combined organic layers were dried over anhydrous Na₂S0 and concentrated. The residue was purified by column chromatography (silica gel, 10 to 50 % ethyl acetate in petroleum ether) to give ethyl 2-(4-(((tert- butoxycarbonyl)amino)methyl)-lH-l,2,3-triazol-l-yl)acetate (850 mg). LC-MS (ESI) found: 285[M+l]⁺.

Step 2: Preparation of 2-(4-(((tert-butoxycarbonyl)amino)methyl)-lH-l,2,3-triazol-l- yl)acetic acid

[0242] To a mixture of ethyl 2-(4-(((tert-butoxycarbonyl)amino)methyl)-lH-l,2,3-triazol-l- yl)acetate (500 mg, 1.75 mmol) in THF/MeOH (6 mL, 1/1, v/v) was added LiOH (2.6 mL, 5.2 mmol, 2M). The reaction mixture was stirred at room temperature for 1 h. TLC showed the reaction was complete. The organic volatiles were removed under vacuum. The aqueous layer was adjusted pH to 3 with diluted H₂S0₄ and extracted with CHCl₃/i-PrOH (5 ml x 10, 3/1, v/v). The combined organic layers were dried over Na₂S0₄ and concentrated to give 2-(4- (((tert-butoxycarbonyl)amino)methyl)-lH-l,2,3-triazol-l-yl)acetic acid (450 mg) as a colorless oil. LC-MS(ESI) found: 257 [M+l]⁺.

Step 3: Preparation of 2-(4-(aminomethyl)-lH-l,2,3-triazol-l-yl)acetic acid

[0243] To a solution of 2-(4-(((tert-butoxycarbonyl)amino)methyl)-lH-l,2,3-triazol-l- yl)acetic acid (450 mg, 1.75 mmol) in DCM (4.5 mL) was added TFA (1.5 mL). After the completion of addition, the reaction mixture was stirred at room temperature overnight. LCMS showed the reaction was complete. The mixture was concentrated to give 2-(4-(aminomethyl)- lH-l,2,3-triazol-l-yl)acetic acid TFA salt (800 mg) as a brown oil. LC-MS (ESI) found: 157 [M+l] ⁺. Step 4: Preparation of sodium 2-(4-((((((J ?, A,5A,6 ?)-5-methoxy-4-((2 ?,J ?)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)methyl)-lH-l,2,3-triazol-l-yl)acetate

[0244] To a solution of 2-(4-(aminomethyl)-lH-l,2,3-triazol-l-yl)acetic acid (105 mg, 0.67 mmol) in CH3CN (3 mL) and water (lmL) was added DIPEA (0.29 mL, 1.78 mmol) drop-wise at 0°C. The mixture was stirred at 0°C for 10 min and Intermediate 1 (200 mg, 0.45 mmol) was added. The reaction mixture was stirred at r.t. overnight. TLC showed the reaction was complete. The mixture was concentrated under vacuum to remove the solvent while keeping the temperature below 40°C. The residue was purified by prep-HPLC (Cl 8, 0 ~ 90 % MeCN in H₂0 with 0.1 % formic acid) to give 2-(4-((((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl- 3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)methyl)- lH-l,2,3-triazol-l-yl)acetic acid (70 mg, 0.15 mmol). The acid was dissolved in MeCN (1 mL) and aq. NaOH (1.4 mL, 0.1M) was added at 0°C. After lyophilization, sodium 2-(4- {[({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl]-l- oxaspiro[2.5]octan-6-yl]oxy}carbonyl)amino]methyl}-lH-l,2,3-triazol-l-yl)acetate (70 mg) was obtained as a white solid. LC-MS (ESI) m/z(M+l): 465. 1H NMR (400 MHz, DMSO-d6 ) d 8.53 (s, 1H), 7.64 (d, J = 27.7 Hz, 1H), 5.31 (s, 1H), 5.20 (t, J = 7.2 Hz, 1H), 4.54 (s, 2H), 4.18 (t, J = 13.1 Hz, 2H), 3.53 (d, J = 10.8 Hz, 1H), 3.29 (s, 3H), 2.83 (dd, J = 7.7, 4.4 Hz, 1H), 2.58 - 2.53 (m, 2H), 2.22 - 2.14 (m, 2H), 1.87 (dd, J = 26.7, 15.9 Hz, 3H), 1.71 (s, 4H), 1.61 (s, 3H), 1.10 - 1.01 (m, 4H).

Example 33: Preparation of sodium 2-(4-((((((3 R,4S,5S,6R)-5-metho\y-4-((2R,3 R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)(methyl)amino)methyl)-lH-l,2,3-triazol-l-yl)acetate

Step 1: Preparation of ethyl 2-(4-((methylamino)methyl)-lH-l,2,3-triazol-l-yl)acetate

[0245] The mixture of methyl(prop-2-yn-l-yl)amine (300 mg, 4.34 mmol), ethyl 2- azidoacetate (728 mg, 5.64 mmol), Cu(OAc)₂ (259 mg, 1.30 mmol) and sodium ascorbate (432 mg, 2.17 mmol) in t-BuOH/H₂0 (6 mL, 1/1, v/v) was stirred at 30°C overnight. TLC showed the reaction was complete. The solvent was removed under vacuum. The residue was purified by flash chromatography (silica gel, 5% ~ 20% MeOH in DCM) to afford ethyl 2-(4- ((methylamino)methyl)-lH-l,2,3-triazol-l-yl)acetate (450 mg) as a brown solid. LC-MS (ESI) m/z(M+l): 199.

Step 2: Preparation of 2-(4-((methylamino)methyl)-lH-l,2,3-triazol-l-yl)acetic acid

[0246] To a mixture of ethyl 2-{4-[(methylamino)methyl]-lH-l,2,3-triazol-l-yl}acetate (450 mg, 2.27 mmol) in THF/MeOH (6 mL, v/v = 1/1) was added LiOH (3.4 mL, 6.8 mmol, 2M). The reaction mixture was stirred at room temperature for 1 h. TLC showed the reaction was complete. The reaction mixture was adjusted pH to 3 with diluted H₂S0_4. The volatile was removed under vacuum to give 2-{4-[(methylamino)methyl]-lH-l,2,3-triazol-l-yl}acetic acid (-386 mg) in water which was used directly in the next step without further purification. LC- MS(ESI) found: 171 [M+l]⁺.

Step 3: Preparation of sodium 2-(4-((((((J ?, A,5A,6 ?)-5-methoxy-4-((2 ?,J ?)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)(methyl)amino)methyl)-lH-l,2,3-triazol-l-yl)acetate

[0247] To a mixture of 2-(4-((methylamino)methyl)-lH-l,2,3-triazol-l-yl)acetic acid (171 mg, 1.01 mmol) in MeCN (6 mL) and water (2 mL) was added DIPEA (0.22 mL, 1.34 mmol) drop-wise at 0°C. The mixture was stirred at 0°C for 10 min and Intermediate 1 (300 mg, 0.67 mmol) was added. The reaction mixture was stirred at r.t. overnight. TLC showed the reaction was complete. The mixture was concentrated under vacuum to remove the solvent while keeping the temperature below 40°C. The residue was purified by prep-HPLC (Cl 8, 0 ~ 90 % MeCN in H₂0 with 0.1 % formic acid) to give 2-(4-((((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)- 2 -methyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6- yl)oxy)carbonyl)(methyl)amino)methyl)-lH-l,2,3-triazol-l-yl)acetic acid (240 mg). It was dissolved in MeCN (2 mL) and added aq. NaOH (4.5 mL, 0.1M) at 0°C. After lyophilization, sodium 2-(4-((((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)(methyl)amino)methyl)-lH-l,2,3- triazol-l-yl)acetate (240 mg) was obtained as a white solid. LC-MS (ESI) m/z(M+l): 479. 1H NMR (400 MHz, DMSO-d6 ) d 7.81 (d, J = 34.3 Hz, 1H), 5.39 (s, 1H), 5.19 (t, J = 7.4 Hz, 1H), 4.64 - 4.34 (m, 4H), 3.56 (d, J = 11.1 Hz, 1H), 3.29 (s, 3H), 2.84 (d, J = 7.0 Hz, 4H), 2.59 - 2.51 (m, 3H), 2.26 - 2.08 (m, 2H), 2.05 - 1.90 (m, 1H), 1.84 - 1.59 (m, 8H), 1.16 - 0.87 (m, 4H).

Example 34: Preparation of sodium (J/?,5/?)-3,5-dihydroxy-7-(((((J/?, V,5.V,6/?)-5- methoxy-4-((2 ?,J ?)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-

6-yl)oxy)carbonyl)amino)heptanoate

Step 1: Preparation of (.?/?, 5/?)-7-amino-3,5-dihydroxyheptanoic acid

[0248] To a mixture of tert-butyl 2-((4i?,6 ?)-6-(2-aminoethyl)-2,2-dimethyl-l,3-dioxan-4- yl)acetate (1.35 g, 4.9 mmol) in DCM (30 mL) was slowly added TFA (10 mL). The resulting mixture was stirred at r.t. for 2h. TLC showed the reaction was complete. The mixture was concentrated to give crude (.V)-2-(pyrrol i di n-3 -yl )propan-2-ol (1.3 g), which was used in the next step without further purification. LC -MS (ESI) m/z (M-l): 178.

Step 2: Preparation of ( S,6A)-6-(2-aminoethyl)-4-hydroxytetrahydro-2H-pyran-2-one

[0249] A mixture of (3i?,5i?)-7-amino-3,5-dihydroxyheptanoic acid (1.3 g) in aq. NaHCO-, solution (50 mL) was stirred at r.t. for lh, and extracted with DCM/MeOH (30 mL x 2). The combined organic layers was dried over anhydrous Na₂S0₄ and concentrated under vacuum to give a yellow residue, which was used in the next step without further purification. LC-MS (ESI) m/z (M-l): 160.

Step 3: Preparation of (3/?,5/?)-3,5-dihydroxy-7-(((((3/?, V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)heptanoic acid

[0250] To a solution of (7S',7V)-6-(2-ami noethyl )-4-hydroxytetrahydro-2H-pyran-2-one (310 mg, 1.9 mmol) in DMF (10 mL) was added DIPEA (0.6 mL, 3.8 mmol) drop-wise at 0°C. The mixture was stirred at 0°C for 10 min, and Intermediate 1 (447 mg, 1.1 mmol) was added. The reaction mixture was stirred at r.t. overnight under a N₂ atmosphere. TLC showed the reaction was complete. The mixture was concentrated, and the residue was purified by prep-HPLC (08,0 ~ 90 % acetonitrile in H₂0 with 0.1 % formic acid) to give (3R, 5/ )-3,5-dihydroxy-7- (((((37?, 4S, 5S, 6R)-5 -m ethoxy-4 -((2/?, 37?)-2-methyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 - oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)heptanoic acid (300 mg) as a white solid. LC-MS (ESI) m/z (M+l): 486.

Step 4: Preparation of sodium (3/?,5/?)-3,5-dih droxy-7-(((((3/?,4S,5A,6/?)-5-methoxy-4-

((2/?,3/?)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)heptanoate

[0251] To a mixture of (3R, 5/ )-3,5-dihydroxy-7-[( { [(3/ ,7V,5,.y, ri/ )-5-methoxy-4-[(2R.,3R.)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6- yl]oxy}carbonyl)amino]heptanoic acid (210 mg, 0.43 mmol) in MeCN (lmL) was added 0.1 M NaOH (3.8 mL) at -60 °C with stirring. The solution was lyophilized to give sodium

(3/?,5/?)-3,5-dihydroxy-7-[({ [(3/?, 4S,5S, 6/?)-5-methoxy-4-[(2/?)-2-methyl-3-(3-methylbut-2-en- l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)amino]heptanoate (215 mg) as a white solid. LC-MS (ESI) m/z (M+l): 486. 1H NMR (400 MHz, DMSO-d6 ) d 7.59 (s, 1H),

7.08 (s, 1H), 5.19 (t, 7= 7.4 Hz, 1H), 4.56 (d, J= 2.6 Hz, 1H), 4.30 (m, 1H), 4.21 (s, 1H), 3.71 (s, 1H), 3.37 (d, J= 2.4 Hz, 1H), 3.30 (s, 3H), 3.14 (dd, J= 9.3, 4.2 Hz, 2H), 2.81 (d, J= 4.4 Hz, 1H), 2.22 - 2.10 (m, 3H), 2.07 - 1.91 (m, 4H), 1.89 - 1.76 (m, 3H), 1.70 (d, 7= 8.3 Hz, 5H), 1.61 (s, 4H), 1.57 - 1.43 (m, 3H), 1.07 (s, 3H), 0.90 - 0.83 (m, 1H). Example 35: Preparation of sodium 3-(3-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)azetidin-l-yl)-2,2-dimethylpropanoate

Step 1: Preparation of 3-bromo-2,2-dimethylpropanoic acid

[0252] To a solution of 3 -hydroxy-2, 2-dimethylpropanoic acid (3.5 g, 30 mmol) and CBr₄ (14.9 g, 45 mmol) in THF (50 mL) was added PPh₃ (11.8 g, 45 mmol) at 0°C. The mixture was stirred at 0°C for 30 min. The reaction was stirred at room temperature overnight under N₂ atmosphere. The reaction mixture was then quenched with water and extracted with DCM. The dichloromethane solution was dried over Na₂S0₄, filtered and concentrated to give crude product. The crude product was purified by chromatography (silica gel, 1 to ~50 % AcOEt in PE) to give 3 -bromo-2, 2-dimethylpropanoic acid (1.3 g) as a colorless oil.

Step 2: Preparation of 3-(3-((tert-butoxycarbonyl)amino)azetidin-l-yl)-2,2- dimethylpropanoic acid

[0253] To a solution of tert-butyl azeti din-3 -ylcarbamate hydrochloride (1.15 g, 5.5 mmol) and K₂C0₃ (1.4 g, 10 mmol) in DMF (20 mL) was added 3 -bromo-2, 2-dimethylpropanoic acid (1.0 g, 5.5 mmol) dropwise at 0°C. The reaction was stirred at room temperature overnight under N₂ atmosphere. TLC showed the reaction was complete. Water was added and the pH of the water layer adjusted to 5~6, then the water layer was extracted with DCM. The

dichloromethane solution was dried over anhydrous Na₂S0 , filtered and concentrated to give crude product. The crude product was purified by column chromatography (silica gel, 1 to -10 % MeOH in DCM) to give 3-(3-((tert-butoxycarbonyl)amino)azetidin-l-yl)-2,2- dimethylpropanoic acid (550 mg) as a white solid. LC-MS (ESI) found: 273 [M+l]⁺.

Step 3: Preparation of 3-(3-aminoazetidin-l-yl)-2,2-dimethylpropanoic acid

trifluoroacetic acid salt

[0254] To a mixture of 3-(3-((tert-butoxycarbonyl)amino)azetidin-l-yl)-2,2- dimethylpropanoic acid (550 mg, 2.0 mmol) in dichloromethane (5 mL) was added

trifluoroacetic acid (3 mL) dropwise at 0°C. The reaction was stirred room temperature for 1 h. TLC showed the reaction was complete. The mixture was concentrated under reduced pressure to give 3-(3-aminoazetidin-l-yl)-2,2-dimethylpropanoic acid trifluoroacetic acid salt (440 mg) as a yellow syrup, which was used in the next step without further purification. LC-MS (ESI) found: 173[M+1]⁺.

Step 4: Preparation of 3-(3-(((((3/?, V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)azetidin- l-yl)-2,2-dimethylpropanoic acid

[0255] 3-(3-aminoazetidin-l-yl)-2,2-dimethylpropanoic acid trifluoroacetic acid salt (440 mg, 1.9 mmol) was coupled with Intermediate 1 (700 mg, l .6mmol) by following the General Procedure for Coupling. The reaction was concentrated under vacuum to remove the solvent while keeping the temperature below 40°C. The residue was diluted with dichloromethane and washed with ammonium acetate buffer (pH 4.0, 20 mL x 2). The combined organic layers was dried with Na₂S0₄, filtered and concentrated to give crude product. The residue was purified by prep-HPLC (Method B: 0.1% NH₄OH) to give 3-(3-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)- 2 -methyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)azetidin-l-yl)-2,2-dimethylpropanoic acid (155 mg) as a white solid. LC-MS (ESI) found: 481 [M+l]⁺.

Step 5: Preparation of sodium 3-(3-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)azetidin-l-yl)-2,2-dimethylpropanoic acid

[0256] To a solution of 3-(3-(((((3i?, S^,,5_<S^,,6/?)-5-methoxy-4-((2i?,3i?)-2-methyl-3-(3- methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)azetidin- 1 - yl)-2,2-dimethylpropanoic acid (155 mg, 0.33 mmol) in acetonitrile (1 mL) was added aq. NaOH (3 mL, 0.3 mmol, 0.1N) dropwise at -20°C. The mixture was lyophilized to give sodium 3 -(3 ~(((((3R, 4S, 5S, 6R)-5-methoxy-4-((2R, 3f?)-2-methyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)azetidin-l-yl)-2,2-dimethylpropanoic acid (160 mg) as a white solid. LC-MS (ESI) found: 481 [M+l]⁺. 1H NMR (400 MHz, DMSO-d6 ) d 7.63 (d, J= 7.5 Hz, 1H), 5.26 (s, 1H), 5.19 (t, J= 7.4 Hz, 1H), 4.06 (dd, J= 14.1, 7.0 Hz, 1H), 3.57 (t, J= 6.4 Hz, 2H), 3.52 (dd, J= 10.8, 2.4 Hz, 1H), 3.27 (s, 3H), 3.00 (dd, J= 14.8, 7.4 Hz,

2H), 2.84 (d, J= 4.2 Hz, 1H), 2.57 (d, J= 4.3 Hz, 1H), 2.16 (dd, J= 13.7, 7.0 Hz, 2H), 1.99 - 1.74 (m, 3H), 1.69 (d, j= 13.4 Hz, 4H), 1.62 (d, j= 7.6 Hz, 4H), 1.08 (s, 3H), 0.99 (s, 8H).

Example 36: Preparation of sodium ( 3R,5R)-3,5-dihydroxy-7-((l-((((3R,4S,5S,6R)-5 - methoxy-4-((2 ?,J ?)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-

6-yl)oxy)carbonyl)azetidin-3-yl)oxy)heptanoate

1. Preparation of tert-butyl 2-((4R, 6 ?)-6-(2-h drox ethyl)-2, 2-dimethyl- 1,3-dioxan- 4-yl)acetate

[0257] tert-Butyl 2-((4R, 6/i)-6-(2-aminoethyl)-2,2-dimethyl- 1 ,3 -dioxan-4-yl (acetate (10 g, 36.6 mmol) was dissolved in 200 mL of 4: 1 H₂0: AcOH and the mixture was placed in ice bath. To this mixture was slowly added aqueous sodium nitrite (150 mL, 2 M) via a syringe pump over 30 min. The reaction was stirred for 4 h at room temperature. TLC showed the reaction was complete. The reaction was extracted with EtOAc (3 ^c 50 mL). The organic layer was dried with anhydrous MgS0₄, filtered, and the filtrate was concentrated via rotary evaporation. The residue was purified by flash chromatography (silica gel, 1 % ~ 10% ethyl acetate in petroleum ether) to give tert-butyl 2-((4f?,6/?)-6-(2-hydroxyethyl)-2,2-dimethyl-l,3-dioxan-4- yl)acetate (1.7 g, 16.9 %) as a colorless oil.

2. Preparation of tert-butyl 2-(( /?,6A)-6-(2-iodoethyl)-2,2-dimethyl-l,3-dioxan-4- yl)acetate

[0258] To a solution of tert-butyl 2-((4R, ri/i)-6-(2-hydroxyethyl)-2,2-dimethyl- 1 ,3-dioxan-4- yl)acetate (1.7 g, 6.2 mmol), PPh₃ (3.3 g, 12.4 mmol) and imidazole (1.0 g, 15 mmol) in THF (30 mL) at 0°C was added I₂ (3.2 g, 12.4 mmol) dropwise. The reaction was stirred at RT overnight. The mixture was washed with water, and the organic layer was separated and washed with brine, dried over anhydrous Na₂S0 and concentrated. The residue was purified by flash chromatography (silica gel, 0 ~ 30 % EtOAc in PE) to give tert-butyl 2-((4R,6S)-6-(2- iodoethyl)-2, 2-dimethyl- 1, 3 -dioxan-4-yl)acetate (1.9 g, 79.8%) as a yellow solid.

3. Preparation of tert-butyl 2-(( /?,6/?)-6-(2-((l-benzhydrylazetidin-3-yl)oxy)ethyl)-

2,2-dimethyl-l,3-dioxan-4-yl)acetate

[0259] A solution of l-benzhydrylazeti din-3 -ol (600 mg, 2.51 mmol) in THF (10 mL) was added sodium hydride (200 mg, 5.02 mmol ) at 0°C under nitrogen atmosphere and the mixture was stirred for 15 mins at 0°C. tert-Butyl 2-((4A, riS)-6-(2-iodoethyl)-2, 2-dimethyl- 1,3- dioxan-4-yl)acetate (1.1 g, 3.01 mmol) was added and the mixture was warmed to room temperature and stirred for 18 hours. TLC showed the reaction was complete. Water (20 mL) was added and the mixture was extracted with ethyl acetate (30 mL). The organic extract was washed with brine, dried over anhydrous Na₂S0₄, and evaporated in vacuo. The residue was purified by flash chromatography (silica gel, 1 % ~ 50 % ethyl acetate in petroleum ether) to give tert-butyl2-((4i?,di?)-6-(2-((l-benzhydrylazetidin-3-yl)oxy)ethyl)-2,2-dimethyl-l,3-dioxan- 4-yl)acetate (200 mg) as a colorless oil. LC-MS (ESI) m/z (M+l): 496. 4. Preparation of (.?/?, 5/?)-7-(azetidin-3-yloxy)-3,5-dihydroxyheptanoic acid

[0260] To a mixture of tert-butyl 2-[{4R, 6R)-6-{2-\ [\ -(diphenyl methyl )azetidin-3- yl]oxy}ethyl)-2,2-dimethyl-l,3-dioxan-4-yl]acetate (200 mg, 0.4 mmol) and Pd/C (100 mg) in THF (3 mL) was added TFA (5 mL). After the air was replaced by hydrogen three times, the reaction mixture was stirred at room temperature for 16 hrs. The resulting mixture

was concentrated to give crude product, which was used in the next step without further purification. LC-MS (ESI) m/z (M+l): 234.

5. Preparation of (.?/?, 5/?)-7-(azetidin-3-yloxy)-3,5-dihydroxyheptanoic acid

[0261] To a solution of (3R, 5//)-7-(azetidin-3-yloxy)-3,5-dihydroxyheptanoicacid (50 mg, 0.21 mmol) in MeCN (5 mL) was added DIPEA (0.2 mL) dropwise at 0 °C. The mixture was stirred at 0 °C for lOmin, and (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut- 2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl (4-nitrophenyl) carbonate (Intermediate 1, 95 mg, 0.21 mmol) was added. The reaction mixture was stirred at RT overnight under N₂ atmosphere. The mixture was concentrated under vacuum to remove the solvent while keeping the temperature below 40 °C. The residue was purified by prep-HPLC (Cl 8,0 ~ 90 % acetonitrile in H₂0 with 0.1 % NH₃Ή₂0) to give (3R, 5/i)-3,5-dihydroxy-7-{ [ 1 - ({[(3i?,4ri',5ri',6/?)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl]-l- oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3-yl]oxy}heptanoic acid (22mg) as a colorless oil. LC-MS (ESI) m/z (M+l): 542.

6. Preparation of sodium (3/?,5/?)-3,5-dihydroxy-7-((l-((((3/?, V,5,V,6/?)-5-methoxy-

4-((2 ?,J ?)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)oxy)heptanoate

[0262] To a mixture of (3R, 5/i)-3,5-dihydroxy-7- { [ l -( J [(3/^-/.S',5.S',/ /^)-5-methoxy-4- [( 2R , 3f?)-2-methyl-3-(3 -methylbut-2-en-l -yl)oxiran-2-yl]- 1 -oxaspiro[2.5]octan-6- yl]oxy}carbonyl)azetidin-3-yl]oxy}heptanoic acid (22mg, 0.04 mmol) in MeCN (1 mL) was added 0.1N NaOH (0.36 mL) at -60°C with stirring. After lyophilization, (3R,4S,5S,6R)-5- methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6- yl3-{[(3f?,5f?)-3,5-dihydroxy-7-oxo-7-(sodiooxy)heptyl]oxy}azetidine-l-carboxylate

(2lmg) was obtained as a colorless oil. LC-MS (ESI) m/z (M-l): 540. 1H NMR (400 MHz, DMSO-d6) d 5.30 (s, 1H), 5.19 (s, 1H), 4.73 (s, 1H), 4.24 (s, 1H), 4.12 - 4.01 (m, 2H), 3.68 (s, 4H), 3.54 - 3.49 (m, 1H), 3.28 (s, 3H), 3.10 (d, J = 7.6 Hz, 1H), 2.89 (d, J = 8.1 Hz, 1H), 2.84

(d, J = 4.3 Hz, 1H), 2.67 (s, 1H), 2.56 (d, J = 4.1 Hz, 2H), 2.33 (s, 1H), 2.17 (d, J = 7.1 Hz, 2H),

1.98 (m, 2H), 1.81 - 1.72 (m, 4H), 1.71 (s, 3H), 1.61 (s, 4H), 1.53 (s, 1H), 1.51 - 1.40 (m, 2H),

1.40 - 1.33 (m, 4H), 1.27 (m, 3H), 1.07 (s, 3H), 1.01 (d, J = 22.2 Hz, 1H). Example 37: Preparation of sodium 2-(6-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2,6- diazaspiro [3.3] heptan-2-yl)acetate

1. Preparation of tert-butyl 6-(2-(tert-butoxy)-2-oxoethyl)-2,6- diazaspiro [3.3] heptane-2-carboxylate

[0263] A mixture of tert-butyl 2,6-diazaspiro[3.3]heptane-2-carboxylate (1.06 g, 5.35 mmol), tert-butyl 2-bromoacetate (1.03 g, 5.28 mmol) and Cs₂C0₃ (3.49 g,l0.7 mmol) in DMF (10 mL) was stirred at 50°C for l6h. TLC showed the reaction was complete. The slurry was diluted with EA, and washed with water and brine. The combined organic layers were dried over anhydrous Na₂S0₄, concentrated under reduced pressure, and purified by flash

chromatography to give tert-butyl 6-(2-(tert-butoxy)-2-oxoethyl)-2,6-diazaspiro[3.3]heptane-2- carboxylate (45 mg) as a white solid. LC-MS (ESI) found: 313 [M+H] ⁺

2. Preparation of 2-(2,6-diazaspiro[3.3]heptan-2-yl)acetic acid

TFA salt

Boa

TFA, DCM

[0264] To a solution of tert-butyl 6-(2-(tert-butoxy)-2-oxoethyl)-2,6-diazaspiro[3.3]heptane- 2-carboxylate (345 mg, 1.10 mmol) in DCM (2 mL) was added TFA (1 mL). After the completion of addition, the reaction mixture was stirred at room temperature for 3 hours. TLC showed the reaction was complete, and the mixture was concentrated to give crude 2-(2,6- diazaspiro[3.3]heptan-2-yl)acetic acid TFA salt (1.3 g) as a brown oil. LC-MS (ESI) found: 157 [M+l] ⁺.

3. Preparation of 2-(6-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2,6- diazaspiro [3.3] heptan-2-yl)acetic acid

TFA salt

Intermediate 1

[0265] Tinder N₂ atmosphere, to a solution of 2-(2,6-diazaspiro[3.3]heptan-2-yl)acetic acid 2,2,2-trifluoroacetate salt (crude, 1.3 g, 1.06 mmol), and diisopropyl ethyl amine (650 mg, 5.0 mmol) in CH₃CN (5 mL) was added a solution of (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl (4-nitrophenyl) carbonate (Intermediate 1, 490 mg, 1.09 mmol) in C¾CN (1 mL) dropwise at 0°C, followed by addition of DMAP (5 mg, 0.067 mmol). Then the mixture was stirred at r.t. for 16 h. TLC showed the reaction was complete. The reaction mixture was concentrated and purified by prep-HPLC (Method A, ¾0 (0.1% FA)/ C¾CN) to give 2-(6-((((3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)-2,6-diazaspiro[3.3]heptan-2-yl)acetic acid (138 mg) as a colorless oil. LC-MS (ESI) found: 465 [M+l] ⁺.

4. Preparation of sodium 2-(6-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2,6- diazaspiro [3.3] heptan-2-yl)acetate

[0266] To a solution of 2-(6-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2,6- diazaspiro[3.3]heptan-2-yl)acetic acid (80 mg, 0.174 mmol) in CH₃CN (1 mL) was added 0.1 N NaOH (1.55 mL, 0.155 mmol) solution at -60°C with stirring. After lypholization, sodium 2-(6- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2,6-diazaspiro[3.3]heptan-2-yl)acetate (80 mg) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6 ) d 5.27 (s, 1H), 5.19 (s, 1H), 3.97 (s, 4H), 3.63 (s, 4H), 3.51 (dd, J = 11.0, 2.4 Hz, 1H), 3.27 (d, J = 6.2 Hz, 3H), 3.11 (s, 2H), 2.85 (d, J = 4.3 Hz, 1H), 2.56 (dd, J = 11.7, 5.3 Hz, 2H), 2. l7 (s, 2H), 1.97 - 1.87 (m, 1H), 1.80— 1.67 (m, 6H), 1.61 (s, 3H), 1.05 (d, J = 17.9 Hz, 4H).

Example 38: Preparation of sodium 2-(4-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2- oxopiperazin-l-yl)acetate

1. Preparation of tert-butyl 4-(2-(tert-butoxy)-2-oxoethyl)-3-oxopiperazine-l- carboxylate

TFA

^ ° O

OH

[0267] To a solution of tert-butyl 3-oxopiperazine-l-carboxylate (3.9 g, 19.5 mmol) in DMF (10 mL), were added tert-butyl 2-bromoacetate (2.84 mL, 19.5 mmol) and sodium hydride

(0.856 g, 21.4 mmol, 60% in mineral oil). The reaction mixture was stirred at 25 °C for 16 hours. TLC showed the reaction was complete. The slurry was diluted with EA, and washed with water and brine. The combined organic layers were dried over anhydrous Na₂S0₄, concentrated under reduced pressure and purified by flash chromatography to give tert-butyl 4- (2-(tert-butoxy)-2-oxoethyl)-3-oxopiperazine-l-carboxylate (3.89 g) as a white solid. LC-MS (ESI) found: 315 [M+H] ⁺.

2. Preparation of 2-(2-oxopiperazin-l-yl)acetic acid

[0268] To a solution of tert-butyl 4-[2-(tert-butoxy)-2-oxoethyl]-3-oxopiperazine-l- carboxylate (310 mg, 0.98 mmol) in DCM (2 mL) was added TFA (1 mL). After addition, the reaction mixture was stirred at room temperature for 3 hours. TLC showed the reaction was reaction was complete. The reaction mixture was then concentrated to give crude 2-(2- oxopiperazin-l-yl)acetic acid (TFA salt, 500 mg) as a brown oil, which was directly used in the next reaction step. LC-MS (ESI) found: 159 [M+l]⁺.

3. Preparation of 2-(4-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2-oxopiperazin- l-yl)acetic acid

Intermediate 1

[0269] To a solution of 2-(2-oxopiperazin-l-yl)acetic acid 2,2,2-trifluoroacetate salt (500 mg, 0.98 mmol) and N,N-diisopropylethylamine (707 mg, 5.5 mmol) in CH₃CN (3 mL) under a N₂ atmosphere was added a solution of (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl (4-nitrophenyl) carbonate

(Intermediate 1, 490 mg, 1.09 mmol) in CH₃CN (1 mL) dropwise at 0°C, followed by addition of DMAP (5 mg, 0.067 mmol). The mixture was then stirred at r.t. for 16 hrs. TLC showed the reaction was complete. The reaction mixture was then concentrated and purified by prep-HPLC (Method A, H₂0 (0.1% FA)/ CH₃CN) to give 2-(4-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2- oxopiperazin-l-yl)acetic acid (150 mg) as a colorless oil. LC-MS (ESI) found: 467 [M+l]⁺.

4. Preparation of sodium 2-(4-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2- oxopiperazin-l-yl)acetate

[0270] To a solution of 2-(4-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2-oxopiperazin- 1 - yl)acetic acid (103 mg, 0.221 mmol) in CH₃CN (1 mL) was added 0.1 N NaOH (1.98 mL, 0.198 mmol) at -60 °C with stirring. After lyophilization, sodium 2-(4-((((3R,4S,5S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)-2-oxopiperazin-l-yl)acetate (105 mg) was obtained as white solid. 1H NMR (400 MHz, DMSO-d6 ) d 5.40 (s, 1H), 5.19 (t, J= 7.4 Hz, 1H), 3.89 (d, J= 21.0 Hz, 2H), 3.69 - 3.49 (m, 5H), 3.35 (s, 3H), 3.29 (s, 3H), 2.86 (d, J= 4.3 Hz, 1H), 2.58 (d, J= 4.3 Hz, 1H), 2.17 (dd, j= 13.5, 6.9 Hz, 2H), 1.99 (td, j= 13.5, 4.4 Hz, 1H), 1.85 - 1.77 (m, 2H), 1.73 (d, j =

17.4 Hz, 4H), 1.61 (s, 3H), 1.08 (s, 3H), 1.01 (d, J= 13.6 Hz, 1H).

Example 39: Preparation of sodium 2-((2-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2- azaspiro [3.3] heptan-6-yl)oxy)acetate

equivalent

1. TFA/DCM,

rt, overnight 0.1 M NaOH(aq)

2. Intermediate 1 0 °C to rt

DIPEA, MeCN equivalent

0 °C to rt 6h

1. Preparation of tert-butyl 6-hydroxy-2-azaspiro[3.3]heptane-2-carboxylate

equivalent

[0271] To a solution of tert-butyl 6-oxo-2-azaspiro[3.3]heptane-2-carboxylate (845 mg) in MeOH (20 mL) was added NaBH₄ (152 mg) at 0 °C. The mixture was stirred at r.t. for 6 hrs. TLC showed the reaction was complete. The mixture was concentrated and the residue was purified by column chromatography (silica gel, 1 % ~ 10 % MeOH in DCM) to give tert-butyl 6-hydroxy-2-azaspiro[3.3]heptane-2-carboxylate (853 mg, quantitive) as white solid. 1H NMR (400 MHz, DMSO-d6 ) d 5.00 (d, J = 6.2 Hz, 1H), 3.92 (q, J = 7.0 Hz, 1H), 3.75 (d, J = 21.0 Hz, 4H), 2.37 (ddd, J = 11.8, 6.1, 2.6 Hz, 2H), 1.95 - 1.86 (m, 2H), 1.35 (s, 9H). LC MS: m/z (M+l)+ 214. 2. Preparation of tert-butyl 6-(2-(tert-butoxy)-2-oxoethoxy)-2-azaspiro[3.3]heptane- 2-carboxylate

[0272] To a solution of NaH (60% wt, 80 mg) in anhydrous THF (5 mL) was added a solution of tert-butyl 6-hydroxy-2-azaspiro[3.3]heptane-2-carboxylate (213 mg) in anhydrous THF (5 mL) while cooling the reaction in an ice-bath. After 1 h, tert-butyl 2-bromoacetate was added to the mixture at the same temperature. The mixture was stirred at r.t overnight. TLC showed the reaction was complete. The reaction was quenched with NH₄Cl (aq) and extracted with EA (3 x 20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (silica gel, 1 % ~ 10 % MeOH in DCM) to give tert-butyl 6-(2-(tert-butoxy)-2-oxoethoxy)-2- azaspiro[3.3]heptane-2-carboxylate (236 mg) as a yellow oil. 1H NMR (400 MHz, CDCl₃) d 3.93 (m, 1H), 3.87 (d, J = 8.4 Hz, 4H), 3.85 (s, 2H), 2.54 - 2.42 (m, 2H), 2.21 - 2.14 (m, 2H), 1.47 (s, 9H), 1.42 (s, 9H). LC MS: m/z (M+l)⁺ 328.

3. Preparation of sodium 2-((2-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2- azaspiro [3.3] heptan-6-yl)oxy)acetate

[0273] To a solution of tert-butyl 6-(2-(tert-butoxy)-2-oxoethoxy)-2-azaspiro[3.3]heptane-2- carboxylate (236 mg) in DCM (2.5 mL) was added TFA (2.5 mL), and the resulting mixture was stirred at room temperature overnight. TLC showed the reaction was complete. The solvent was removed under vacuum. The residue was dissolved in MeCN (10 mL), and DIPEA (0.5 mL) was added dropwise while cooling the reaction in an ice-bath. To the resulting solution was added (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2- yl)-l-oxaspiro[2.5]octan-6-yl (4-nitrophenyl) carbonate (Intermediate 1, 280 mg), and the reaction was allowed to warm to r.t. and be stirred at r.t. for 6 hrs. TLC showed the reaction was complete. The reaction mixture was quenched with aqueous NaHCO, (1 M) and extracted with EA (3 x 20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (silica gel, 1 % ~ 10 % MeOH in DCM) and prep-HPLC (Cl 8, 0 ~ 90 % acetonitrile in H₂0 with 0.1 % NH₃Ή₂0) to give 2-((2-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2- en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2-azaspiro[3.3]heptan-6- yl)oxy)acetic acid (100 mg ) as a white solid. LC MS: m/z (M-l) 478. To a solution of 2-((2- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2-azaspiro[3.3]heptan-6-yl)oxy)acetic acid (100 mg) in H₂0/MeCN (v/v, 10: 1, 5 mL) was added aqueous NaOH (0.1 M, 2 mL) at -40 °C. The solvent was lyophilized to give sodium 2-((2-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2- azaspiro[3.3]heptan-6-yl)oxy)acetate (100 mg) as a white solid. 'H NMR (400 MHz, DMSO- d6) d 5.27 (dd, J = 4.2, 2.5 Hz, 1H), 5.19 (td, J = 7.4, 3.9 Hz, 1H), 3.91 (s, 1H), 3.83 (d, J = 17.0 Hz, 4H), 3.54 - 3.47 (m, 2H), 3.27 (s, 3H), 2.84 (d, J = 4.4 Hz, 1H), 2.56 (d, J = 4.3 Hz, 1H), 2.53 (d, J = 6.6 Hz, lH),2.36 (t, J = 9.5 Hz, 2H), 2.17 (q, J = 6.4 Hz, 2H), 2.03 - 1.94 (m, 2H), 1.90 (dd, J = 13.3, 4.6 Hz, 1H), 1.80 - 1.72 (m, 1H), 1.74 (d, J = 11.0 Hz, 1H), 1.70 (s, 3H), 1.61 (s, 3H), 1.07 (s, 3H), 1.04 - 0.96 (m, 1H), 0.94 (d, J = 6.5 Hz, 1H). LC MS: m/z (M- 1) 478.

Example 40: Preparation of sodium 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-3- methylazetidin-3-yl)oxy)acetate

, ,

0 C to rt 6h

1. Preparation of tert-butyl 3-hydroxy-3-methylazetidine-l-carboxylate

[0274] To a solution of 3-methylazetidin-3-ol hydrochloride (500 mg) in DCM (10 mL) was added Et₃N (1.63 g) at r.t. After 5 mins, Boc₂0 (1.77 g) was added, and the reaction was stirred at r.t. for 6hrs. The mixture was then concentrated, and the residue was purified by column chromatography (silica gel, 1 % ~ 10 % MeOH in DCM) to give tert-butyl 3 -hydroxy-3 - methylazetidine-l-carboxylate (757 mg, quantitive) as a yellow oil. LC MS: m/z (M+l)⁺ 188.

2. Preparation of tert-butyl 3-(2-(tert-butoxy)-2-oxoethoxy)-3-methylazetidine-l- carboxylate

g

[0275] To a solution of NaH (60% wt, 80 mg) in anhydrous THF (5 mL) was added a solution of tert-butyl 3 -hydroxy-3 -methylazeti dine- l-carboxylate (187 mg) in anhydrous THF (5 mL) while cooling the reaction in an ice-bath. After 1 h, tert-butyl 2-bromoacetate was added to the mixture at the same temperature. The mixture was then allowed to stir at r.t overnight. The reaction was quenched with NH₄Cl (aq) and extracted with EA (3 x 20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and

concentrated. The residue was purified by column chromatography (silica gel, 1 % ~ 10 % MeOH in DCM) to give tert-butyl 3 -(2-(tert-butoxy)-2-oxoethoxy)-3 -methylazeti dine- 1- carboxylate (220 mg) as a yellow oil. LC MS: m/z (M+l)⁺ 302.

3. Preparation of sodium 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-3- methylazetidin-3-yl)oxy)acetate

[0276] To a solution of tert-butyl 3-(2-(tert-butoxy)-2-oxoethoxy)-3-methylazetidine-l- carboxylate (220 mg) in DCM (2.5 mL) was added TFA (2.5 mL), and the resulting mixture was stirred at room temperature overnight. The solvent was removed under vacuum. The residue was dissolved in MeCN (10 mL), and DIPEA (0.5 mL) was added dropwise while cooling in an ice-bath. To the resulting solution was added (3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6-yl (4- nitrophenyl) carbonate (Intermediate 1, 282 mg), and the reaction was allowed to stir at room temperature for 6 hrs. The reaction mixture was quenched with aqueous NaHC0₃ (1 M) and extracted with EA (3 x 20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (silica gel, 1 % ~ 10 % MeOH in DCM) and prep-HPLC (Cl 8,0 ~ 90 % acetonitrile in ¾0 with 0.1 % NH₃Ή₂O) to give 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut- 2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-3 -methylazeti din-3 - yl)oxy)acetic acid (210 mg) as a white solid. LC MS: m/z (M-l) 474. To a solution of 2-((l- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-3-methylazetidin-3-yl)oxy)acetic acid in

H₂0/MeCN(v/v, 10: 1, 5 mL) was added aqueous NaOH (0.1 M, 0.9 eq) at -40 °C. The solvent was lyophilized to give sodium 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-3-methylazetidin-3- yl)oxy)acetate (220 mg) as a white solid. 1H NMR (400 MHz, DMSO-d6 ) d 5.30 (s, 1H), 5.23 - 5.11 (m, 1H), 3.86 (d, J = 8.9 Hz, 2H), 3.60 (d, J = 8.9 Hz, 2H), 3.54 - 3.49 (m, 3H), 3.28 (s,

3H), 2.84 (d, J = 4.3 Hz, 1H), 2.55 (dd, J = 7.5, 5.3 Hz, 2H), 2.17 (q, J = 7.3 Hz, 2H), 1.93 (s, 1H), 1.77 (dd, J = 14.4, 10.5 Hz, 2H), 1.72 - 1.69 (m, 4H), 1.61 (d, J = 1.2 Hz, 3H), 1.37 (s, 3H), 1.07 (s, 3H), 1.02 (dd, J = 13.7, 3.7 Hz, 1H), 0.94 (d, J = 6.5 Hz, 1H). LC MS: m/z (M-l) 474.

Example 41: Preparation of sodium 2-(4-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)piperazin-l-yl)acetate

Intermediate 1 1. Preparation of tert-butyl 4-(2-(tert-butoxy)-2-oxoethyl)piperazine-l-carboxylate

[0277] To a solution of tert-butyl piperazine- l-carboxylate (1.5 g, 8.05 mmol) in DMF (10 mL), were added tert-butyl 2-bromoacetate (1.68 g, 8.70 mmol) and Cs₂C0₃ (3.49 g, 10.7 mmol). The reaction mixture was stirred at 25 °C for 16 hours. TLC showed the reaction was complete. The slurry was diluted with EA, and washed with water and brine. The combined organic layers were dried over anhydrous Na₂S0₄, concentrated under reduced pressure and purified by flash chromatography to give tert-butyl 4-(2-(tert-butoxy)-2-oxoethyl)piperazine-l- carboxylate (2.08 g) as a white solid. LC-MS (ESI) found: 301 [M+H] ⁺

2. Preparation of 2-(piperazin-l-yl)acetic acid lt

[0278] To a solution of tert-butyl 4-[2-(tert-butoxy)-2-oxoethyl]piperazine- l-carboxylate (230 mg, 0.766 mmol) in DCM (2 mL) was added TFA (1 mL). After the addition, the reaction mixture was stirred at room temperature for 3 hours. TLC showed the reaction was complete. The mixture was concentrated to give crude 2-(piperazin-l-yl)acetic acid TFA salt (500 mg ) as a brown oil. LC-MS (ESI) found: 145 [M+l]⁺.

3. Preparation of 2-(4-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)piperazin-l- yl)acetic acid

Intermediate 1

[0279] Under N₂ atmosphere, to a solution of 2-(piperazin-l-yl)acetic acid 2,2,2- trifluoroacetate salt (110 mg, 0.65 mmol), ethyl diisopropylamine (500 mg, 3.9 mmol) in

CH₃CN (3 mL) was added a solution of (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl (4-nitrophenyl) carbonate

(Intermediate 1, 290 mg, 0.65 mmol) in CH₃CN (1 mL) dropwise at 0°C, followed by adding DMAP (5 mg, 0.067 mmol). Then the mixture was stirred at r.t. for 16 h. The reaction mixture was concentrated and purified by prep-HPLC (Method A, H₂0 (0.1% FA)/ CH₃CN) to give 2- (4-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)piperazin-l-yl)acetic acid (150 mg) as a colorless oil. LC-MS (ESI) found: 453 [M+l]⁺.

4. Preparation of sodium 2-(4-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)piperazin-l- yl)acetate

[0280] To a solution of 2-(4-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)piperazin- 1 -yl)acetic acid (80 mg, 0.18 mmol) in CH₃CN (1 mL) was added 0.1 N NaOH (1.59 mL, 0.159 mmol) solution at -60 °C with stirring. After lyophilization, sodium 2-(4-((((3R,4S,5S,6R)-5-methoxy- 4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)piperazin-l-yl)acetate (82 mg) was obtained as white solid. 1H NMR (400 MHz, DMSO-d6) d 5.36 (s, 1H), 5.19 (t, J= 7.5 Hz, 1H), 3.54 (dd, J= 11.0, 2.5 Hz, 1H), 3.28 (s, 7H), 2.85 (d, j= 4.3 Hz, 1H), 2.66 (s, 2H), 2.58 (dd, j= 7.2, 5.4 Hz, 2H), 2.40 (d, j= 17.4 Hz, 4H), 2.23 - 2.09 (m, 2H), 1.94 (td, 7 = 13.3, 4.3 Hz, 1H), 1.79 (d, 7= 1 1.0 Hz, 2H), 1.71 (s, 4H), 1.61 (s, 3H), 1.08 (s, 3H), 1.02 (d, J= 13.7 Hz, 1H).

Example 42: Preparation of sodium l-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)-lH-pyrazole-4-carboxylate

X TFA

1. Preparation of tert-butyl 3-(methanesulfonyloxy)azetidine-l-carboxylat

[0281] To a solution of tert-butyl 3-hydroxyazetidine-l-carboxylate (1.0 g, 5.77 mmol) and TEA (1.15 g, 11.5 mmol) in DCM (15 mL) was added methanesulfonyl chloride (0.67 mL,

8.66 mmol) at 0 °C. After 2 h, TLC showed the reaction was complete, and the reaction was diluted with ice brine. The organic layer was separated, dried over anhydrous MgS0₄ and concentrated to give tert-butyl 3-(methanesulfonyloxy)azetidine-l-carboxylate (1.2 g) as a colorless oil. LC-MS(ESI) found: 196 [M+H-56]⁺

2. Preparation of methyl l-{l-[(tert-butoxy)carbonyl]azetidin-3-yl}-lH-pyrazole-4- carboxylate

[0282] To a mixture of tert-butyl 3-(methanesulfonyloxy)azetidine-l-carboxylate (750 mg, 2.98 mmol) in DMF (10 mL) was added K₂C0₃ (1.2 g, 8.95 mmol) and methyl lH-pyrazole-4- carboxylate (376. 4 mg, 2.98 mmol). The reaction was stirred at 100 °C overnight. The mixture was washed with water and extracted with EA. The organic layer was dried over anhydrous Na₂S0₄ and concentrated under reduced pressure to give methyl l-{ l-[(tert- butoxy)carbonyl]azetidin-3-yl}-lH-pyrazole-4-carboxylate (550 mg). LC-MS (ESI) found: 236 [M+H-56]⁺.

3. Preparation of l-{l-[(tert-butoxy)carbonyl]azetidin-3-yl}-lH-pyrazole-4- carboxylic acid

[0283] To a stirred suspension of methyl l-{ l-[(tert-butoxy)carbonyl]azetidin-3-yl}-lH- pyrazole-4-carboxylate (550 mg, 1.95 mmol) in THF (10 mL) and H₂0 (5 mL) was

added LiOH (93.65 mg, 3.91 mmol). The reaction mixture was stirred at r.t. overnight. The reaction mixture was concentrated to remove the solvent, and 5 mL of water and 5 mL of EtOAc were added. The water layer was adjusted to pH = 3 with 1N HC1 and then extracted with DCM. The organic layer was concentrated to give l-{ l-[(tert-butoxy)carbonyl]azetidin-3- yl}-lH-pyrazole-4-carboxylic acid (430 mg) as a colorless oil. LC-MS(ESI) found: 268

[M+l]⁺.

4. Preparation of l-(azetidin-3-yl)-lH-pyrazole-4-carboxylic acid trifluoroacetic acid salt

X TFA

[0284] To a mixture of l-{ l-[(tert-butoxy)carbonyl]azetidin-3-yl}-lH -pyrazole-4- carboxylic acid (430 mg, 1.60 mmol) in DCM (2 mL) was added TFA (1 mL) dropwise at 0 °C. The reaction was stirred at room temperature for 1 h. The mixture was concentrated under reduced pressure to give l-(azetidin-3-yl)-lH-pyrazole-4-carboxylic acid trifluoroacetic acid salt (210 mgas a yellow syrup, which was directly used in the next step without

purification. LC-MS (ESI) found: 168 [M+l]⁺.

5. Preparation of l-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)- lH-pyrazole-4-carboxylic acid

[0285] To a solution of l-(azetidin-3-yl)-lH-pyrazole-4-carboxylic acid (210 mg, 1.25 mmol) in MeCN (10 mL) was added DIPEA (638.15 mg, 5.03 mmol) dropwise at 0 °C. The mixture was stirred at 0 °C for 10 min, and (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl (4-nitrophenyl) carbonate

(Intermediate 1, 562.12 mg, 1.26 mmol) was added at the same temperature. The reaction was then stirred at room temperature overnight under N₂ atmosphere. The mixture was then concentrated under vacuum to remove the solvent while keeping external temperature below 40°C. The residue was diluted with dichlorom ethane and washed with ammonium acetate buffer (pH 4.0, 20 mL). The dichloromethane solution was dried over anhydrous Na₂S0₄, filtered and concentrated to give the crude product. The residue was purified by column chromatography on silica gel (dichloromethane: methanol = 100 : 1 to 10 : 1) and further purified by preparative HPLC (C18, 0 ~ 90 % acetonitrile in H₂0 with 0.1 % NH₃ H₂0) to give l-[l-({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl]- l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3-yl]-lH-pyrazole-4-carboxylic acid (230 mg) as a white solid. LC-MS (ESI) found: 476 [M+l]⁺.

6. Preparation of sodium l-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)- lH-pyrazole-4-carboxylate

[0286] To a solution of l-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-lH- pyrazole-4-carboxylic acid (230 mg, 0.48 mmol) in acetonitrile (1 mL) was added 5 mL of 0.1 N aq. NaOH solution at -50 °C. The mixture was lyophilized overnight to give sodium l-(l- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-lH-pyrazole-4-carboxylate (230 mg) as a white solid. LC-MS (ESI) found: 476 [M+l]⁺. 1H NMR (400 MHz, DMSO-d6 ) d 8.07 (d, ./ = 2.6 Hz, 1H), 7.72 (s, 1H), 5.34 (s, 1H), 5.25 (dd, J= 9.3, 3.9 Hz, 1H), 5.18 (t, J= 7.4 Hz, 1H), 4.31 (t, J= 8.4 Hz, 2H), 4.18 (s, 2H), 3.55 (dd, 7= 11.0, 2.6 Hz, 1H), 3.30 (s, 3H), 2.84 (d, J = 4.3 Hz, 1H), 2.55 (dd, 7= 13.6, 5.3 Hz, 2H), 2.17 (s, 2H), 2.03 - 1.89 (m, 1H), 1.80 (dd, 7 = 19.7, 10.8 Hz, 3H), 1.70 (s, 3H), 1.60 (s, 3H), 1.08 (s, 3H), 1.03 (d, J= 13.2 Hz, 1H).

Example 43: Preparation of sodium 5-((((((J ?, »V,5»V,6 ?)-5-methoxy-4-((2 ?,J ?)-2-methyl-

3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)methyl)picolinate

1. Preparation of sodium 5-(aminomethyl)picolinate

[0287] To a solution of 5-cyanopicolinic acid (200 mg, 1.35 mmol) in MeOH (2 mL) and 1M NaOH (2 mL) was added Pd(OH)₂/C (100 mg). Then the reaction was stirred under hydrogen at r.t. overnight. TLC showed the reaction was complete. The reaction mixture was filtered and concentrated under vacuum to give crude sodium 5-(aminomethyl)picolinate (280 mg, crude) as brown oil, which was used in the next step without further purification. LC-MS (ESI) found: l53[M+l]⁺.

2. Preparation of 5-((((((3/?, V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)methyl)picolinic acid

[0288] To a solution of sodium 5-(aminomethyl)picolinate (280 mg, crude) in MeCN/H₂0 (2 mL /2 mL) was added (3f?, ,S^,,5S^,, d/?)-5-methoxy-4-((2f?,3f?)-2-methyl-3-(3-methylbut-2-en- l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl (4-nitrophenyl) carbonate (Intermediate 1, 300 mg, 0.68 mmol), and the reaction was then stirred at r.t. overnight. TLC showed the reaction was complete. The resulting mixture was concentrated to give a residue, which was purified by prep-HPLC (Method B: 0.1% NH₄OH) to afford 5-((((((3R,4S,5S, 6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)methyl)picolinic acid (140 mg) as a colorless oil. LC-MS (ESI) found: 461 [M+l]⁺.

3. Preparation of sodium 5-((((((3/?, V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)methyl)picolinate

[0289] To a solution of 5-((((((3f?,4,S^,,5S^,, d/?)-5-methoxy-4-((2f?,3f?)-2-methyl-3-(3- methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)methyl)picolinic acid (140 mg, 0.3 mmol) in MeCN (1.0 mL) was added 0.1 N NaOH solution (2.7 mL, 0.27 mmol) at -60°C with stirring, followed by lyophilization to give sodium 5-((((((3i?,4ri^,,5ri',6/?)-5-methoxy-4-((2i?,3i?)-2-methyl-3-(3- methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)methyl)picolinate (124.7 mg) as a white solid. LC-MS (ESI) found: 46l [M+l]⁺. 1H NMR (400 MHz, DMSO-d6 ) d 8.46 - 8.38 (m, 1H), 7.90 (d, J = 8.0 Hz, 1H), 7.77 (s, 1H), 7.68 (d, J = 7.9 Hz, 1H), 5.33 (s, 1H), 5.20 (s, 1H), 4.25 (d, J = 6.0 Hz, 2H), 3.29 (s, 4H), 2.85 (d, J = 4.3 Hz, 1H), 2.58 (d, J = 4.3 Hz, 1H), 2.19 (dd, J = 13.0, 6.5 Hz, 2H), 1.96 - 1.78 (m, 3H), 1.71 (s, 4H), 1.61 (s, 3H), 1.09 (d, J = 10.1 Hz, 4H). Example 44: Preparation of sodium l-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)-lH-pyrazole-3-carboxylate

1. Preparation of tert-butyl 3-(methanesulfonyloxy)azetidine-l-carboxylate

Boc — N^>— OH Boc -O OMs

[0290] To a solution of tert-butyl 3-hydroxyazetidine-l-carboxylate (1.0 g, 5.77 mmol) and TEA (1.15 g, 11.5 mmol) in DCM (15 mL) was added methanesulfonyl chloride (0.67 mL, 8.66 mmol) at 0 °C. After 2 h, TLC showed the reaction was complete, and the reaction was diluted with ice brine. The organic layer was dried over anhydrous MgS0₄ and

concentrated to give tert-butyl 3-(methanesulfonyloxy)azetidine-l-carboxylate (l.2g) as a colorless oil. LC-MS(ESI) found: 196 [M+H-56]⁺

2. Preparation of l-(l-(tert-butoxycarbonyl)azetidin-3-yl)-lH-pyrazole-3- carboxylate

[0291] To a mixture of tert-butyl 3-(methanesulfonyloxy)azetidine-l-carboxylate (750 mg, 2.98 mmol) in DMF (10 mL) was added K₂C0₃ (1.2 g, 8.95 mmol) and methyl lH-pyrazole- 3-carboxylate (376.4 mg, 2.98 mmol). The reaction was stirred at 100 °C overnight. TLC showed the reaction was complete. The mixture was washed with water and extracted with EA. The organic layer was dried over anhydrous Na₂S0₄ and concentrated under reduced pressure to give methyl l-(l-(tert-butoxycarbonyl)azetidin-3-yl)-lH-pyrazole-3-carboxylate (650 mg). LC-MS (ESI) found: 236 [M+H-56]⁺.

3. Preparation of l-{l-[(tert-butoxy)carbonyl]azetidin-3-yl}-lH-pyrazole-3- carboxylic acid

[0292] To a stirred suspension of methyl l-{ l-[(tert-butoxy)carbonyl]azetidin-3-yl}-lH- pyrazole-3-carboxylate (650 mg, 2.31 mmol) in THF (10 mL) and H₂0 (5 mL) was added LiOH (110.66 mg, 4.62 mmol). The reaction mixture was stirred at r.t. overnight. TLC showed the reaction was complete. The reaction mixture was concentrated to remove the solvent, and 5 mL of water and 5 mL of EtOAc were added. The water layer was adjusted to pH = 3 with 1N HC1 and then extracted with DCM. The organic layer was concentrated to give l-{ l-[(tert- butoxy)carbonyl]azetidin-3-yl}-lH-pyrazole-3-carboxylic acid (470 mg) as a colorless oil. LC- MS(ESI) found: 268 [M+l]⁺.

4. Preparation of l-(azetidin-3-yl)-lH-pyrazole-3-carboxylic acid trifluoroacetic acid salt

[0293] To a mixture of l-{ l-[(tert-butoxy)carbonyl]azetidin-3-yl}-lH -pyrazole-3- carboxylic acid (470 mg, 1.76 mmol) in DCM (2 mL) was added TFA (1 mL) dropwise at 0 °C. The reaction was stirred at room temperature for 1 h. Then the mixture was concentrated under reduced pressure to give l-(azetidin-3-yl)-lH-pyrazole-3-carboxylic acid trifluoroacetic acid salt (347 mg) as a yellow syrup, which was directly used in the next step without further purification. LC-MS (ESI) found: 168 [M+l]⁺.

5. Preparation of l-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)- lH-pyrazole-3-carboxylic acid

[0294] To a solution of l-(azetidin-3-yl)-lH-pyrazole-3-carboxylic acid (347 mg, 1.38 mmol) in MeCN (10 mL) was added DIPEA (890.1 mg, 6.9 mmol) dropwise at 0 °C to pH = 8. The mixture was stirred at 0°C for 10 min and (3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl- 3-(3-methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl 4-nitrophenyl carbonate

(Intermediate 1, 562.12 mg, 1.26 mmol) was added. After addition, the reaction was stirred at room temperature overnight under N₂ atmosphere. TLC showed the reaction was complete. The mixture was then concentrated under vacuum to remove the solvent while keeping external temperature below 40°C. The residue was diluted with dichloromethane and washed with ammonium acetate buffer (pH 4.0, 20 mL). The dichloromethane solution was dried over anhydrous Na₂S0₄, filtered and concentrated to give the crude product. The residue was purified by column chromatography on silica gel (dichloromethane: methanol = 100 : 1 to 10 : 1) and further purified by preparative HPLC (C18, 0 ~ 90 % acetonitrile in H₂0 with 0.1 %

NH₃ H₂0) to give l-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2- en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-lH-pyrazole-3- carboxylic acid (210 mg as a white solid. LC-MS (ESI) found: 476 [M+l]⁺.

6. Preparation of sodium l-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)- lH-pyrazole-3-carboxylate

[0295] To a solution of l-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-lH- pyrazole-3 -carboxylic acid (210 mg, 0.44 mmol) in acetonitrile (1 mL) was added 5 mL of 0.1 N aq. NaOH solution at -50 °C. The mixture was lyophilized overnight to give sodium l-[l- ({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl]-l- oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3-yl]-lH-pyrazole-4-carboxylate (200 mg) as a white solid. LC-MS (ESI) found: 476 [M+l]⁺. 1H NMR (400 MHz, DMSO-d6 ) d 7.77 (s, 1H), 6.45 (s, 1H), 5.35 (s, 1H), 5.24 (d, J= 5.7 Hz, 1H), 5.18 (t, J= 7.4 Hz, 1H), 4.32 (t, J= 8.4 Hz, 2H), 4.24 - 4.15 (m, 2H), 3.55 (dd, 7= 11.0, 2.5 Hz, 1H), 3.31 (s, 3H), 2.84 (d, J= 4.3 Hz, 1H), 2.56 (dd, J = 11.5, 5.3 Hz, 2H), 2.25 - 2.10 (m, 2H), 1.95 (dd, 7= 13.4, 3.9 Hz, 1H), 1.83 (s, 1H), 1.76 (t, J= 14.2 Hz, 2H), 1.70 (s, 3H), 1.60 (s, 3H), 1.08 (s, 3H), 1.03 (d, J= 12.9 Hz,

1H). Example 45: Preparation of sodium 6-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)nicotinate

1. Preparation of tert-butyl 3-(5-bromopyridin-2-yl)-3-hydroxyazetidine-l- carboxylate

1 . isopropyl magnesium chloride,

[0296] To a stirred solution of 5-bromo-2-iodopyridine (5.0 g, 17.6 mmol) in dry THF (30 mL) was added isopropylmagmesium chloride (2.0 M in THF, 9.69 mL, 19.3 mmol) dropwise at -20 °C over a period of 10 min. Then a solution of tert-butyl 3-oxoazetidine-l-carboxylate (3.62 g, 21.13 mmol) in dry THF (10 mL) was added at -20°C to the above reaction mixture. The reaction mixture was stirred at r.t. for 16 hrs under N_2. and the starting material was consumed completely by TLC. The reaction mixture was cooled to -l0°C and quenched with 10% citric acid solution, extracted with EA and washed with NaHCO, (aq.). The organic layer was dried over anhydrous Na₂S0₄ and concentrated in vacuo. The residue was purified by flash chromtography on silica gel eluting with PE/EtOAc 10-20/1 to give tert-butyl 3-(5- bromopyridin-2-yl)-3-hydroxyazetidine-l-carboxylate (730 mg) as a white solid. LC-MS (ESI) found: 329 [M+H] ⁺ 2. Preparation of methyl 6-(l-(tert-butoxycarbonyl)-3-hydroxyazetidin-3- yl)nicotinate

CO PdCI₂(dppf)

[0297] To a stirred solution of tert-butyl 3-(5-bromopyridin-2-yl)-3-hydroxyazetidine-l- carboxylate (200 mg, 0.60 mmol) in MeOH (5mL), Pd(dppf)Cl₂ (22.66 mg, 0.031 mmol) was added in one portion at 25 °C, followed by TEA (2 mL). The mixture is purged with carbon monoxide at room temperature for 10 minutes, then it was heated to 90 °C and stirred overnight under 40 psi of carbon monoxide. TLC and LC-MS showed the reaction was complete. The reaction solution is cooled to room temperature, quenched with saturated brine and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over magnesium sulfate, and concentrated under reduced pressure. The resulting residue was purified by column chromatography (silica gel: hexane: ethyl acetate = 15: 1) to give methyl 6-( 1 -(tert- butoxycarbonyl)-3-hydroxyazetidin-3-yl)nicotinate (155 mg) as a brown oil. LC-MS (ESI) found: 309 [M+l]⁺.

3. Preparation of methyl 6-(l-(tert-butoxycarbonyl)-3-

((methylsulfonyl)oxy)azetidin-3-yl)nicotinate

[0298] To a stirred solution of methyl 6-{ l-[(tert-butoxy)carbonyl]-3-hydroxyazetidin-3- yl}pyridine-3-carboxylate (300 mg, 0.97 mmol) in dry DCM (5 mL), was added triethylamine (0.21 mL, 1.46 mmol) dropwise. Five minutes later, methanesulfonyl chloride (0.083 mL, 1.07 mmol) was added at 0 °C over a period of 10 min. The reaction mixture was stirred at r.t. for 1.0 h under N₂ and the starting material was completely consumed by TLC. The reaction mixture was cooled to 0°C and diluted with 10% NaCl solution, extracted with DCM and washed with NaHCO, (aq.). The organic layer was dried over anhydrous

Na₂S0₄ and concentrated in vacuo to give crude methyl 6-(l-(tert-butoxycarbonyl)-3- ((methylsulfonyl)oxy)azetidin-3-yl)nicotinate (410 mg) as a colorless oil. LC-MS (ESI) found: 387 [M+l]⁺. 4. Preparation of methyl 6-(l-(tert-butoxycarbonyl)azetidin-3-yl)nicotinate

[0299] To a stirred solution of methyl 6-{ l-[(tert-butoxy)carbonyl]-3- (methanesulfonyloxy)azetidin-3-yl}pyridine-3-carboxylate (410 mg, 1.06 mmol) in dry MeOH (5 mL), was added Pd/C (20 mg, 0.188 mmol) in one portion at 25°C. The reaction mixture was stirred at r.t. for 1.0 h under N₂ and the starting material was completely consumed by TLC.

The reaction mixture was filtered. To the filtrate was added 10% NaCl solution, and the aqueous layer was extracted with EA. The organic layer was dried over anhydrous

Na₂S0₄ and concentrated in vacuo to give methyl 6-(l-(tert-butoxycarbonyl)azetidin-3- yl)nicotinate (105 mg) as a colorless oil. LC-MS (ESI) found: 293 [M+l]⁺.

5. Preparation of lithium 6-(l-(tert-butoxycarbonyl)azetidin-3-yl)nicotinate

[0300] To a stirred solution of methyl 6-{ l-[(tert-butoxy)carbonyl]azetidin-3-yl}pyridine-3- carboxylate (105 mg, 0.36 mmol) in THF (3 mL), was added lithium hydroxide (39.5 mg, 0.89 mmol) in H₂0 (1 mL) in one portion at 25°C. The reaction mixture was stirred at r.t. for 1.0 h and the starting material was completely consumed TLC. The reaction mixture was concentrated in vacuo to give lithium 6-(l-(tert-butoxycarbonyl) azeti din-3 -yl) nicotinate (crude, 150 mg) as a colorless oil. LC-MS (ESI) found: 279 [M+l]⁺.

6. Preparation of methyl 6-(azetidin-3-yl)nicotinic acid

TFA salt

[0301] To a solution of lithium 6-{ l-[(tert-butoxy)carbonyl]azetidin-3-yl}pyridine-3- carboxylate (crude, 150 mg, 0.35 mmol) in DCM (2 mL) was added TFA (1 mL). After the addition, the reaction mixture was stirred at room temperature for 3 hours. TLC showed the reaction was complete. The mixture was concentrated to give 6-(azeti din-3 -yl) nicotinic acid TFA salt (crude, 300 mg) as a brown oil. LC-MS (ESI) found: 177 [M-l] . 7. Preparation of 6-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)nicotinic acid

Intermediate 1

[0302] Under a N₂ atmosphere, to a solution of 6-(azetidin-3-yl)nicotinic acid 2,2,2- trifluoroacetate salt (crude, 300 mg) and, ethyldiisopropylamine (462 mg, 3.5 mmol) in CH₃CN (3 mL) was added a solution of (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl (4-nitrophenyl) carbonate

(Intermediate 1, 160 mg, 0.36 mmol) in CH₃CN (1 mL) dropwise at 0°C, followed by addition of DMAP (5 mg, 0.067 mmol). The mixture was then stirred at r.t. for 16 hrs. The reaction mixture was concentrated and purified by prep-HPLC (Method A, H₂0 (0.1% FA)/ CH₃CN) to give 6-( 1 -((((3//,4.V,5.V,6//)-5-methoxy-4-((2//,3//)-2- ethyl-3-(3- ethylbut-2-en- l -yl)oxiran-2- yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)nicotinic acid (130 mg) as a colorless oil. LC-MS (ESI) found: 487 [M+l]⁺.

8. Preparation of sodium 6-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)nicotinate

[0303] To a solution of 6-( 1 -((((3//,4.V,5.V,6//)-5-methoxy-4-((2//,3//)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)nicotinic acid (53 mg, 0.11 mmol) in CH₃CN (0.5 mL) was added 0.1 N NaOH (0.98 mL, 0.098 mmol) solution at -60°C with stirring. Sodium 6-(l-((((3i?,4ri^,,5ri',6i?)-5-methoxy-4- ((2//,3//)-2-methyl-3-(3-methylbut-2-en- l -yl)oxiran-2-yl)- l -oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azeti din-3 -yl)nicotinate (55 mg) was obtained as white solid after

lyophilization. 1H MR (400 MHz, DMSO-d6 ) d 8.96 (s, 1H), 8.06 (dd, J= 7.9, 1.7 Hz, 1H), 7.21 (d, J= 7.9 Hz, 1H), 5.34 (s, 1H), 5.19 (t, j= 7.4 Hz, 1H), 4.23 (t, 7= 8.2 Hz, 2H), 4.14 - 3.93 (m, 3H), 3.55 (dd, 7 = 11.0, 2.4 Hz, 1H), 3.31 (s, 3H), 2.85 (d, 7= 4.3 Hz, 1H), 2.56 (dd, J = 10.3, 5.2 Hz, 2H), 2.17 (d, J= 4.9 Hz, 2H), 2.02 - 1.92 (m, 1H), 1.88 - 1.73 (m, 3H), 1.71 (s, 3H), 1.61 (s, 3H), 1.09 (s, 3H), 1.03 (d, J= 12.9 Hz, 1H).

Example 46: Preparation of sodium 5-(3-hydroxy-l-((((3R,4S,5S,6R)-5-methoxy-4-

((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)picolinate

1. Preparation of tert-butyl 3-(6-bromopyridin-3-yl)-3-hydroxyazetidine-l- carboxylate

[0304] To a stirred solution of 2-bromo-5-iodopyridine (5.0 g, 17.6 mmol) in dry THF (30 mL), was added isopropylmagnesium chloride (9.69 mL, 19.37 mmol) dropwise over a period of 10 min at -20°C. Then a solution of tert-butyl 3-oxoazetidine-l-carboxylate (3.62 g, 21.1 mmol) in dry THF (10 mL) was added at -20°C to the above reaction mixture. The reaction mixture was then stirred at r.t. for 16 hrs under N₂. The starting material was completely consumed by TLC . The reaction mixture was cooled to -l0°C and quenched with 10% citric acid solution, extracted with EA and washed with NaHC0₃ (aq.). The organic layer was dried over anhydrous Na₂S0₄ and concentrated in vacuo. Then the crude product was purified by flash chromatography on silica gel eluting with PE/ EtOAc 10-20/1 to give tert-butyl 3-(6- brom opyri din-3 -yl)-3-hydroxyazeti dine- l-carboxylate (3.56 g) as a white solid. LC-MS (ESI) found: 329 [M+H] ⁺

2. Preparation of methyl 5-{l-[(tert-butoxy)carbonyl]-3-hydroxyazetidin-3- yl}pyridine-2-carboxylate

CO Pd(dppf)CI

[0305] To a stirred solution of tert-butyl 3-(6-bromopyridin-3-yl)-3-hydroxyazetidine-l- carboxylate (1.1 g, 3.34 mmol) in MeOH (5mL), was added Pd(dppf)Cl₂ (0.023 g, 0.031 mmol) in one portion at 25°C, followed by the addition of TEA (2 mL). The mixture was purged with carbon monoxide at room temperature for 10 minutes, and it was then heated to 90 °C and stirred overnight under 40 psi of carbon monoxide. TLC showed the reaction was complete. The reaction solution was cooled to room temperature, and diluted with saturated brine and extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The resulting residue was purified by column chromatography (silica gel; hexane: ethyl acetate = 15: 1) to give methyl 5-{ l-[(tert-butoxy)carbonyl]-3-hydroxyazetidin-3-yl}pyridine-2-carboxylate (650 mg) as a brown oil. LC-MS (ESI) found: 309 [M+l]⁺.

3. Preparation of lithium 5-(l-(tert-butoxycarbonyl)-3-hydroxyazetidin-3-yl)picolinate

[0306] To a stirred solution of methyl 5-(l-(tert-butoxycarbonyl)-3-hydroxyazetidin-3- yl)picolinate (120 mg, 0.39 mmol) in THF (3 mL), was added lithium hydroxide (32.9 mg, 0.78 mmol) in H₂0 (lmL) in one portion at 25 °C. The reaction mixture was stirred at r.t. for 1.0 h and the starting material was completely consumed by TLC. The reaction mixture was concentrated in vacuo to give lithium 5-(l-(tert-butoxycarbonyl)-3-hydroxyazetidin-3- yl)picolinate (crude, 150 mg) as a colorless oil. LC-MS (ESI) found: 295 [M+l]⁺.

4. Preparation of 5-(3-hydroxyazetidin-3-yl)picolinic acid

TFA salt

COOH

[0307] To a solution of lithium 5-(l-(tert-butoxycarbonyl)-3-hydroxyazetidin-3-yl)picolinate (crude, 150 mg) in DCM (2 mL) at r.t. was added TFA (1 mL). After the addition, the reaction mixture was stirred at room temperature for 3 hours. The mixture was concentrated to give 5- (3-hydroxyazetidin-3-yl)picolinic acid (crude, TFA salt, 150 mg) as a brown oil. LC-MS (ESI) found: 195 [M+l]⁺.

5. Preparation of 5-(3-hydroxy-l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl- 3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)picolinic acid

Intermediate 1

[0308] Under a N₂ atmosphere, to a solution of 5-(3-hydroxyazetidin-3-yl)pyridine-2- carboxylic acid (78.1 mg, 0.4 mmol), DIPEA (0.53 mL, 3.22 mmol) in CH₃CN (3 mL) was added dropwise a solution of (3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut- 2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl 4-nitrophenyl carbonate (Intermediate 1, 180 mg, 0.40 mmol) in CH₃CN (1 mL) at 0°C, followed by adding DMAP (5 mg, 0.067 mmol). Then the mixture was stirred at r.t. for 16 h. TLC showed the reaction was complete. The reaction mixture was concentrated and purified by prep-HPLC (Method A, H₂0 (0.1% FA)/ CH₃CN) to give 5-[3-hydroxy-l-({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3- yl]pyridine-2-carboxylic acid (123 mg) as a colorless oil. LC-MS (ESI) found: 503 [M+l]⁺.

6. Preparation of sodium 5-(3-hydroxy-l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)picolinate

[0309] To a solution of 5-[3-hydroxy-l-({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl- 3-(3-methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3- yl]pyridine-2-carboxylic acid (123 mg, 0.25 mmol) in CH₃CN (0.3 mL) was added 0.1 N NaOH (2.20 mL, 0.22 mmol) solution at -60°C with stirring. After lyophilization, sodium 5- [3-hydroxy- l-({[(3R,4S, 5 S,6R)-5-methoxy-4-[(2R,3R)-2 -methyl-3 -(3-methylbut-2-en-l - yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3-yl]pyridine-2-carboxylate (122 mg) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6 ) d 8.61 (s, 1H), 7.87 (dd, J= 20.4, 8.1 Hz, 2H), 6.62 (s, 1H), 5.37 (s, 1H), 5.18 (s, 1H), 4.12 (s, 4H), 3.56 (dd, J =

11.0, 2.5 Hz, 1H), 3.31 - 3.29 (m, 3H), 2.85 (d, J= 4.3 Hz, 1H), 2.57 (d, J= 4.3 Hz, 1H), 2.54 (d, j= 6.3 Hz, 1H), 2.17 (s, 2H), 1.96 (dd, j= 13.3, 4.6 Hz, 1H), 1.86 - 1.72 (m, 3H), 1.69 (s, 3H), 1.60 (s, 3H), 1.09 (s, 3H), 1.02 (d, j= 12.8 Hz, 1H). Example 47: Preparation of sodium 6-(3-hydroxy-l-((((3R,4S,5S,6R)-5-methoxy-4- ((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)nicotinate

1. Preparation of 6-(l-(tert-butoxycarbonyl)-3-hydroxyazetidin-3-yl)nicotinic acid

[0310] To a stirred solution of methyl 6-(l-(tert-butoxycarbonyl)-3-hydroxyazeti din-3 - yl)nicotinate (120 mg, 0.39 mmol) in THF (3 mL), was added lithium hydroxide (32.9 mg, 0.78 mmol) in H₂0 (1 mL) in one portion at 25°C. The reaction mixture was stirred at r.t. for 1.0 h and the starting material was completely consumed by TLC. The reaction mixture was concentrated in vacuo to give lithium 6-(l-(tert-butoxycarbonyl)-3-hydroxyazetidin-3- yl)nicotinate (crude, 150 mg) as a colorless oil. LC-MS (ESI) found: 295 [M+l]⁺.

2. Preparation of 6-(3-hydroxyazetidin-3-yl)nicotinic acid TFA salt

Boc

[0311] To a solution of lithium 6-(l-(tert-butoxycarbonyl)-3-hydroxyazetidin-3-yl)nicotinate (crude, 150 mg) in DCM (2 mL) was added TFA (1 mL). After the addition, the reaction mixture was stirred at room temperature for 3 hours. TLC showed the reaction was complete. The mixture was concentrated to give 6-(3-hydroxyazeti din-3 -yl)nicotinic acid (crude, TFA salt, 150 mg) as a brown oil. LC-MS (ESI) found: 195 [M+l]⁺.

3. Preparation of 6-(3-hydroxy-l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl- 3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)nicotinic acid

TFA salt

Intermediate 1

[0312] Under a N₂ atmosphere, to a solution of 6-(3-hydroxyazeti din-3 -yl)pyridine-3- carboxylic acid (78.12 mg, 0.40 mmol), DIPEA (0.40 mL, 2.42 mmol) in MeCN (3 mL) was added a solution of (3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl 4-nitrophenyl carbonate (Intermediate 1, 180 mg, 0.40 mmol) in CH₃CN (1 mL) dropwise at 0°C, followed by adding DMAP (5 mg, 0.067 mmol). Then the mixture was stirred at r.t. for 16 h. TLC showed the reaction was complete. The reaction mixture was concentrated and purified by prep-HPLC (Method A, H₂0 (0.1% FA)/ CH₃CN) to give 6-[3-hydroxy-l-({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3- yl]pyridine-3 -carboxylic acid (25 mg) as a colorless oil. LC-MS (ESI) found: 503 [M+l]⁺.

4. Preparation of sodium 6-(3-hydroxy-l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)nicotinate

[0313] To a solution of 6-(3-hydroxy-l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl- 3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6-yl)oxy)carbonyl)azetidin-3 - yl)nicotinic acid (25 mg, 0.05 mmol) in CH₃CN (0.3 mL) was added 0.1 N NaOH

solution(0.45 mL, 0.05 mmol) at -60°C with stirring. After lyophilization, sodium 6-(3- hydroxy- 1 -((((3R,4S,5 S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3 -methylbut-2-en- 1 -yl)oxiran- 2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)nicotinate (26 mg) was obtained as white solid. 1H MR (400 MHz, DMSO-d6 ) d 8.92 (s, 1H), 8.09 (dd, J= 8.0, 1.7 Hz, 1H), 7.49 (d, j= 7.9 Hz, 1H), 6.62 (s, 1H), 5.35 (s, 1H), 5.18 (t, j= 7.4 Hz, 1H), 4.29 (d, j= 26.8 Hz, 2H), 3.97 (s, 2H), 3.55 (dd, j= 11.0, 2.6 Hz, 1H), 3.30 (s, 3H), 2.84 (d, j= 4.3 Hz, 1H), 2.55 (dd, j= 7.6, 5.3 Hz, 2H), 2.16 (d, j= 5.0 Hz, 2H), 1.97 (s, 1H), 1.87 - 1.73 (m, 3H), 1.70 (s, 3H), 1.60 (s, 3H), 1.08 (d, j= 4.6 Hz, 3H), 1.03 (s, 1H).

Example 48: Preparation of sodium 3-fluoro-l-((((.?/?, S,5A,6/?)-5-methoxy-4-((2/?,.?/?)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidine-3-carboxylate

1. Preparation of l-(tert-butoxycarbonyl)azetidine-3-carboxylic acid

[0314] Di-tert-butyldi carbonate (18 g, 83 mmol) was added to a solution of 3- azetidinecarboxylic acid (7.6 g, 75 mmol) in THF (150 mL), water (75 mL) and 2M NaOH (75 mL) while cooling in an ice-bath. The reaction mixture was then stirred at r.t. for 24 h. TLC showed the reaction was complete. The organic solvent was removed in vacuo and the residual water mixture was acidified to pH = 1 by the addition of 4 M HC1. The water mixture was then extracted with EtOAc and the combined organic layers were washed with brine, dried over anhydrous MgS0₄ and concentrated in vacuo to give l-(tert-butoxycarbonyl)azetidine-3- carboxylic acid (11 g) as a white solid. LC-MS (ESI) m/z (M+l): 202.

2. Preparation of l-tert-butyl 3-methyl azetidine-l,3-dicarboxylate

[0315] To a solution of azetidine-l,3-dicarboxylic acid mono-tert-butyl ester (11 g, 54.7 mmol) in DMF (75 mL), were added K₂C0₃ (15 g, 109.4 mmol) and CH₃I (9.3 g, 65.6 mmol). The reaction mixture was stirred at r.t. for 24 h. TLC showed the reaction was complete. Water was added. The water phase was extracted with EtOAc and the combined organic layers were washed with brine, dried over anhydrous MgS0₄ and concentrated in vacuo to give azetidine- l,3-dicarboxylic acid l-tert-butyl ester 3-methyl ester (8 g) as a white solid. LC-MS (ESI) m/z (M+l): 216.

3. Preparation of l-tert-butyl3-methyl3-fluoroazetidine-l,3-dicarboxylate

N-Fluorobenzenesulfonimide

LiHMDS

[0316] To a solution of l-tert-butyl 3-methyl azetidine-l,3-dicarboxylate (8 g, 37.2 mmol) in THF (80 mL), was added LiHMDS (44 mL, 44.6 mmol, 1 M in THF) at -78°C. 3 N- fluorobenzenesulfonimide (14 g, 44.6 mmol) was added 30 min later. The reaction mixture was stirred at -78°C for 4 hrs. TLC showed the reaction was complete. Water was added. The water phase was extracted with EtOAc and the combined organic layers were washed with brine, dried over anhydrous MgS0₄ and concentrated in vacuo to give l-tert-butyl3 -methyl 3- fluoroazetidine-l,3-dicarboxylate (6 g) as a colorless oil. LC-MS (ESI) m/z (M+l): 234.

4. Preparation of l-(tert-butoxycarbonyl)-3-fluoroazetidine-3-carboxylic acid

[0317] To a stirred suspension of l-tert-butyl 3-methyl 3-fluoroazetidine-l,3-dicarboxylate (6 g, 25.7 mmol) in MeOH:H₂0 (1 : 1, 60 mL) was added lithium hydroxide (10 g, 257 mmol) at room temperature and the mixture was stirred at room temperature for 24 hours. The reaction mixture was concentrated, water (20 mL) was added to the residue, and the resulting mixture was acidified to pH~4 with 1 N HCI. The acidified aqueous layer was extracted with ethyl acetate (5 x 50 mL). The combined organic layers were washed with brine (150 mL), dried over anhydrous Na₂S0₄ and evaporated under reduced pressure to give l-(tert-butoxycarbonyl)-3- fluoroazetidine-3 -carboxylic acid (5.5 g) as a white solid. LC-MS (ESI) m/z (M+l): 220.

5. Preparation of 3-fluoroazetidine-3-carboxylic acid

[0318] To a mixture of l-(tert-butoxycarbonyl)-3-fluoroazetidine-3 -carboxylic acid (300 mg, 1.36 mmol) in DCM (2 mL) was added TFA (1 mL). The reaction mixture was stirred at room temperature for 2hrs. TLC showed the reaction was complete. The resulting mixture was concentrated to give crude product, which was used in the next step without further purification. LC-MS (ESI) m/z (M+l): 120.

6. Preparation of 3-fluoro-l-((((J/?, V,5,V,6/?)-5-methoxy-4-((2/?,J/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidine-3- carboxylic acid

[0319] To a solution of 3-fluoroazetidine-3-carboxylic acid (150 mg, 1.25 mmol) in MeCN (5 mL) was added DIPEA (1 mL, 5.03 mmol) dropwise at 0 °C. The mixture was stirred at 0 °C for 10 min, then (3f?, ri',5ri',6/?)-5-methoxy-4-[(2f?,3f?)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl-4-nitrophenylcarbonate (Intermediate 1, 563 mg,

1.25 mmol) was added. The reaction mixture was stirred at r.t. overnight under a N₂

atmosphere. The mixture was concentrated under vacuum to remove the solvent while keeping the temperature below 40 °C. The residue was purified by column chromatography (silica gel, 1 % ~ 10 % MeOH in DCM) and prep-HPLC (Cl 8,0 ~ 90 % acetonitrile in H₂0 with 0.1 % NH₃Ή₂O) to give 3-fluoro-l-({[(3i?, ,S^,,5S^,, 6/?)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidine-3- carboxylicacid (300 mg) as a colorless oil. LC-MS (ESI) m/z (M+l): 428.

7. Preparation of sodium 3-fluoro-l-(((( /?, S,5»S,6/?)-5-methoxy-4-((2/?, /?)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidine-3-carboxylate

[0320] To a mixture of 3-fluoro-l-({[(3i?,4,S^,,5S^,, d/?)-5-methoxy-4-[(2R,3R) -2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidine-3- carboxylic acid (210 mg, 0.49 mmol) in MeCN (1 mL) was added aq. NaOH (4.4 mL, 0.1 mol/L) at -60 °C with stirring. The reaction mixture was lyophilized to give sodium 3-fluoro-l- ((((3f?, 4S, 5S, 6// )- 5 -m ethoxy-4 -((2//, 3f?)-2-methyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 - oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidine-3-carboxylate (195 mg) as a white solid. LC- MS (ESI) m/z (M+l): 428. 1H NMR (400 MHz, DMSO-d6 ) 5 5.31 (s, 1H), 5.19 (t, J = 7.3 Hz, 1H), 4.21 (s, 2H), 3.85 (d, J = 9.1 Hz, 2H), 3.53 (dd, J = 10.9, 2.5 Hz, 1H), 3.29 (s, 3H), 2.84

(d, J = 4.3 Hz, 1H), 2.57 (dd, J = 7.8, 4.6 Hz, 2H), 2.17 (dd, J = 12.8, 6.4 Hz, 2H), 1.99 - 1.88 (m, 1H), 1.78 (dd, J = 18.5, 7.2 Hz, 3H), 1.70 (s, 3H), 1.61 (s, 3H), 1.08 (s, 3H), 1.02 (d, J = 13.6 Hz, 1H).

Example 49: Preparation of sodium 6-(3-fluoro-l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-

2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)nicotinate

l. Preparation of methyl 6-(l-(tert-butoxycarbonyl)-3-fluoroazetidin-3-yl)nicotinate

[0321] To a stirred solution of methyl 6-{ l-[(tert-butoxy)carbonyl]-3-hydroxyazetidin-3- yl}pyridine-3-carboxylate (205 mg, 0.665 mmol) in dry DCM (5 mL), was added

(diethylamino)sulfur trifluoride (0.163 mL, 1.33 mmol) at 0°C over a period of 10 min. The reaction mixture was stirred at r.t. for 2 h under N₂, and the starting material was completely consumed by TLC. The reaction mixture was cooled to -l0°C and quenched with water, extracted with EA and washed with NaCl (aq.). The organic layer was dried over anhydrous Na₂S0₄ and concentrated in vacuo, then the crude product was purified by flash

chromatography on silica gel eluting with PE/EtOAc = 20-15/1 to give methyl 6-(l-(tert- butoxycarbonyl)-3-fluoroazeti din-3 -yl)nicotinate (185 mg) as a colorless oil. LC-MS (ESI) found: 311 [M+H] ⁺

2. Preparation of lithium 6-(l-(tert-butoxycarbonyl)-3-fluoroazetidin-3- yl)nicotinate

[0322] To a stirred solution of methyl 6-(l-(tert-butoxycarbonyl)-3-fluoroazeti din-3 - yl)nicotinate (185 mg, 0.59 mmol) in THF (3 mL), was added lithium hydroxide (52.44 mg, 1.19 mmol) in H₂0 (1 mL) in one portion at 25°C. The reaction mixture was stirred at r.t. for 1.0 h, and the starting material was completely consumed by TLC. The reaction mixture was concentrated in vacuo to give lithium 6-(l-(tert-butoxycarbonyl)-3-fluoroazetidin-3- yl)ninotinate (crude, 256 mg) as a colorless oil. LC-MS (ESI) found: 297 [M+l]⁺.

3. Preparation of 6-(3-fluoroazetidin-3-yl)nicotinic acid

TFA salt

[0323] To a solution of lithium 6-(l-(tert-butoxycarbonyl)-3-fluoroazetidin-3-yl)nicotinate (crude, mg, 0.57 mmol) in DCM (2 mL) was added TFA (1 mL). After the addition, the reaction mixture was stirred at room temperature for 3 hours. TLC showed the reaction was complete. The reaction was concentrated to give 6-(3-fluoroazeti din-3 -yl)nicotinic acid (crude, TFA salt, 200 mg) as a brown oil. LC-MS (ESI) found: 197 [M+l]⁺.

4. Preparation of sodium 6-(3-fluoro-l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)nicotinate

[0324] Stepl : Under a N₂ atmosphere, to a solution of 6-(3-fluoroazeti din-3 -yl)pyridine-3- carboxylic acid (200 mg, 0.51 mmol, 2,2,2-trifluoroacetate salt) and DIPEA (0.42 mL, 2.55 mmol) in CH₃CN (3 mL) was added a solution of (3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl 4-nitrophenyl carbonate (Intermediate 1, 228.0 mg, 0.50 mmol) in CH₃CN (1 mL) dropwise at 0°C, followed by addition of DMAP (5 mg, 0.067 mmol). The mixture was then stirred at r.t. for 16 h. TLC showed the reaction was complete. The reaction mixture was concentrated and purified by prep-HPLC (Method A, H₂0 (0.1% FA)/ CH₃CN) to give 6-(3-fluoro-l-((((3R,4S,5S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)nicotinic acid (96 mg) as a colorless oil. LC-MS (ESI) found:

505 [M+l]⁺. Step2: To a solution of 6-(3-fluoro-l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)nicotinic acid (96 mg, 0.19 mmol) in CH₃CN (0.5 mL) was added 0.1 N NaOH (1.73 mL) solution at -60°C with stirring. After lypholization, sodium 6-(3-fluoro-l-((((3R,4S,5S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azeti din-3 -yl)nicotinate (lOOmg) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6 ) d 8.99 (s, 1H), 8.17 (dd, J= 8.0, 1.7 Hz, 1H), 7.46 (d, J= 7.9 Hz, 1H), 5.36 (s, 1H), 5.18 (t, J = 7.3 Hz, 1H), 4.46 (s, 2H), 4.29 (dd, j= 22.4, 10.3 Hz, 2H), 3.56 (dd, j = 11.0, 2.5 Hz, 1H), 3.31 (s, 3H), 2.84 (d, 7= 4.3 Hz, 1H), 2.56 (dd, 7= 9.3, 5.3 Hz, 2H), 2.17 (s, 2H), 2.02 - 1.93 (m, 1H), 1.81 (dt, J= 29.8, 14.9 Hz, 3H), 1.70 (s, 3H), 1.60 (s, 3H), 1.08 (s,

3H), 1.03 (d, J= 12.7 Hz, 1H).

Example 50: Preparation of sodium 5-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl) picolinate

Intermediate 1

1. Preparation of methyl 5-(l-(tert-butoxycarbonyl)-3-

((methylsulfonyl)oxy)azetidin-3-yl)picolinate

[0325] To a stirred solution of methyl 5-(l-(tert-butoxycarbonyl)-3-hydroxyazetidin-3- yl)picolinate (240 mg, 0.778 mmol) and triethylamine (0.163 mL, 1.17 mmol) in dry DCM (5mL) was added methanesulfonyl chloride (0.066 mL, 1.856 mmol) dropwise over a period of 10 min at 0°C. The reaction mixture was stirred at r.t. for 1.0 h under N₂ and the starting material was completely consumed by TLC. The reaction mixture was cooled to 0°C, quenched with 10% NaCl solution and extracted with DCM. The combined organic layers were washed with NaHCO, (aq.), dried over anhydrous Na₂S0₄ and concentrated in vacuo to give methyl 5- (l-(tert-butoxycarbonyl)-3-((methylsulfonyl)oxy)azetidin-3-yl)picolinate (294 mg) as a colorless oil. LC-MS (ESI) found: 387 [M+l]⁺.

2. Preparation of methyl 5-(l-(tert-butoxycarbonyl)azetidin-3-yl)picolinate

[0326] To a stirred solution of methyl 5-(l-(tert-butoxycarbonyl)-3-

((methylsulfonyl)oxy)azetidin-3-yl)picolinate (290 mg, 0.751 mmol) in dry MeOH (5 mL), was added Pd/C (20 mg, 0.188 mmol, 10%) in one portion at 25 °C. The reaction mixture was stirred at r.t. for 1.0 h under N₂, and the starting material was completely consumed by TLC. The reaction mixture was filtered, and 10% NaCl solution was added to the filtrate. The resulting mixture was extracted with EA. The organic layer was dried over anhydrous Na₂S0₄ and concentrated in vacuo to give methyl 5-(l-(tert-butoxycarbonyl)azetidin-3-yl)picolinate (200 mg) as a colorless oil. LC-MS (ESI) found: 293 [M+l]⁺.

3. Preparation of lithium 5-(l-(tert-butoxycarbonyl)azetidin-3-yl) picolinate

[0327] To a stirred solution of methyl 5-(l-(tert-butoxycarbonyl)azetidin-3-yl)picolinate (200 mg, 0.68 mmol) in THF 3 (mL), was added lithium hydroxide (33 mg, 0.89 mmol) in H₂0 (1 mL) in one portion at 25°C. The reaction mixture was stirred at r.t. for 1.0 h and the starting material was completely consumed by TLC. The reaction mixture was concentrated in vacuo to give lithium 5-(l-(tert-butoxycarbonyl)azetidin-3-yl)picolinate (crude, 300 mg) as a colorless oil. LC-MS (ESI) found: 279 [M+l]⁺.

4. Preparation of 5-(azetidin-3-yl)picolinic acid

[0328] To a solution of lithium 5-(l-(tert-butoxycarbonyl)azetidin-3-yl)picolinate (crude, 200 mg, 0.72 mmol) in DCM (2 mL) was added TFA (1 mL). After the addition, the reaction mixture was stirred at room temperature for 3 hours. Then the mixture was concentrated to afford 5-(azetidin-3-yl)picolinic acid (crude, TFA salt, 200 mg) as a brown oil. LC-MS (ESI) found: 179 [M+l]⁺.

5. Preparation of sodium 5-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)picolinate

Intermediate 1

[0329] Stepl : Under N₂ atmosphere, to a solution of 5-(azetidin-3-yl)pyridine-2-carboxylic acid (51.44 mg, 0.29 mmol, TFA salt), DIPEA (0.239 mL, 1.4 mmol) in CH₃CN (3 mL) was added a solution of (3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl 4-nitrophenyl carbonate (Intermediate 1 300 mg, 0.67 mmol) in CH₃CN (1 mL) dropwise at 0°C, followed by adding DMAP (5 mg, 0.067 mmol). Then the mixture was stirred at r.t. for 16 hrs. TLC showed the reaction was complete. The reaction mixture was concentrated and purified by prep-HPLC (Method A, H₂0 (0.1% FA)/ CH₃CN) to give 5-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut- 2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azeti din-3 -yl)picolinic acid (140 mg) as a colorless oil. LC-MS (ESI) found: 487 [M+l]⁺. Step2: To a solution of 5-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut- 2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)picolinic acid (70 mg, 0.144 mmol) in CH₃CN (0.5 mL) was added 0.1 N NaOH (1.30 mL) solution at -60°C with stirring. After lypholization, sodium 5-[l-({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3- yl]pyridine-2-carboxylate (75 mg) was obtained as a white solid. 1H NMR (400 MHz, DMSO- d6) d 8.39 (s, 1H), 7.88 (d, 7= 8.1 Hz, 1H), 7.82 - 7.77 (m, 1H), 5.35 (s, 1H), 5.19 (d, J= 7.0 Hz, 1H), 4.32 (s, 2H), 3.91 (s, 3H), 3.55 (dd, 7= 11.0, 2.4 Hz, 1H), 3.31 (s, 3H), 2.85 (d, 7 =

4.3 Hz, 1H), 2.56 (dd, J= 11.1, 5.3 Hz, 2H), 2. l7 (s, 2H), 2.02 - 1.91 (m, 1H), 1.79 (q, J= 19.0 Hz, 3H), 1.69 (s, 3H), 1.60 (s, 3H), 1.09 (s, 3H), 1.03 (d, J= 13.2 Hz, 1H).

Example 51: Preparation of sodium l-[l-({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6- yl]oxy}carbonyl)azetidin-3-yl]-lH-imidazole-4-carboxylate

1. Preparation of tert-butyl 3-(methanesulfonyloxy)azetidine-l-carboxylate

[0330] To a stirred solution of tert-butyl 3-hydroxyazetidine-l-carboxylate (5.0 g, 28.87 mmol) and TEA (5.83 g, 57.74 mmol) in DCM (100 mL) was added methanesulfonyl chloride (3.35 mL, 43.31 mmol) at 0 °C. The reaction mixture was stirred at r.t. overnight. TLC showed the reaction was complete. The mixture was washed with aq. NaHCO,, and the organic layer was separated. The solvent was removed under reduced pressure to obtain tert-butyl 3- (methanesulfonyloxy)azetidine-l-carboxylate (6.3 g) as a colorless oil. LC-MS(ESI) found: 196 [M+H-56]⁺

2. Preparation of methyl l-{l-[(tert-butoxy)carbonyl]azetidin-3-yl}-lH-imidazole- 4-carboxylate

[0331] To a mixture of tert-butyl 3-(methanesulfonyloxy)azetidine-l-carboxylate (1.5 g, 5.97 mmol) in DMF (28 mL) was added Cs₂C0₃ (5.83 g, 17.90 mmol) and methyl 1H- imidazole-4-carboxylate (1.51 g, 11.94 mmol). The reaction was stirred at 100 °C overnight. TLC showed the reaction was complete. The mixture was washed with water and extracted with EA, and the organic layer was dried over anhydrous Na₂S0₄ and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with hexanes : ethyl acetate = 1 : 1 to give methyl l-{ l-[(tert-butoxy)carbonyl]azetidin-3-yl}-lH- imidazole-4-carboxylate (410 mg) and methyl l-(l-(tert-butoxycarbonyl)azetidin-3-yl)-lH- imidazole-5-carboxylate (130 mg). LC-MS (ESI) found: 282 [M+H]⁺. methyl l-{ l-[(tert- butoxy)carbonyl]azetidin-3-yl}-lH-imidazole-4-carboxylate: 1H NMR (400 MHz, CDCl₃) d 8.84 (s, 1H), 7.99 (s, 1H), 5.38 (s, 1H), 4.57 (t, J = 8.5 Hz, 2H), 4.10 (d, J = 7.1 Hz, 2H), 3.97 (s, 3H), 1.49 (s, 9H). methyl l-(l-(tert-butoxycarbonyl)azetidin-3-yl)-lH-imidazole-5- carboxylate: 1H NMR (400 MHz, CDCI3) d 9.27 (s, 1H), 7.96 (s, 1H), 5.67 - 5.59 (m, 1H), 4.63 - 4.51 (m, 2H), 4.30 (dd, J= 9.9, 5.0 Hz, 2H), 3.93 (s, 3H), 1.48 (s, 9H).

3. Preparation of l-{l-[(tert-butoxy)carbonyl]azetidin-3-yl}-lH-imidazole-4- carboxylic acid

[0332] To a stirred suspension of methyl l-{ l-[(tert-butoxy)carbonyl]azetidin-3-yl}-lH- imidazole-4-carboxylate (410 mg, 1.46 mmol) in THF (10 mL) and H₂0 (5 mL) was added Li OH (93.65 mg, 3.91 mmol). The reaction mixture was stirred at r.t. overnight. TLC showed the reaction was complete. The reaction mixture was concentrated to remove the solvent, then diluted with 5 mL of water and 5 mL of EtOAc. The water phase was adjusted to pH = 3 with 1N HC1 and then extracted with DCM. The organic layer was concentrated to give l-{ 1- [(tert-butoxy)carbonyl]azetidin-3-yl}-lH-imidazole-4-carboxylic acid (250 mg) as a colorless oil. LC-MS(ESI) found: 268 [M+l]⁺. 4. Preparation of l-(azetidin-3-yl)-lH-imidazole-4-carboxylic acid trifluoroacetic acid salt

[0333] To a solution of l-{ l-[(tert-butoxy)carbonyl]azetidin-3-yl}-lH-imidazole-4- carboxylic acid (250 mg, 0.94 mmol) in DCM (2 mL) was added TFA (1 mL) dropwise at 0 °C The reaction was stirred at room temperature for 1 h. TLC showed the reaction was complete. The mixture was concentrated under reduced pressure to give l-(azetidin-3-yl)-lH-imidazole- 4-carboxylic acid trifluoroacetic acid salt (240 mg) as a yellow syrup, which was used directly in the next step without purification. LC-MS (ESI) found: 168 [M+l]⁺.

1. Preparation of l-[l-({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3-yl]- lH-pyrazole-4-carboxylic acid

[0334] To a solution of l-(azetidin-3-yl)-lH-imidazole-4-carboxylic acid (240 mg, 0.75 mmol) in MeCN (5 mL) was added DIPEA (385.86 mg, 2.99 mmol) dropwise at 0 °C. The mixture was stirred at 0°C for 10 min, and (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl (4-nitrophenyl) carbonate (Intermediate 1, 334.62 mg, 0.75 mmol) was added. The reaction was then stirred at room temperature overnight under N₂ atmosphere. TLC showed the reaction was complete. The mixture was then concentrated under vacuum to remove the solvent while keeping external temperature below 40°C. The residue was diluted with dichloromethane and washed with ammonium acetate buffer (pH 4.0, 20 mL). The organic layer was dried over anhydrous Na₂S0₄, filtered and concentrated to give the crude product. The residue was purified by column chromatography on silica gel column (eluent: dichloromethane: methanol = 100 : 1 to 10 : 1) and further purified by preparative HPLC (C18, 0 ~ 90 % acetonitrile in H₂0 with 0.1 % NH₃ H₂0) to give l-[l-({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2- en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3-yl]-lH-imidazole-4- carboxylic acid (186 mg) as a white solid. LC-MS (ESI) found: 476 [M+l]⁺.

5. Preparation of sodium l-[l-({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3- yl]-lH-imidazole-4-carboxylate

[0335] To a solution of l-[l-({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3-yl]-lH- imidazole-4-carboxylic acid (186 mg, 0.39 mmol) in MeCN (2 mL) was added 0.1 N aq. NaOH (3.52 mL) at -50 °C. The mixture was lyophilized overnight to give sodium l-[l- ({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl]-l- oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3-yl]-lH-imidazole-4-carboxylate (190 mg) as a white solid. 1H NMR (400 MHz, DMSO-d6 ) d 7.85 (s, 1H), 7.07 (s, 1H), 5.86 (s, 1H), 5.33 (s, 1H), 5.19 (t, J= 7.3 Hz, 1H), 4.30 (t, J= 8.7 Hz, 2H), 4.17 - 4.03 (m, 2H), 3.54 (dd, J= 11.0, 2.4 Hz, 1H), 3.30 (s, 3H), 2.85 (d, j= 4.3 Hz, 1H), 2.56 (t, j= 5.9 Hz, 2H), 2.17 (dq, j= 15.3, 7.8 Hz, 2H), 2.01 - 1.91 (m, 1H), 1.80 (dd, J= 20.2, 10.9 Hz, 2H), 1.70 (s, 4H), 1.60 (s, 3H), 1.08 (s, 3H), 1.01 (d, J= 13.3 Hz, 1H). Example 52: Preparation of sodium 5-(3-fluoro-l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)- 2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)picolinate

Intermediate 1

1. Preparation of methyl 5-(l-(tert-butoxycarbonyl)-3-fluoroazetidin-3-yl)picolinate

[0336] To a stirred solution of methyl 5-(l-(tert-butoxycarbonyl)-3-hydroxyazetidin-3- yl)picolinate (250 mg, 0.81 mmol) in dry DCM (5 mL), was added (diethylamino)sulfur trifluoride (0.198 mL, 1.62 mmol) drop wise at 0°C over a period of 10 min. The reaction mixture was stirred at r.t. for 2 h under N₂, and the starting material was completely consumed by TLC. The reaction mixture was cooled to -l0°C and quenched with water, extracted with

EA, and the combined organic layers washed with NaCl (aq.). The organic layer was dried over anhydrous Na₂S0₄ and concentrated in vacuo, then the crude product was purified by flash chromatography on silica gel eluting with PE/EtOAc 20-15/1 to give methyl 5-(l-(tert- butoxycarbonyl)-3-fluoroazeti din-3 -yl)picolinate (240 mg) as a colorless oil. LC-MS (ESI) found: 311 [M+H] ⁺

2. Preparation of lithium 5-(l-(tert-butoxycarbonyl)-3-fluoroazetidin-3- yl)picolinate acid

[0337] To a stirred solution of methyl 5-(l-(tert-butoxycarbonyl)-3-fluoroazetidin-3- yl)picolinate (240 mg, 0.774 mmol) in THF (3 mL), was added lithium hydroxide (52.0 mg, 1.19 mmol) in H₂0 (1 mL) in one portion at 25°C. The reaction mixture was stirred at r.t. for 1.0 h and the starting material was completely consumed by TLC. The reaction mixture was concentrated in vacuo to give lithium 5-(l-(tert-butoxycarbonyl)-3-fluoroazetidin-3- yl)picolinate(crude, 226 mg) as a colorless oil. LC-MS (ESI) found: 297 [M+l]⁺.

3. Preparation of 5-(3-fluoroazetidin-3-yl)picolinic acid

TFA salt

[0338] To a solution of lithium 5-(l-(tert-butoxycarbonyl)-3-fluoroazetidin-3-yl)picolinate (crude, 226 mg, 0.77 mmol) in DCM (2 mL) was added TFA (1 mL). After the addition, the reaction mixture was stirred at room temperature for 3 hours. TLC showed the reaction was complete. The solvent ws removed to give 5-(3-fluoroazetidin-3-yl)picolinic acid (crude, TFA salt, 200 mg) as a brown oil. LC-MS (ESI) found: 197 [M+l]⁺.

4. Preparation of sodium 5-(3-fluoro-l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)picolinate

Intermediate 1

[0339] Step 1 : To a solution of 5-(3-fluoroazetidin-3-yl)picolinic acid 2,2,2-trifluoroacetate salt (131 mg, 0.67 mmol) and ethyldiisopropylamine (700 mg, 5.4 mmol) in CH₃CN (3 mL) under N₂ was added a solution of (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl (4-nitrophenyl) carbonate

(Intermediate 1, 300 mg, 0.67 mmol) in CH₃CN (1 mL) dropwise at 0°C, followed by addition of DMAP (5 mg, 0.067 mmol). The mixture was then stirred at r.t. for 16 h. TLC showed the reaction was complete. The reaction mixture was concentrated and purified by prep-HPLC (Method A, H₂0 (0.1% FA)/ CH₃CN) to give 5-(3-fluoro-l-((((3i?,4ri^,,5ri^,,6i?)-5-methoxy-4- ((2//,3//)-2-methyl-3-(3-methylbut-2-en- l -yl)oxiran-2-yl)- l -oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)picolinic acid (169 mg) as a colorless oil. LC-MS (ESI) found: 505 [M+l]⁺.

Step 2: To a solution of 5-(3-fluoro-l-((((3i?,4ri^,,5ri^,,6i?)-5-methoxy-4-((2i?,3i?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)picolinic acid (69 mg, 0.137 mmol) in CH₃CN (0.5 mL) was added 0.1 N NaOH (1.23 mL, 0.123 mmol) solution at -60°C with stirring. After lypholization, sodium 5-(3-fluoro-l- ((((3//,4,V,5ri,6//)-5-methoxy-4-((2//,3//)-2- ethyl-3-(3- ethylbut-2-en- l -yl)oxiran-2-yl)- l - oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)picolinate (66 mg) was obtained as white solid. 1H NMR (400 MHz, DMSO-d6 ) d 8.58 (s, 1H), 7.91 (dd, J = 17.2, 7.5 Hz, 2H),

5.38 (s, 1H), 5.18 (s, 1H), 4.40 (dd, J = 21.3, 7.3 Hz, 4H), 3.57 (d, J = 10.8 Hz, 1H), 3.32 - 3.30 (m, 3H), 2.85 (d, J = 4.1 Hz, 1H), 2.58 (d, J = 4.2 Hz, 1H), 2.54 (d, J = 6.4 Hz, 1H), 2.17 (s,

2H), 1.97 (d, J = 9.2 Hz, 1H), 1.84 (s, 1H), 1.82 - 1.73 (m, 2H), 1.70 (s, 3H), 1.60 (s, 3H), 1.09 (s, 3H), 1.03 (d, J = 13.3 Hz, 1H). Example 53: Preparation of sodium 2-(l-((((J ?, »V,6 ?)-5-methoxy-4-((2 ?,J ?)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- ylidene)acetate

1. Preparation of tert-butyl 3-(2-(tert-butoxy)-2-oxoethyl)-3-hydroxyazetidine-l- carboxylate

[0340] TMSC1 (0.5 mL, 6.4 mmol) was added to a suspension of Zn (6.1 g, 93.4 mmol) in THF (200 mL) under N_2. The mixture was stirred for 15 mins at room temperature and then heated to reflux (75 °C). The heating was stopped, and tert-butyl 2-bromoacetate (7.9 mL, 71.2 mmol) was added at such a rate that the THF boiled gently. After being heated to reflux for 1 h, the mixture was stirred for 1 h at room temperature. A solution of tert-butyl 3-oxoazetidine-l- carboxylate (10 g, 58.4 mmol) in THF (30 mL) while cooling the reaction in an ice bath. After being stirred for 1 h at room temperature, TLC showed the reaction was complete. The mixture was quenched by dropwise addition of aq. NH₄Cl (50 mL) at 0°C and then extracted with EA (50 mL). The organic layer was dried over anhydrous Na₂S0₄ and concentrated under vacuum to give a yellow residue, which was purified by flash chromatography (silica gel, 1 %~ 20 % ethyl acetate in petroleum ether) to give tert-butyl3-(2-(tert-butoxy)-2-oxoethyl)-3- hydroxyazetidine-l-carboxylate (8.5 g) as a colorless oil. LC-MS (ESI) m/z (M+l): 288. 2. Preparation of tert-butyl 3-(2-(tert-butoxy)-2-oxoethylidene)azetidine-l- carboxylate

[0341] To a solution of tert-butyl 3-(2-(tert-butoxy)-2-oxoethyl)-3-hydroxyazetidine-l- carboxylate (300 mg, 1.1 mmol) in DCM (5 mL) was added (diethylamino)sulfur trifluoride (0.3 mL, 2.1 mmol) under ice-bath. The reaction was stirred at r.t. for 16 hrs. The resulting mixture was washed with a NaHCO, solution and brine, dried over anhydrous Na₂S0₄, filtered and concentrated to give crude product, which was used in the next step without further purification. LC-MS (ESI) m/z (M+l): 270.

3. Preparation of 2-(azetidin-3-ylidene)acetic acid

[0342] To a mixture of tert-butyl 3-(2-(tert-butoxy)-2-oxoethylidene)azetidine-l-carboxylate (260 mg, 0.89 mmol) in DCM (1 mL) was added TFA (2 mL). The reaction mixture was stirred at room temperature for 2 hrs. The resulting mixture was concentrated to give crude product, which was used in the next step without further purification.

4. Preparation of 2-(l-((((3/?, V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- ylidene)aceticacid

[0343] To a solution of 2-(azeti din-3 -ylidene)acetic acid (300 mg, 1.74 mmol) in MeCN (5 mL) was added DIPEA (1.2 mL, 6.9 mmol) drop wise at 0 °C. The mixture was stirred at 0 °C for 10 min, then (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl (4-nitrophenyl) carbonate (Intermediate 1, 779 mg, 1.74 mmol) was added. The reaction mixture was stirred at r.t. overnight under N₂ atmosphere. The mixture was concentrated under vacuum to remove the solvent while keeping the temperature below 40 °C. The residue was purified by column chromatography (silica gel, 1 % ~ 10 % MeOH in DCM) and prep-HPLC (Cl 8, 0 ~ 90 % acetonitrile in H₂0 with 0.1 % NH₃ Ή₂O) to give (3/^-/.V,5_*SA/ -5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-en- l - yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl4-[(l,3-dioxolan-2-yl)methyl]piperazine-l- carboxylate (150 mg) as a colorless oil. LC-MS (ESI) m/z (M+l): 422.

5. Preparation of sodium 2-(l-((((.?/?, S,6/?)-5-methoxy-4-((2/?,.?/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- ylidene)acetate

[0344] To a mixture of 2-(l-((((3i?, S',6 ?)-5-methoxy-4-((2i?,3i?)-2-methyl-3-(3-methylbut- 2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-ylidene)acetic acid (100 mg, 0.23 mmol) in MeCN (1 mL) was added aq. NaOH (2.1 mL, 0.1 M) at -60 °C with stirring. The reaction mixture was lyophilized to give sodium 2-(l-((((3f?, ri', 6/?)-5-methoxy-4- ((27?, 37?)-2-methyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azeti din-3 -ylidene)acetate (103 mg) as a white solid. LC-MS (ESI) m/z (M+l): 422. 1H NMR (400 MHz, DMSO-d6 ) d 5.50 (s, 1H), 5.32 (s, 1H), 5.19 (t, J = 7.4 Hz, 1H), 4.53 (d, J = 81.6 Hz, 4H), 3.53 (m, 1H), 3.29 (d, J = 3.8 Hz, 4H), 2.85 (d, J = 4.3 Hz, 1H),

2.60 - 2.54 (m, 2H), 2.17 (m, 2H), 2.01 - 1.91 (m, 1H), 1.77 (d, J = 11.0 Hz, 2H), 1.71 (s, 4H), 1.61 (s, 3H), 1.08 (s, 3H), 1.01 (d, J = 13.3 Hz, 1H).

Example 54: Preparation of sodium 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)thio)acetate

Intermediate 1 1. Preparation of tert-butyl 3-((2-ethoxy-2-oxoethyl)thio)azetidine-l-carboxylate

[0345] To a stirred solution of ethyl 2-mercaptoacetate (, 889 mg, 7.38 mmol) and tert-butyl 3-iodoazetidine-l-carboxylate (1.9 g, 6.711 mmol) in dry DMF (15 mL), was added K₂C0₃ (1.85 g, 13.4 mmol) at 0°C over a period of 10 min. The reaction mixture was stirred at 50 °C for 16 hrs under N₂, and the starting material was completely consumed by TLC. The reaction mixture was cooled to 0°C, quenched with water, extracted with EA and washed with

NaCl (aq.). The organic layer was dried over anhydrous Na₂S0₄ and concentrated in vacuo. Then the resulting crude product was purified by flash chromatography on silica gel eluting with PE/EtOAc 10-5/1 to give tert-butyl 3-((2-ethoxy-2-oxoethyl)thio)azetidine-l-carboxylate (1.7 g) as a colorless oil. LC-MS (ESI) found: 276 [M+H] ⁺

2. Preparation of lithium 2-((l-(tert-butoxycarbonyl)azetidin-3-yl)thio)acetate

[0346] To a stirred solution of tert-butyl 3 -((2-ethoxy -2-oxoethyl)thio)azeti dine- 1- carboxylate (240 mg, 0.872 mmol) in THF (3 mL), lithium hydroxide (109 mg, 2.62 mmol) in H₂0 (1 mL) was added in one portion at 25°C. The reaction mixture was stirred at r.t. for 1.0 h, and the starting material was completely consumed by TLC. The reaction mixture was concentrated in vacuo to give lithium 2-((l-(tert-butoxycarbonyl)azeti din-3 -yl)thio)acetate (crude, 400 mg) as a colorless oil. LC-MS (ESI) found:248 [M+l]⁺.

3. Preparation of 2-(azetidin-3-ylthio)acetic acid

TFA salt

[0347] To a solution of lithium 2-((l-(tert-butoxycarbonyl)azeti din-3 -yl)thio)acetate (crude, 400 mg, 0.87 mmol) in DCM (2 mL) was added TFA (1 mL). After the addition, the reaction mixture was stirred at room temperature for 3 hours. It was concentrated to give 2-(azeti din-3 - ylthio)acetic acid (crude, TFA salt, 500 mg) as a brown oil. LC-MS (ESI) found: 148 [M+l]⁺.

4. Preparation of sodium 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)thio)acetate TFA salt

Intermediate 1

[0348] Stepl : To a solution of 2-(azeti din-3 -ylthio)acetic acid 2,2,2-trifluoroacetate salt (500 mg, 0.87 mmol), DIPEA (700 mg, 5.4 mmol) in CH₃CN (3 mL) under N₂ was added a solution of (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl (4-nitrophenyl) carbonate (Intermediate 1, 390 mg, 0.87 mmol) in CH₃CN (1 mL) dropwise at 0°C, followed by addition of DMAP (5 mg, 0.067 mmol). The mixture was then stirred at r.t. for 16 h. TLC showed the reaction was complete. The reaction mixture was concentrated and purified by prep-HPLC (Method A, H₂0 (0.1% FA)/ CH₃CN) to give 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran- 2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)thio)acetic acid (220 mg) as a colorless oil. LC-MS (ESI) found: 456 [M+l]⁺.

Step2: To a solution of 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)thio)acetic acid (86 mg, 0.189 mmol) in CH₃CN (0.5 mL) was added 0.1 N NaOH (1.7 mL, 0.17 mmol) solution at -60°C with stirring. After lypholization, sodium 2-((l-((((3R,4S,5S,6R)-

5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-

6-yl)oxy)carbonyl)azetidin-3-yl)thio)acetate 85 mg) was obtained as a white solid. ¹H NMR (400 MHz, DMSO-£¾) d 5.29 (dt, J= 4.6, 2.7 Hz, 1H), 5.19 (td, J= 7.4, 3.7 Hz, 1H), 4.16 (t, J = 8.2 Hz, 2H), 3.79 - 3.61 (m, 3H), 3.52 (dd, 7= 11.0, 2.6 Hz, 1H), 3.28 (s, 3H), 2.93 (s, 2H),

2.84 (d, j= 4.4 Hz, 1H), 2.57 (t, j= 5.5 Hz, 2H), 2.18 (hept, j= 7.6, 7.1 Hz, 2H), 1.93 (td, j = 13.1, 12.2, 6.7 Hz, 1H), 1.82 - 1.67 (m, 6H), 1.61 (d, J= 1.3 Hz, 3H), 1.07 (s, 3H), 1.04 - 0.97 (m, 1H). Example 55: Preparation of sodium 2-((l-((((3/?, S,5»S,6/?)-5-methoxy-4-((2/?,3/?)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)sulfonyl)acetate

5. Preparation of tert-butyl 3-((2-ethoxy-2-oxoethyl)sulfonyl)azetidine-l- carboxylate

[0349] To a solution of tert-butyl 3-[(2-ethoxy-2-oxoethyl)sulfanyl]azetidine-l-carboxylate (305 mg, 1.11 mmol) in DCM (4 mL), was added 3-chloroperoxybenzoic acid (477.85 mg, 2.77 mmol). The reaction mixture was stirred at 25 °C for 16 hours. TLC showed the reaction was complete. The slurry was diluted with DCM and washed with water and brine. The combined organic layers were dried over anhydrous Na₂S0₄ and concentrated under reduced pressure.

The residue was purified by flash chromatography to give tert-butyl 3-(2-ethoxy-2- oxoethanesulfonyl)azetidine-l-carboxylate (300 mg) as a white solid. LC-MS (ESI) found: 308 [M+H] ⁺

6. Preparation of lithium 2-((l-(tert-butoxycarbonyl)azetidin-3-yl)sulfonyl)acetate

LiOH*H₂0 ^OLi

THF/H₂O

.N'T 'O

Boc Boc

[0350] To a stirred solution of tert-butyl 3-(2-ethoxy-2-oxoethanesulfonyl)azetidine-l- carboxylate (100 mg, 0.32 mmol) in THF (3 mL) was added lithium hydroxide monohydrate (68.31 mg, 1.63 mmol) in H₂0 (1 mL) in one portion at 25°C. The reaction mixture was stirred at r.t. for 1.0 h, and the starting material was completely consumed by TLC. The reaction mixture was concentrated in vacuo to give crude lithium 2-({ l-[(tert-butoxy)carbonyl]azetidin- 3-yl}sulfonyl)acetate (100 mg) as a colorless oil. LC-MS (ESI) found:280 [M+l]⁺.

7. Preparation of 2-(azetidin-3-ylsulfonyl)acetic acid

Boc

[0351] To a solution of lithium 2-({ l-[(tert-butoxy)carbonyl]azetidin-3- yl}sulfonyl)acetate (100 mg, 0.3 mmol) in DCM (2 mL) was added TFA (1 mL). After the addition, the reaction mixture was stirred at room temperature for 3 hours. TLC showed the reaction was complete. It was concentrated under vacuum to give 2-(azeti din-3 - ylsulfonyl)acetic acid (70 mg) as a brown oil. LC-MS (ESI) found: 180[M+1]⁺.

8. Preparation of sodium 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)sulfonyl)acetate

[0352] Step 1 : To a solution of 2-(azetidine-3-sulfonyl)acetic acid 2,2,2-trifluoroacetate salt (75.08 mg, 0.33 mmol), and DIPEA (0.33 mL, 2.0 mmol) in CH₃CN (3 mL) under N₂ was added dropwise a solution of (3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut- 2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl 4-nitrophenyl carbonate (Intermediate 1, 150 mg, 0.34 mmol) in CH₃CN (1 mL) at 0°C, followed by addition of DMAP (4.1 mg, 0.033 mmol). The mixture was then stirred at r.t. for 16 h. The reaction mixture was concentrated under vacuum and purified by prep-HPLC (Method A, H₂0 (0.1% FA)/ CH₃CN) to give 2-((l- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)sulfonyl)acetic acid (72 mg) as a colorless oil. LC-MS (ESI) found: 488 [M+l]⁺.

Step 2: To a solution of 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)sulfonyl)acetic acid (72 mg, 0.15 mmol) in CH₃CN (0.5 mL) was added 0.1 N NaOH (1.34 mL) at -60°C with stirring. After lyophilization, sodium 2-((l-((((3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)azetidin-3-yl)sulfonyl)acetate (75.3 mg) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) d 5.31 (s, 1H), 5.20 (t, J= 7.5 Hz, 1H), 4.65 (dd, J= 14.1, 6.8 Hz, 1H), 4.08 (d, j= 9.0 Hz, 4H), 3.63 (s, 2H), 3.54 (dd, j= 11.0, 2.6 Hz, 1H), 3.28 (d, j= 4.6 Hz, 3H), 2.86 (d, j= 4.3 Hz, 1H), 2.59 (dd, j= 9.6, 5.2 Hz, 2H), 2.18 (dd, j= 14.9, 7.5 Hz, 2H), 1.96 (dd, J= 13.4, 9.2 Hz, 1H), 1.80 (dd, 7= 16.6, 7.1 Hz, 2H), 1.73 - 1.66 (m, 4H), 1.62 (s, 3H), 1.08 (s, 3H), 1.02 (d, J= 13.6 Hz, 1H).

Example 56: Preparation of sodium 2-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2- azaspiro [3.3] heptane-6-carboxylate

1. Preparation of 2-((((3/?,4,V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2- azaspiro [3.3] heptane-6-carboxylic acid

[0353] To a solution of 2-azaspiro[3.3]heptane-6-carboxylic acid 2,2,2-trifluoroacetate salt (94.64 mg, 0.67 mmol), and DIPEA (0.69 mL, 4.02 mmol) in C¾CN (4 mL) under N₂ was added a solution of (3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl 4-nitrophenyl carbonate (Intermediate 1, 300 mg, 0.67 mmol) in C¾CN (1 mL) dropwise at 0°C, followed by adding DMAP (8.24 mg, 0.067 mmol). The mixture was then stirred at r.t. for 16 hrs. The reaction mixture was concentrated and purified by prep-HPLC (Method A, H₂0 (0.1% FA)/ CH₃CN) to give 2-({[(3R,4S,5S,6R)-

5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-

6-yl]oxy}carbonyl)-2-azaspiro[3.3]heptane-6-carboxylic acid (209 mg) as a colorless oil. LC- MS (ESI) found: 450 [M+l]⁺.

2. Preparation of sodium 2-((((3/?,4,V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2- azaspiro [3.3] heptane-6-carboxylate

[0354] To a solution of 2-({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)-2- azaspiro[3.3]heptane-6-carboxylic acid (100 mg, 0.223 mmol) in CH₃CN (0.3 mL) was added 0.1 N NaOH (2.00 mL, 0.20 mmol) solution at -60°C with stirring. After lyophilization, sodium 2-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran- 2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2-azaspiro[3.3]heptane-6-carboxylate (100 mg) was obtained as white solid. 1H NMR (400 MHz, DMSO-d6) 85.28 (s, 1H), 5.20 (t, J = 7.5 Hz, 1H), 3.80 (d, J= 31.8 Hz, 4H), 3.51 (dd, J= 1.0, 2.5 Hz, 1H), 3.27 (s, 3H), 3.26 - 3.22

(m, 1H), 2.85 (d, J= 4.3 Hz, 1H), 2.56 (t, J= 5.8 Hz, 2H), 2.25 - 2.09 (m, 6H), 1.99 - 1.87 (m, 1H), 1.80 - 1.66 (m, 6H), 1.62 (s, 3H), 1.08 (s, 3H), 1.01 (d, J= 13.5 Hz, 1H).

Example 57: Preparation of sodium 2,2-difluoro-3-(l-((((3R,4S,5S,6R)-5-methoxy-4- ((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)propanoate

1. Preparation of tert-butyl 3-formylazetidine-l-carboxylate

[0355] To the solution of tert-butyl 3-(hydroxymethyl)azetidine-l-carboxylate (7.0 g) in DCM (100 mL) was added Dess-Martin periodinane (24.0 g). The reaction was allowed to be stirred at r.t overnight. TLC showed the reaction was complete. Then the reaction was quenched with 10% Na₂S₂0₃(aq), and extracted with EA. The organic layer was separated, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (silica gel, 20 % ~ 50 % EA in PE) to give tert-butyl 3- formylazetidine-l-carboxylate (S-2, 6.0 g) as colorless oil. 1H NMR (400 MHz, CDCI₃) d 9.78

(t, J = 1.7 Hz, 1H), 4.09 - 3.92 (m, 4H), 3.28 (dddd, J = 12.8, 8.1, 4.6, 2.3 Hz, 1H), 1.37 (d, J = 1.5 Hz, 9H).

2. Preparation of (Z)-3-(2-((tert-butyldimethylsilyl)oxy)-3-ethoxy-3-oxoprop-l-en- l-yl)azetidine-l-carboxylate

[0356] To the solution of ethyl 2-((tert-butyldimethylsilyl)oxy)-2-(dimethoxyphosphoryl) acetate (15.9 g, prepared using literature procedure) in anhydrous THF (100 mL) was added LiHMDS (2 mol/L in THF) dropwise under N₂ at -70 °C. After 30 min, tert-butyl 3- formylazetidine-l-carboxylate (S2, 6.0 g) in anhydrous THF (50 mL) was added to the mixture drop-wise at the same temperature. After 4 hours, TLC showed the reaction was complete. The reaction was quenched with NH₄Cl (aq), and extracted with EA. The organic layer was separated, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (silica gel, 5 % ~ 10 % EA in PE) to give (Z)-3 -^-((tert- butyl dimethylsilyl)oxy)-3 -ethoxy-3 -ox oprop-l-en-l -yl)azeti dine- l-carboxylate (S-3, 4.5 g) as a colorless oil. 1H NMR (400 MHz, CDCl₃) d 6.06 (d, J = 9.4 Hz, 1H), 4.07 (q, J = 7.1 Hz, 2H), 4.00 (t, J = 8.4 Hz, 2H), 3.59 (dd, J = 8.3, 5.9 Hz, 2H), 3.54 - 3.40 (m, 1H), 1.30 (s, 9H), 1.18 (t, J = 7.1 Hz, 3H), 0.81 (s, 9H), 0.00 (s, 6H).

3. Preparation of tert-butyl 3-(3-ethoxy-2,3-dioxopropyl)azetidine-l-carboxylate

[0357] A solution of (Z)-3-(2-((tert-butyldimethylsilyl)oxy)-3-ethoxy-3-oxoprop-l-en-l- yl)azetidine-l-carboxylate (800 mg) in DCM (20 mL) was treated with TBAF (1 M in THF, 4.0 mL) and stirred at r.t. for 4 hours. TLC showed the reaction was complete. The reaction was extracted with EA and washed with water. The organic layer was separated, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (silica gel, 5 % ~ 10 % EA in PE) to give tert-butyl 3 -(3 -ethoxy-2,3 - dioxopropyl)azetidine-l-carboxylate (600 mg) as a colorless oil. 1H NMR (400 MHz,

Chloroform-d) d 4.21 (qd, J = 7.2, 1.1 Hz, 2H), 4.02 (td, J = 8.6, 1.1 Hz, 2H), 3.55 - 3.36 (m, 2H), 3.08 (dd, J = 7.5, 0.9 Hz, 2H), 2.81 (ddt, J = 13.3, 7.8, 3.8 Hz, 1H), 1.31 (d, J = 1.2 Hz, 9H), 1.26 (td, J = 7.1, 1.1 Hz, 3H).

4. Preparation of tert-butyl 3-(3-ethoxy-2,2-difluoro-3-oxopropyl)azetidine-l-carboxylate

[0358] A solution of tert-butyl 3 -(3 -ethoxy-2,3 -di oxopropyl)azeti dine- l-carboxylate (500 mg) in DCM (10 mL) was treated with (diethylamino)sulfur triflouride (594 mg) at 0 °C. The reaction was allowed to stir at r.t for 6 hrs, and was monitored with TLC till complete. The reaction was quenched with NaHCO,(aq) and extracted with EA (3 x 20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and concentrated. The crude product, was used in the next step without further purification.

2. Preparation of lithium 3-(l-(tert-butoxycarbonyl)azetidin-3-yl)-2,2- difluoropropanoate

[0359] The residue from last step was dissolved in MeOH/H₂0(v/v, 3/1, 10 mL). To the solution was added LiOH H₂0 (77 mg) and the mixture was stirred at r.t for 6 hrs. TLC showed the reaction was complete. The solvent was removed under vacuum to give lithium 3-(l-(tert- butoxycarbonyl)azeti din-3 -yl)-2,2-difluoropropanoate (300 mg) as a yellow solid. LC MS: m/z (M-Li) 264.

3. Preparation of 2,2-difluoro-3-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)propanoic acid

[0360] To a solution of lithium 3-(l-(tert-butoxycarbonyl)azetidin-3-yl)-2,2- difluoropropanoate (300 mg) in DCM(5 mL) was added TFA (2.5 mL). The resulting mixture was stirred at room temperature for 3 hrs. TLC showed the reaction was complete. The solvent was removed under vacuum. The residue was dissolved in MeCN (10 mL), and DIPEA (0.75 mL) was added dropwise while cooling the reaction in an ice bath. To the resulting solution was added (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2- yl)-l-oxaspiro[2.5]octan-6-yl (4-nitrophenyl) carbonate (Intermediate 1, 440 mg), and the mixture was stirred at r.t. for 6 hrs. TLC showed the reaction was complete. The reaction mixture was quenched with aqueous NaHC0₃ (1 M) and extracted with EA (3 x 20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (silica gel, 1 % ~ 10 % MeOH in DCM) to give 2,2-difluoro-3-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2- en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)propanoic acid (36 mg). LC MS: (M-H) 472.

4. Preparation of sodium 2,2-difluoro-3-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-

2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)propanoate

[0361] To a solution of 2,2-difluoro-3-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)propanoic acid (36 mg) in MeCN (20 mL) was added aqueous NaOH (0.1 M) at -20 °C. The solvent was removed by lyophilization to give sodium 2,2- difluoro-3-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)propanoate (36 mg) as a white solid. LC MS: m/z (M-Na) 472. 1H MR (400 MHz, DMSO-d6) d 5.28 (s, 1H), 5.19 (ddq, J= 8.9, 7.3, 1.5 Hz, 1H), 3.93 (t, J= 8.4 Hz, 2H), 3.58 (t, J= 7.4 Hz, 2H), 3.51 (dd, J = 11.0, 2.6 Hz, 1H), 3.27 (s, 3H), 2.84 (d, J= 4.4 Hz, 1H), 2.74 (hept, J= 7.5 Hz, 1H), 2.58 - 2.53 (m, 2H), 2.16 (td, J= 16.3, 8.5 Hz, 5H), 1.93 (td, = 13.3, 4.5 Hz, 1H), 1.74 (d, J= 11.0 Hz, 2H), 1.70 (d, j= 1.4 Hz, 3H), 1.61 (d, j= 1.3 Hz, 3H), 1.07 (s, 3H), 1.00 (dt, j= 14.2, 3.6 Hz, 1H). ¹⁹F NMR (377 MHz, DMSO-d6) d -101.45, -101.53. Examples 58(a) and 58(b): Preparation of sodium 2-((l-((((3/?, S,5»S,6/?)-5-methoxy-4-

((2 ?,3 ?)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)sulfinyl)acetate

1. Preparation of tert-butyl 3-((2-ethoxy-2-oxoethyl)sulfinyl)azetidine-l-carboxylate

[0362] To a solution of tert-butyl 3-[(2-ethoxy-2-oxoethyl)sulfanyl]azetidine-l-carboxylate (lg, 3.6 mmol) in DCM (10 mL), was added 3-chloroperoxybenzoic acid (0.63 g, 3.6 mmol). The reaction mixture was stirred at 25 °C for 16 hours. TLC showed the reaction was complete.

The slurry was diluted with DCM and washed with water and brine. The combined organic layers were dried over anhydrous Na₂S0₄, concentrated under reduced pressure and purified by flash chromatography to give tert-butyl 3-(2-ethoxy-2-oxoethanesulfmyl)azetidine-l- carboxylate (830 mg) as a white solid. LC-MS (ESI) found: 292 [M+H] ⁺

2. Preparation of lithium 2-((l-(terf-butoxycarbonyl)azetidin-3-yl)sulfinyl)acetic acid

[0363] To a stirred solution of tert-butyl 3-(2-ethoxy-2-oxoethanesulfmyl)azetidine-l- carboxylate (350 mg, 1.20 mmol) in THF (3 mL), was added lithium hydroxide monohydrate (151.3 mg, 3.60 mmol) in H₂0 (1 mL) in one portion at 25°C. The reaction mixture was stirred at r.t. for 1.0 h and the starting material was completely consumed by TLC. The reaction mixture was concentrated in vacuo to give crude lithium 2-({ l-[(tert-butoxy)carbonyl]azetidin- 3-yl}sulfmyl)acetate (mg) as a colorless oil. LC-MS (ESI) found:264 [M+l]⁺. 3. Preparation of 2-(azetidin-3-ylsulfinyl)acetic acid

k OH

Bocri .

[0364] To a solution of lithium 2-({l-[(tert-butoxy)carbonyl]azetidin-3-yl}sulfmyl)acetate (300 mg, 1.14 mmol) in DCM (5 mL) was added TFA (1 mL). After the addition, the reaction mixture was stirred at room temperature for 3 hours. TLC showed the reaction was complete. The mixture was concentrated to give 2-(azetidine-3-sulfmyl)acetic acid (200 mg) as a brown oil. LC-MS (ESI) found: 164 [M+l]⁺.

4. Preparation of sodium 2-((l-((((3/?,4A,5A,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)sulfinyl)acetate

[0365] Step 1 : To a solution of 2-(azetidine-3-sulfmyl)acetic acid 2,2,2-trifluoroacetate salt (200 mg, 1.10 mmol), DIPEA (1.10 mL, 6.62 mmol) in MeCN (5 mL) under N₂ was added dropwise a solution of (3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl 4-nitrophenyl carbonate (Intermediate 1, 494 mg, 1.10 mmol) in CH₃CN (1 mL) at 0°C, followed by addition of DMAP (13.6 mg, 0.110 mmol). The mixture was then stirred at r.t. for 16 h. TLC showed the reaction was complete. The reaction mixture was concentrated. The residue was purified by prep-HPLC (Method A, H₂0 (0.1% FA)/ CH₃CN) and chiral SFC resolution (Instrument: Waters Thar 80 preparative SFC; Column: ChiralPak IG, 250x21.2mm I.D., 5pm; Mobile phase: C0₂ for A and methanol (0.1%NH₃Ή₂0) for B; Gradient: B 30%; Flow rate: 50 mL /min; Back pressure: 100 bar; Column temperature: 35°C; Wavelength: 220nm) to give separated diasteriomers of 2-((l- ((((3//,4k,5k,6//)-5-methoxy-4-((2//,3//)-2-methyl-3-(3-methylbut-2-en- l -yl)oxiran-2-yl)- l - oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)sulfmyl)acetic acid (70 mg and 65 mg) as colorless oils. LC-MS (ESI) found: 472 [M+l]⁺.

Step 2: To a solution of 2-(( l -((((3//,4k,5k,6//)-5-methoxy-4-((2//,3//)-2- ethyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)sulfmyl)acetic acid (70 mg, 0.14 mmol) in CH₃CN (0.5 mL) was added 0.1 N NaOH (1.34 mL) solution at -60°C with stirring. After lyophilization, sodium 2-((l-((((3R,4S,5S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)sulfmyl)acetate (70 mg) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6 ) d 5.29 (s, 1H), 5.20 (t, J= 7.3 Hz, 1H), 4.25 (d, J= 27.4 Hz, 1H), 4.13 (s, 1H), 4.00 (dd, j= 11.5, 6.1 Hz, 2H), 3.92 (s, 1H), 3.53 (dd, j= 11.0, 2.4 Hz, 1H), 3.28 (s, 3H), 3.25 (s, 1H), 3.15 (d, J= 14.0 Hz, 1H), 2.85 (d, J= 4.2 Hz, 1H), 2.59 (t, J= 6.0 Hz, 2H), 2.24 - 2.11 (m, 2H), 1.97 (s, 1H), 1.78 (d, 7= 10.4 Hz, 2H), 1.71 (s, 4H), 1.62 (s, 3H), 1.07 (d,

J= 9.0 Hz, 3H), 1.02 (d, J= 13.9 Hz, 1H).

Step 3 : To a solution of 2-(( l -((((3/^4.V,5.V,6/^)-5-methoxy-4-((2/^3/^)-2- ethyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)sulfmyl)acetic acid (65 mg, 0.13 mmol) in CH₃CN (0.5 mL) was added 0.1 N NaOH (1.20 mL) solution at -60°C with stirring. After lyophilization, sodium 2-((l-((((3R,4S,5S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)sulfmyl)acetate (65 mg) was obtained as a white solid. 1H MR (400 MHz, DMSO-d6) d 5.29 (s, 1H), 5.20 (t, J = 7.5 Hz, 1H), 4.23 (s, 1H), 4.13 (t, J = 8.1 Hz, 1H), 4.01 (s, 3H), 3.53 (dd, J = 11.0, 2.5 Hz, 1H), 3.29 (s, 3H), 3.13 (d, J = 13.8 Hz, 2H), 2.85 (d, J = 4.3 Hz, 1H), 2.57 (d, J = 4.3 Hz, 2H), 2.18 (dd, J = 14.4, 7.4 Hz, 2H), 1.99 - 1.91 (m,

1H), 1.78 (d, J = 10.7 Hz, 2H), 1.69 (d, J = 19.1 Hz, 4H), 1.62 (s, 3H), 1.08 (s, 3H), 1.02 (d, J = 12.0 Hz, 1H).

Example 59: Preparation of sodium l-(l-((((3/?, S,5A,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl- 3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)-6-oxo- 1 ,6-dih dropyridine-3-carboxylate

1. Preparation of methyl l-(l-(terf-butoxycarbonyl)azetidin-3-yl)-6-oxo-l,6- dihydropyridine-3-carboxylate

[0366] To a solution of tert-butyl 3-iodoazetidine-l-carboxylate (1.25 g, 4.41 mmol) and methyl 6-hydroxypyridine-3-carboxylate (0.676 g, 4.42 mmol) in DMF (5 mL), was added Cs₂C0₃ (2.16 g, 6.62 mmol). The reaction mixture was stirred at 25 °C for 16 hours. TLC showed the reaction was complete. The slurry was diluted with EA, and washed with water and brine. The combined organic layers were dried over anhydrous Na₂S0₄, concentrated under reduced pressure and purified by flash chromatography to give methyl l-{ l-[(tert- butoxy)carbonyl]azetidin-3-yl}-6-oxo-l,6-dihydropyridine-3-carboxylate (270 mg) as a white solid. LC-MS (ESI) found: 309 [M+H] ⁺.

2. Preparation of l-(azetidin-3-yl)-6-oxo-l,6-dihydropyridine-3-carboxylic acid, trifluoroacetate salt

[0367] Step 1 : To a stirred solution of methyl l-{ l-[(tert-butoxy)carbonyl]azetidin-3-yl}-6- oxo-l,6-dihydropyridine-3-carboxylate (217 mg, 0.71 mmol) in THF (3 mL), was added LiOH H₂0 (88.68 mg, 2.11 mmol) in H₂0 (lmL) in one portion at 25°C. The reaction mixture was stirred at r.t. for 1.0 h and the starting material was completely consumed by TLC. The reaction mixture was concentrated in vacuo to give crude target product as a colorless oil. LC- MS (ESI) found: 295 [M+l]⁺. Used in the next reaction step without further purification.

Step 2: The crude target product was dissolved in DCM (3 mL), then TFA (1 mL) was added. After the addition, the reaction mixture was stirred at room temperature for 3 hours. TLC showed the reaction was complete. The reaction mixture was concentrated to give l-(azetidin- 3-yl)-6-oxo-l,6-dihydropyridine-3-carboxylic acid, TFA salt (150 mg) as a brown oil. LC-MS (ESI) found: 195 [M+l]⁺.

3. Preparation of l-(l-((((3/?,4A,5A,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-6- oxo-1, 6-dihydropyridine-3-carboxylic acid

[0368] To a solution of l-(azetidin-3-yl)-6-oxo-l,6-dihydropyridine-3-carboxylic acid trifluoroacetate salt (150 mg, 0.69 mmol), and DIPEA (0.69 mL, 4.03 mmol) in CH₃CN (4 mL) was added dropwise a solution of (3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl 4-nitrophenyl carbonate

(Intermediate 1, 300 mg, 0.67 mmol) in CH₃CN (2 mL) at 0°C, followed by the addition of DMAP (8.24 mg, 0.067 mmol). Then the mixture was then stirred at r.t. for 16 h. TLC showed the reaction was complete. The reaction mixture was concentrated and purified by prep-HPLC (Method A, H₂0 (0.1% FA)/ CH₃CN) to give l-[l-({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6- yl]oxy}carbonyl)azetidin-3-yl]-6-oxo-l,6-dihydropyridine-3-carboxylic acid (160 mg) as a colorless oil. LC-MS (ESI) found: 503 [M+l]⁺.

4. Preparation of sodium l-(l-((((3/?,4A,5A,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)- 6-oxo- 1 ,6-dih dropyridine-3-carboxylate

[0369] To a solution of l-[l-({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3-yl]-6- oxo-l,6-dihydropyridine-3 -carboxylic acid (lOOmg, 0.199 mmol) in CH₃CN (0.3 mL) was added 0.1 N NaOH (1.79 mL, 0.179 mmol) solution at -60°C with stirring. After

lyophilization, sodium l-[l-({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut- 2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3-yl]-6-oxo-l,6- dihydropyridine-3-carboxylate (100 mg) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) d 8.07 (s, 1H), 7.84 (dd, J= 9.2, 2.2 Hz, 1H), 6.24 (d, J= 9.3 Hz, 1H), 5.31 (d, J = 20.8 Hz, 2H), 5.19 (t, J= 7.4 Hz, 1H), 4.30 (t, J= 8.6 Hz, 2H), 4.09 (dd, J= 9.1, 5.7 Hz, 2H), 3.55 (dd, J = 10.9, 2.6 Hz, 1H), 3.31 (d, 7= 3.4 Hz, 3H), 2.85 (d, 7= 4.4 Hz, 1H), 2.58 (t, j = 5.9 Hz, 2H), 2.16 (dt, J= 14.3, 6.9 Hz, 2H), 1.98 (t, J= 11.6 Hz, 1H), 1.86 (d, 7= 11.5 Hz, 1H), 1.79 (d, J= 10.9 Hz, 1H), 1.73 (d, J= 15.8 Hz, 4H), 1.61 (s, 3H), 1.09 (d, J= 3.5 Hz, 3H),

1.03 (d, J= 12.8 Hz, 1H).

Example 60: Preparation of sodium l-(l-((((3/?, S,5»S,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl- 3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)-2-oxo- 1 ,2-dih dropyridine-4-carboxylate

1. Preparation of methyl l-(l-(terf-butoxycarbonyl)azetidin-3-yl)-2-oxo-l,2- dihydropyridine-4-carboxylate

[0370] To a solution of tert-butyl 3-iodoazetidine-l-carboxylate (1.0 g, 3.5 mmol) and methyl 2-hydroxypyridine-4-carboxylate (0.54 g, 3.53 mmol) in DMF (15 mL), was added CS2CO3 (1.73 g, 5.30 mmol). The reaction mixture was stirred at 60 °C for 16 hours. TLC showed the reaction was complete. The slurry was diluted with EA and washed with water and brine. The combined organic layers were dried over anhydrous Na₂S0₄, concentrated under reduced pressure and purified by flash chromatography to give methyl l-{ l-[(tert- butoxy)carbonyl]azetidin-3-yl}-2-oxo-l,2-dihydropyridine-4-carboxylate (208 mg) as a white solid. LC-MS (ESI) found: 309 [M+H] ⁺

2. Preparation of l-(azetidin-3-yl)-6-oxo-l,6-dihydropyridine-3-carboxylic acid

[0371] Step 1 : To a stirred solution of methyl l-{ l-[(tert-butoxy)carbonyl]azetidin-3-yl}-2- oxo-l,2-dihydropyridine-4-carboxylate (200 mg, 0.65 mmol) in THF (3 mL), was added Li0H H₂0 (81.74 mg, 1.95 mmol) in H₂0 (1 mL) in one portion at 25°C. The reaction mixture was stirred at r.t. for 1.0 h and the starting material was completely consumed by TLC. The reaction mixture was concentrated in vacuo to give the crude product as a colorless oil which was used in the next reaction step without further purification. LC-MS (ESI) found: 295

[M+l]⁺.

Step 2: The crude product was dissolved in DCM (3 mL), TFA (1 mL) was added and the reaction mixture was stirred at room temperature for 3 hours. TLC showed the reaction was complete. The reaction mixture was concentrated to give l-(azeti din-3 -yl)-2-oxo- 1,2- dihydropyridine-4-carboxylic acid, TFA salt (150 mg) as a brown oil. LC-MS (ESI) found: 195 [M+l]⁺.

3. Preparation of sodium l-(l-((((3/?,4A,5A,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)- 2-oxo- 1 ,2-dih dropyridine-4-carboxylate

[0372] Step 1 : Under N₂ atmosphere, to a solution of l-(azeti din-3 -yl)-2-oxo- 1,2- dihydropyridine-4-carboxylic acid trifluoroacetate salt (141.03 mg, 0.58 mmol) and DIPEA (524.6 mg, 4.07 mmol) in MeCN (8 mL) was added dropwise a solution of (3R,4S,5S,6R)-5- methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6- yl 4-nitrophenyl carbonate (Intermediate 1, 260 mg, 0.58 mmol) in CH₃CN (1 mL) at 0°C, followed by adding DMAP (3.58 mg, 0.029 mmol). The mixture was then stirred at r.t. for 16 h. TLC showed the reaction was complete. The reaction mixture was concentrated under vacuum and purified by prep-HPLC (Method A, H₂0 (0.1% FA)/ CH₃CN) to give l-(l- ((((3//,4,V,5ri,6//)-5-methoxy-4-((2//,3//)-2-methyl-3-(3-methylbut-2-en- l -yl)oxiran-2-yl)- l - oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-2-oxo-l,2-dihydropyridine-4-carboxylic acid (100 mg) as a colorless oil. LC-MS (ESI) found: 503 [M+l]⁺.

Step 2: To a solution of l-(l-((((3i?,4ri',5ri^,,6i?)-5-methoxy-4-((2i?,3i?)-2-methyl-3-(3-methylbut- 2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azeti din-3 -yl)-2-oxo- 1 ,2- dihydropyridine-4-carboxylic acid (100 mg, 0.199 mmoL) in CH₃CN (0.5 mL) was added 0.1 N NaOH (1.8 mL) solution at -60°C with stirring. After lyophilization, sodium l-[l- ({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl]-l- oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3-yl]-2-oxo-l,2-dihydropyridine-4-carboxylate (100 mg) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) d 7.63 (d, J= 7.4 Hz, 1H), 6.64 - 6.57 (m, 2H), 5.31 (d, J= 18.2 Hz, 2H), 5.19 (t, J= 7.4 Hz, 1H), 4.26 (t, = 8.3 Hz, 2H), 4.12 (s, 2H), 3.55 (dd, J= 11.0, 2.5 Hz, 1H), 3.31 (s, 3H), 2.85 (d, J= 4.3 Hz, 1H), 2.57 (dd, J= 9.8, 5.3 Hz, 2H), 2.18 (dd, J= 12.9, 6.3 Hz, 2H), 2.02 - 1.92 (m, 1H), 1.89 - 1.73 (m, 3H), 1.71 (s, 3H), 1.61 (s, 3H), 1.09 (d, j= 4.1 Hz, 3H), 1.03 (d, j= 13.0 Hz, 1H).

Example 61: Preparation of sodium l-(l-((((3/?,4A,5A,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl- 3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)-2-oxo- 1 ,2-dih dropyridine-3-carboxylate

1. Preparation of methyl l-(l-(terf-butoxycarbonyl)azetidin-3-yl)-2-oxo-l,2- dihydropyridine-3-carboxylate

[0373] Step 1 : A heterogeneous mixture of methyl 2-oxo-2H-pyran-3-carboxylate (100 mg, 0.65 mmol) and tert-butyl 3-aminoazetidine-l-carboxylate (111 mg, 0.65 mmol) in DMF (5 mL) were stirred at room temperature for 3 h.

Step 2: The reaction mixture was then treated with 1 -Ethyl-3 -(3

dimethylaminopropyl)carbodiimide (172.97 mg, 0.90 mmol) and DMAP (5.5 mg, 0.045 mmol) at room temperature and the resulting solution was stirred for 12 h. TLC showed the reaction was complete. The reaction mixture was quenched with 1N aqueous HC1 and the solution was extracted with ethyl acetate (4 x 5 mL). The combined organic extracts were washed with 10% aqueous LiCl (3 x 7 mL), dried over anhydrous Na₂S0₄ and filtered. The filtrate

was concentrated under reduced pressure, and the residue was purified by flash

chromatography (silica gel, 20 % ~ 50 % EA in PE) to afford methyl l-{ l-[(tert- butoxy)carbonyl]azetidin-3-yl}-2-oxo-l,2-dihydropyridine-3-carboxylate (130 mg) as a solid. 2. Preparation of lithium l-(l-(ter#-butoxycarbonyl)azetidin-3-yl)-2-oxo-l,2- dihydropyridine-3-carboxylate

[0374] To a stirred solution of methyl l-{ l-[(tert-butoxy)carbonyl]azetidin-3-yl}-2-oxo-l,2- dihydropyridine-3-carboxylate (130 mg, 0.42 mmol) in THF (3 mL), was added LiOH H₂0 (53.0 mg, 1.27 mmol) in H₂0 (1 mL) in one portion at 25°C. The reaction mixture was stirred at r.t. for 1.0 h, and the starting material was completely consumed by TLC. The reaction mixture was concentrated in vacuo to give crude lithium l-{ l-[(tert-butoxy)carbonyl]azetidin- 3-yl}-2-oxo-l,2-dihydropyridine-3-carboxylate (150 mg) as a colorless oil. LC-MS (ESI) found: 295 [M+l]⁺.

3. Preparation of l-(azetidin-3-yl)-2-oxo-l,2-dihydropyridine-3-carboxylic acid, trifluoroacetate salt

[0375] The crude lithium l-{ l-[(tert-butoxy)carbonyl]azetidin-3-yl}-2-oxo-l,2- dihydropyridine-3-carboxylate (150 mg, 0.51 mmol) was dissolved in DCM (3 mL), and TFA (1 mL) was added. After the addition, the reaction mixture was stirred at room temperature for 3 hours. TLC showed the reaction was complete. The reaction mixture was concentrated under vacuum to give l-(azeti din-3 -yl)-2-oxo-l,2-dihydropyridine-3 -carboxylic acid, TFA salt (100 mg) as a brown oil. LC-MS (ESI) found: 195 [M+l]⁺.

4. Preparation of l-(l-((((3/?,4A,5A,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-2- oxo-1, 2-dihydropyridine-3-carboxylic acid

[0376] To a solution of l-(azeti din-3 -yl)-2-oxo-l,2-dihydropyridine-3 -carboxylic acid, trifluoroacetate salt (86.78 mg, 0.45 mmol) and DIPEA (0.46 mL, 2.68 mmol) in C¾CN (4 mL) was added dropwise a solution of (3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl 4-nitrophenyl carbonate

(Intermediate 1, 200 mg, 0.447 mmol) in CH₃CN (1 mL) at 0°C, followed by addition of DMAP (5.50 mg, 0.045 mmol). Then the mixture was stirred at r.t. for 16 h. The reaction mixture was concentrated, and the residue was purified by prep-HPLC (Method A, H₂0 (0.1% FA)/ CH₃CN) to give l-[l-({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut- 2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3-yl]-2-oxo-l,2- dihydropyridine-3 -carboxylic acid (69 mg) as a colorless oil. LC-MS (ESI) found: 503 [M+l]⁺.

5. Preparation of sodium l-(l-((((3/?,4A,5A,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)- 2-oxo- 1 ,2-dih dropyridine-3-carboxylate

[0377] To a solution of l-[l-({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3-yl]-2- oxo-l,2-dihydropyridine-3 -carboxylic acid (69 mg, 0.14 mmol) in CH₃CN (0.3 mL) was added 0.1 N NaOH (1.24 mL, 0.124 mmol) solution at -60°C with stirring. After lyophilization, sodium l-[l-({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3-yl]-2-oxo-l,2- dihydropyridine-3-carboxylate (60 mg) was obtained as a white solid. 1H NMR (400 MHz, DMSO-d6) d 7.68 (d, J= 24.2 Hz, 2H), 6.28 (s, 1H), 5.34 (s, 1H), 5.19 (s, 2H), 4.26 (t, J= 8.5 Hz, 2H), 4.17 - 4.10 (m, 2H), 3.55 (d, J= 10.6 Hz, 1H), 3.31 (s, 3H), 2.85 (d, J= 4.2 Hz, 1H), 2.59 - 2.55 (m, 2H), 2.18 (d, J= 5.2 Hz, 2H), 1.95 (d, J= 9.2 Hz, 1H), 1.80 (d, 7= 11.9 Hz, 2H), 1.76 (s, 1H), 1.71 (s, 4H), 1.61 (s, 3H), 1.09 (s, 3H), 1.02 (d, J= 12.5 Hz, 1H).

Example 62: Preparation of sodium 4-[l-({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6- yl] oxy} carbonyl)azetidin-3-yl]- 1 ,3-thiazole-2-carboxylate

1. Preparation of benzyl 3-(carbonochloridoyl)azetidine-l-carboxylate

[0378] To a stirred solution of l-[(benzyloxy)carbonyl]azetidine-3-carboxylic acid (2.35 g, 9.99 mmol) in DCM (5 mL) was added oxalyl chloride (4.75 g, 29.97 mmol) at 0 °C, followed by a catalytic amount of DMF (2 drops). The reaction mixture was stirred at 0°C for 15 min and then stirred at room temperature for 1 h. The solvent was removed under reduced pressure to afford benzyl 3-(carbonochloridoyl)azetidine-l-carboxylate (2.4 g) as a colorless oil.

2. Preparation of benzyl 3-(2-diazoacetyl)azetidine-l-carboxylate

[0379] To a stirred solution of benzyl 3-(carbonochloridoyl)azetidine-l-carboxylate (2.1 g, 8.28 mmol) in THF (50 mL) was added (diazomethyl)trimethylsilane (12.41 mL, 12.42 mmol) at 0 °C. The reaction mixture was stirred at 0°C for 15 min and then stirred at room temperature overnight. TLC showed the reaction was complete. After removal of the solvent under reduced pressure, EtOAc and sat. aq. NaHC0₃ were added. The aqueous layer was extracted with EtOAc and the combined organic layers was washed with brine and dried over anhydrous Na₂S0_4. The solvent was removed under reduced pressure to afford benzyl 3-(2- diazoacetyl)azetidine-l-carboxylate (1.8 g) as a colorless oil.

3. Preparation of benzyl 3-(2-bromoacetyl)azetidine-l-carboxylate

[0380] To a stirred solution of benzyl 3-(2-diazoacetyl)azetidine-l-carboxylate (1.8 g, 6.92 mmol) in Et₂0 (5 mL) was added HBr (1.43 g, 8.30 mmol) at 0 °C . The reaction mixture was stirred at 0°C for 1 hr. TLC showed the starting material had disappeared. The reaction mixture was neutralized with NaHCO, aq, and the aqueous layer was extracted with EtOAc. The combined organic layers was washed with brine and dried over anhydrous Na₂S0_4. The solvent was removed under reduced pressure to afford benzyl 3-(2-bromoacetyl)azetidine-l- carboxylate (1.9 g) as a colorless oil.

4. Preparation of benzyl 3-propanoylazetidine-l-carboxylate

[0381] To a stirred solution of benzyl 3-(2-bromoacetyl)azetidine-l-carboxylate (1.1 g, 3.52 mmol) in ethanol (20 mL) was added ethyl 2-amino-2-thioxoacetate (0.46 g, 3.52 mmol). The reaction mixture was stirred at l00°C for 3 hr. TLC showed the reaction was complete. After removal of the solvent under reduced pressure, the residue was partitioned between water and EtOAc. The aqueous layer was extracted with EtOAc, and the combined organic layer was washed with brine and dried over anhydrous Na₂S0_4. The solvent was removed under reduced pressure, and the residue was purified by silica gel column eluting with PE/EtOAc (100:1 to 1 : 1) to afford ethyl 4-{ l-[(benzyloxy)carbonyl]azetidin-3-yl}-l,3-thiazole-2-carboxylate (330 mg) as a colorless oil. LC-MS (ESI) found: 347 (M+H)⁺.

5. Preparation of ethyl 4-(azetidin-3-yl)-l,3-thiazole-2-carboxylate

[0382] To a solution of ethyl 4-{ l-[(benzyloxy)carbonyl]azetidin-3-yl}-l,3-thiazole-2- carboxylate (330 mg, 0.95 mmol) in DCM (10 mL) was added trichloroborane (2.85 mL,

2.85 mmol, 1 M DCM solution) at 0°C under N₂ atmosphere, and the reaction was stirred at room temperature overnight. TLC showed the reaction was complete. The reaction mixture was neutralized with aq. NaHCO, and the solvent was removed under reduced pressure to give ethyl 4-(azetidin-3-yl)-l,3-thiazole-2-carboxylate (180 mg) as a colorless oil. LC-MS (ESI) found: 213 (M+H)⁺.

6. Preparation of 4-{l-[(tert-butoxy)carbonyl]azetidin-3-yl}-l,3-thiazole-2- carboxylic acid

[0383] To a stirred solution of ethyl 4-(azetidin-3-yl)-l,3-thiazole-2-carboxylate (180 mg, 0.85 mmol) in THF (5 mL) was added di-tert-butyl dicarbonate (0.27 mL, 1.27 mmol) followed by NaOH (1.27 mL, 2.54 mmol). The mixture was stirred at room temperature for 30 min.

TLC showed the reaction was complete. EtOAc and water were added to the reaction mixture, and the two layers were separated. The aqueous layer was was acidified with 1N HC1 to pH=3 and then extracted with DCM. The combined organic layer was washed with brine and dried over anhydrous Na₂S0_4. The solvent was removed under reduced pressure and the residue was purified by silica gel chromatography eluting with DCM/MeOH (40 : 1) to afford 4-{ l-[(tert-butoxy)carbonyl]azetidin-3-yl}-l,3-thiazole-2-carboxylic acid (160 mg) as a colorless oil.

7. Preparation of 4-(azetidin-3-yl)-l,3-thiazole-2-carboxylic acid, trifluoroacetic acid salt

TFA

[0384] To a solution of 4-{ l-[(tert-butoxy)carbonyl]azetidin-3-yl}-l,3-thiazole-2-carboxylic acid (150 mg, 0.52 mmol) in DCM (2 mL) was added TFA (1 mL) dropwise at 0 °C. The reaction was stirred at room temperature for lhr. TLC showed the reaction was complete. The mixture was concentrated under reduced pressure to give 4-(azeti din-3 -yl)- 1,3 -thiazole-2- carboxylic acid trifluoroacetic acid salt (150 mg) as a yellow syrup, which was directly used in the next step without further purification. LC-MS (ESI) found: 185 (M+H)⁺. 8. Preparation of (3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2- en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl 8-ethyl-5-oxa-2,8-diazaspiro[3.5]nonane-2- carboxylate

[0385] To a solution of 4-(azetidin-3-yl)-l,3-thiazole-2-carboxylic acid (150 mg, 0.49 mmol) in MeCN (4 mL) was added DIPEA (315.14 mg, 2.44 mmol) dropwise at 0°C. The mixture was stirred at 0°C for 10 min, and (3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl 4-nitrophenyl carbonate

(Intermediate 1, 218.64 mg, 0.49 mmol) was added. After addition was complete, the reaction was stirred at room temperature overnight under a N₂ atmosphere. TLC showed the reaction was complete. The mixture was then concentrated under vacuum to remove the solvent while keeping the temperature below 40°C. The residue was diluted with dichloromethane, and the DCM solution was washed with ammonium acetate buffer (pH 4.0, 20 mL) and 5% sodium bicarbonate solution (20 mL X 1). The organic layer was dried over anhydrous Na₂S0₄, filtered and concentrated to give the crude product. The residue was purified by column

chromatography on silica gel (eluent: dichloromethane: methanol = 100 : 1 to 10 : 1) and further purified by prep-HPLC (Cl 8, 0 ~ 90 % acetonitrile in H₂0 with 0.1 % NH₃ H₂0) to give 4-[ 1 -({ [(3R,4S, 5 S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3 -(3 -methylbut-2-en- 1 -yl)oxiran- 2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3-yl]-l,3-thiazole-2-carboxylic acid (51 mg) as a colorless oil. LC-MS (ESI) found: 493 [M+l]⁺.

9. Preparation of sodium 4-[l-({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3- yl]-l ,3-thiazole-2-carboxylate

[0386] To a solution of 4-[l-({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3-yl]-l,3- thiazole-2-carboxylic acid (51 mg, 0.10 mmol) in acetonitrile (1 mL) was added 5 mL of 0.1 N aq. NaOH (0.93 mL, 0.09 mmol) solution at -50 °C. The mixture was lyophilized overnight to give sodium 4-[l-({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3-yl]-l,3-thiazole-2- carboxylate(50 mg) as a white solid. 1H NMR (400 MHz, DMSO-d6) d 7.52 (d, J = 31.7 Hz, 2H), 5.87 (s, 1H), 5.32 (s, 1H), 5.18 (s, 2H), 4.03 (d, J = 5.4 Hz, 2H), 3.54 (dd, J = 10.7, 2.3 Hz, 1H), 3.29 (s, 3H), 2.84 (d, J = 4.3 Hz, 1H), 2.57 (d, J = 4.3 Hz, 1H), 2.23 - 2.12 (m, 2H), 1.99 - 1.78 (m, 3H), 1.78 - 1.63 (m, 5H), 1.61 (s, 3H), 1.14 - 0.99 (m, 4H).

Example 63: Preparation of sodium 3-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)-2-oxopropanoate

1 . TFA, DCM 0.1 M NaOH(aq)

2. DIPEA, Intermediate 1

MeCN

1. Preparation of tert-butyl 3-(3-ethoxy-2,3-dioxopropyl)azetidine-l-carboxylate

[0387] A solution of (Z)-3-(2-((tert-butyldimethylsilyl)oxy)-3-ethoxy-3-oxoprop-l-en-l- yl)azetidine-l-carboxylate (800 mg) in DCM (20 mL) was treated with TBAF (1 M in THF, 4.0 mL) and stirred at r.t. for 4 hours. TLC showed the reaction was complete. The reaction was extracted with EA and washed with water. The organic layer was separated, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (silica gel, 5 % ~ 10 % EA in PE) to give tert-butyl 3 -(3 -ethoxy-2,3 - dioxopropyl)azetidine-l-carboxylate (600 mg) as a colorless oil. 1H NMR (400 MHz,

Chloroform-d) d 4.21 (qd, J = 7.2, 1.1 Hz, 2H), 4.02 (td, J = 8.6, 1.1 Hz, 2H), 3.55 - 3.36 (m, 2H), 3.08 (dd, J = 7.5, 0.9 Hz, 2H), 2.81 (ddt, J = 13.3, 7.8, 3.8 Hz, 1H), 1.31 (d, J = 1.2 Hz, 9H), 1.26 (td, J = 7.1, 1.1 Hz, 3H). 2. Preparation of lithium 3-(l-(tert-butoxycarbonyl)azetidin-3-yl)-2-oxopropanoate

[0388] To a solution of tert-butyl 3 -(3 -ethoxy-2,3 -di ox opropyl)azeti dine- l-carboxylate (135 mg) in MeOH/H₂0(v/v, 3/1, 10 mL) was added LiOH H₂0 (22 mg). The mixture was stirred at r.t for 6 hrs. TLC showed the reaction was complete. The solvent was removed under vacuum to give lithium 3-(l-(tert-butoxycarbonyl)azetidin-3-yl)-2-oxopropanoate (123 mg) as a white solid. LC MS: m/z (M-Li) 242.

3. Preparation of 3-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-2- oxopropanoic acid

[0389] To a solution of lithium 3-(l-(tert-butoxycarbonyl)azetidin-3-yl)-2-oxopropanoate (123 mg) in DCM (2.5 mL) was added TFA (2.5 mL). The resulting mixture was stirred at room temperature overnight. TLC showed the reaction was complete. The solvent was removed under vacuum. The residue was dissolved in MeCN (10 mL), and DIPEA (0.5 mL) was added dropwise while cooling the reaction in an ice-bath. To the resulting solution was added

(3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl (4-nitrophenyl) carbonate (Intermediate 1, 282 mg), and the mixture was stirred at room temperature for 6 hrs. TLC showed the reaction was complete. The reaction mixture was quenched with aqueous NaHCO, (1 M) to pH = 6-7 and extracted with EA (3 x 20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (silica gel, 1 % ~ 10 % MeOH in DCM) to give 3-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-2- oxopropanoic acid (42 mg) as a colorless oil. LC MS: m/z (M+l)⁺ 452.

4. Preparation of sodium 3-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-

2-oxopropanoate

[0390] To a solution of 3-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-2- oxopropanoic acid (42 mg) in MeCN (20 mL) was added aqueous NaOH (0.1 M, ) at -20 °C. The solvent was removed by lyophilization to give sodium 3-(l-((((3R,4S,5S,6R)-5-methoxy- 4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)-2-oxopropanoate (46 mg) as a white foam. LC MS: m/z (M-Na) 450. 1H NMR (400 MHz, DMSO-d6) d 5.28 (s, 1H), 5.20 (t, J= 7.6 Hz, 1H), 3.97 (t, J= 8.1 Hz, 2H), 3.52 (dd, j= 11.0, 2.6 Hz, 3H), 3.28 (s, 3H), 2.85 (d, j= 4.4 Hz, 1H), 2.80 (s, 2H), 2.57 (dd, J= 8.1, 5.1 Hz, 2H), 2.17 (q, J= 15.3, 1 1.3 Hz, 3H), 1.93 (dt, J= 13.6, 6.8 Hz, 1H), 1.75 (d, 7= 11.0 Hz, 2H), 1.71 (s, 4H), 1.61 (d, 7= 1.3 Hz, 3H), 1.08 (s, 3H), 1.01 (d, 7= 13.6 Hz, 1H).

Example 64: Preparation of sodium l-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)-4-oxo-l,4-dihydropyridine-3-carboxylate

1. Preparation of tert-butyl (Z)-2-((dimethylamino)methylene)-3-oxobutanoate

[0391] To a mixture of tert-butyl 3-oxobutanoate (5.4 g, 6.3 mmol) in dioxane (30 mL) was added l, l-dimethoxy-N,N-dimethylmethanamine (19.5 g, 164 mmol). The reaction mixture was stirred at r.t. for 16 h under N₂ atmosphere. After completion of the reaction, the resulting mixture was added to water and extracted with EA. The combined organic layers was washed with brine, dried over anhydrous Na₂S0₄, and concentrated to give crude tert-butyl (Z)-2- ((dimethylamino)methylene)-3-oxobutanoate (, 7.2 g), which was used in the next step without further purification.

2. Preparation of tert-butyl 4-oxo-4H-pyran-3-carboxylate

[0392] To a solution of tert-butyl (2Z)-2-[(dimethylamino)methylidene]-3-oxobutanoate (2.4 g, 11.25 mmol) in 70 mL THF was added potassium /-butoxide (3.15 g, 28.1 mmol) at 0°C. The mixture was stirred at 0°C for 30 mins. Then ethyl formate (0.9 mL, 11.3 mmol) was added dropwise to the mixture. The reaction mixture was stirred at r.t. overnight. The reaction was monitored by TLC and LCMS. It showed the start material was not entirely consumed. The mixture was concentrated under vacuum to remove the solvent while keeping the temperature below 40°C. The residue was diluted with EA and washed with water and brine, dried over anhydrous Na₂S0₄ and concentrated under reduced pressure to give a residue. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 20: 1 to 10: 1) to give tert-butyl 3-[N-methyl(2R,3R)-oxolane-2-amido]azetidine-l-carboxylate (145 mg).

3. Preparation of tert-butyl l-(l-(tert-butoxycarbonyl)azetidin-3-yl)-4-oxo-l,4- dihydropyridine-3-carboxylate

[0393] To a solution of tert-butyl 4-oxo-4H-pyran-3-carboxylate (145 mg, 0.74 mmol) in 5 mL of EtOH was added tert-butyl 3-aminoazetidine-l-carboxylate (127.2 mg, 0.74 mmol) at r.t. The reaction mixture was then stirred at 80 °C for 16 hours. The reaction was monitored by TLC and LCMS and showed the start material was consumed. The mixture was concentrated under vacuum to remove the solvent while keeping the temperature below 40°C. The residue was diluted with EA, washed with water and brine, dried over anhydrous Na₂S0 and concentrated under reduced pressure to give a residue, which was

purified by column chromatography (DCM/ MeOH, 20: 1) on silica gel to afford tert-butyl 1- (l-(tert-butoxycarbonyl)azetidin-3-yl)-4-oxo-l,4-dihydropyridine-3-carboxylate (200 mg) as a colorless oil. 4. Preparation of l-(azetidin-3-yl)-4-oxo-l,4-dihydropyridine-3-carboxylic acid

[0394] To a solution of tert-butyl l-{ l-[(tert-butoxy)carbonyl]azetidin-3-yl}-4-oxo-l,4- dihydropyridine-3-carboxylate (200 mg, 0.57 mmol) in DCM (4 mL) was added TFA (2 mL) at 0 °C. The reaction mixture was then slowly warmed to r.t. and stirred for 1.5 h. TLC showed the reaction was complete. The reaction mixture was concentrated to give a residue, which was used in the next step without further purification.

5. Preparation of l-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-4- oxo-1, 4-dihydropyridine-3-carboxylic acid

[0395] To a solution of l-(azeti din-3 -yl)-4-oxo-l,4-dihydropyridine-3 -carboxylic acid (200 mg, 0.65 mmol) in MeCN (5 mL) was added DIPEA (0.4 mL, 2.4 mmol) dropwise at 0 °C. The mixture was stirred at 0 °C for 10 min, then (3i?,4ri',5ri', 6/?)-5-methoxy-4-[(2i?,3i?)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl 4-nitrophenylcarbonate

(Intermediate 1, 232.5 mg, 0.6 mmol) was added. The reaction mixture was stirred at r.t. overnight under N₂ atmosphere. The mixture was concentrated under vacuum to remove the solvent while keeping the temperature below 40 °C. The residue was purified by column chromatography (silica gel, 1 % ~ 10 % MeOH in DCM) and prep-HPLC (Cl 8, 0 ~ 90 % acetonitrile in H₂0 with 0.1 % NH₃Ή₂0) to give l-(l-((((3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)azeti din-3 -yl)-4-oxo-l,4-dihydropyridine-3 -carboxylic acid (65 mg) as a colorless oil.

6. Preparation of sodium l-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)- 4-oxo- 1 ,4-dih dropyridine-3-carboxylate

[0396] To a mixture of l-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-4-oxo- l,4-dihydropyridine-3 -carboxylic acid (65 mg, 0.12 mmol) in MeCN (1 mL) was added 0.1 M NaOH (1.2 mL) at -60 °C with stirring. The reaction mixture was lyophilized to give sodium 1- (l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-4-oxo-l,4-dihydropyridine-3-carboxylate (51 mg) as a white solid. LC-MS (ESI) m/z (M+l): 503. 1H NMR (400 MHz, DMSO-d6) d 8.27 (s, 1H), 7.87 (s, 1H), 6.24 (s, 1H), 5.35 (s, 1H), 5.19 (t, J= 7.3 Hz, 1H), 5.07 (m, 1H), 4.35 (t, J = 8.9 Hz, 2H), 4.08 (m, 2H), 3.56 (dd, J= 11.0, 2.4 Hz, 1H), 3.31 (s, 4H), 2.85 (d, j= 4.3

Hz, 1H), 2.58 (d, J= 4.3 Hz, 1H), 2.55 (t, J= 6.3 Hz, 1H), 2.17 (t, J= 5.5 Hz, 2H), 1.97 (m, 1H), 1.86 (m, 1H), 1.79 (d, = 11.0 Hz, 1H), 1.70 (s, 3H), 1.61 (s, 3H), 1.09 (s, 3H), 1.03 (d, J = 13.3 Hz, 1H). Examples 65(a) and 65(b): Preparation of sodium 2-hydroxy-3-(l-((((3R,4S,5S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-

6-yl)oxy)carbonyl)azetidin-3-yl)propanoate

1. Preparation of tert-butyl 3-(2-((tert-butyldimethylsilyl)oxy)-3-ethoxy-3- oxopropyl)azetidine-l-carboxylate

[0397] To a solution of (Z)-3-(2-((tert-butyldimethylsilyl)oxy)-3-ethoxy-3-oxoprop-l-en-l- yl)azetidine-l-carboxylate (500 mg) in MeOH (20 mL) was added Pd/C and the mixture was stirred at r.t. under H₂ overnight. TLC showed the reaction was complete. The reaction mixture was filtered and concentrated, and the concentrate was purified by column chromatography (silica gel, 1 % ~ 10 % MeOH in DCM) to give tert-butyl 3-(2-((tert-butyldimethylsilyl)oxy)-3- ethoxy-3-oxopropyl)azetidine-l-carboxylate (400 mg) as a colorless oil. 1H NMR (400 MHz, Chloroform-ύ d 4.21 - 4.12 (m, 3H), 4.01 (td, 7= 8.4, 5.2 Hz, 2H), 3.60 (ddd, J= 14.0, 8.6,

5.7 Hz, 2H), 2.68 (ddt, j= 10.1, 7.9, 4.0 Hz, 1H), 2.00 (dd, j= 7.5, 6.1 Hz, 2H), 1.43 (s, 9H), 1.28 (t, J= 7.1 Hz, 3H), 0.89 (s, 9H), 0.04 (d, J= 12.8 Hz, 6H).

2. Preparation of tert-butyl 3-(3-ethoxy-2-hydroxy-3-oxopropyl)azetidine-l- carboxylate

[0398] A solution of tert-butyl 3-(2-((tert-butyldimethylsilyl)oxy)-3-ethoxy-3- oxopropyl)azetidine-l-carboxylate (400 mg) in DCM (20 mL) was treated with TBAF (1 M in THF, 4.0 mL) and stirred at r.t. for 4 hours. TLC showed the reaction was complete. The reaction was extracted with EA and washed with water. The organic layer was separated, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (silica gel, 5 % ~ 10 % EA in PE) to give tert-butyl 3-(3-ethoxy-2-hydroxy-3- oxopropyl)azetidine-l-carboxylate (209 mg) as a colorless oil. 1H NMR (400 MHz,

Chloroform-d) d 4.18 (qd, J = 7.1, 2.7 Hz, 2H), 4.06 (dd, J = 8.1, 4.0 Hz, 1H), 3.97 (td, J = 8.4, 2.4 Hz, 2H), 3.55 (ddd, J = 16.0, 8.6, 5.7 Hz, 2H), 2.67 (ddt, J = 10.2, 7.9, 4.1 Hz, 1H), 2.03 (ddd, J = 13.9, 8.1, 4.1 Hz, 1H), 1.85 (ddd, J = 13.9, 8.1, 6.9 Hz, 2H), 1.36 (s, 9H), 1.25 (t, J = 7.1 Hz, 3H).

3. Preparation of lithium 3-(l-(tert-butoxycarbonyl)azetidin-3-yl)-2- hydroxypropanoate

[0399] To a solution of tert-butyl 3-(3-ethoxy-2-hydroxy-3-oxopropyl)azetidine-l- carboxylate (209 mg) in MeOH/H₂0(v/v, 3/1, 10 mL) was added LiOH H₂0 (22 mg) and the mixture was stirred at r.t for 6 hrs. TLC showed all the starting material consumed. The solvent was removed under vacuum to give lithium 3-(l-(tert-butoxycarbonyl)azetidin-3-yl)-2- hydroxypropanoate (200 mg) as a white solid. LC MS: m/z (M-Li) 244.

4. Preparation of 2-hydroxy-3-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl- 3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)propanoic acid

[0400] To lithium 3-(l-(tert-butoxycarbonyl)azetidin-3-yl)-2-hydroxypropanoate (200 mg) in DCM(2.5 mL) was added TFA (2.5 mL). The resulting mixture was stirred at room temperature overnight. TLC showed the reaction was complete. The solvent was removed under vacuum. The residue was dissolved in MeCN (10 mL), and DIPEA (0.5 mL) was added dropwise while cooling the reaction in an ice bath. To the resulting solution was added

(3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl (4-nitrophenyl) carbonate (Intermediate 1, 282 mg), and the reaction was stirred at room temperature for 6 hrs. TLC showed the reaction was complete. The reaction mixture was quenched with aqueous NaHC0₃ (1 M) to pH = 6-7 and extracted with EA (3 x 20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (silica gel, 1 % ~ 10 % MeOH in DCM) and SFC (IG - 40% MEOH + 0.1% NH₃ H₂0) to give 2-hydroxy-3-(l- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)propanoic acid (29 mg) as a white solid and 2-hydroxy-3-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)propanoic acid 54 mg) as a white solid. LC MS: m/z (M+H)⁺ 454.

5. Preparation of sodium 3-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-

2-oxopropanoate

[0401] To a solution of 2-hydroxy-3-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl- 3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)propanoic acid in MeCN (20 mL) was added aqueous NaOH (0.1 M, 0.9 eq, 0.57 mL/l.07 mL) at -20 °C. The solvent was removed via lyophilization to give sodium 2-hydroxy-3-(l- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)propanoate (29 mg) as white solid. LC MS: m/z (M-Na) 452. 1H NMR (400 MHz, DMSO-d6) d 5.28 (s, 2H), 5.20 (t, J= 7.7 Hz, 2H), 4.42 (d, j= 3.2 Hz, 2H), 3.91 (t, j= 8.4 Hz, 3H), 3.51 (dd, j= 11.0, 2.6 Hz, 2H), 3.28 (s, 5H), 2.85 (d, J= 4.4 Hz, 2H), 2.70 - 2.61 (m, 2H), 2.62 - 2.53 (m, 2H), 2.18 (s, 2H), 2.23 - 2.10 (m, 1H), 1.94 (s, 1H), 1.79 (dd, J = 26.6, 11.6 Hz, 2H), 1.71 (s, 5H), 1.61 (s, 6H), 1.08 (s, 5H), 1.01 (d, J = 13.5 Hz, 2H). and sodium 2-hydroxy-3-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)propanoate (55 mg) as white solid. LC MS: m/z (M-Na) 452. 1H NMR (400 MHz, DMSO-d6) d 5.28 (s, 1H), 5.20 (t, J= 7.6 Hz, 1H), 4.40 (s, 1H), 3.60 - 3.47 (m, 2H), 3.28 (s, 3H), 2.85 (d, J= 4.4 Hz, 1H), 2.71 - 2.60 (m, 1H), 2.60 - 2.53 (m, 2H), 2.23 - 2.13 (m, 2H), 1.99 - 1.88 (m, 1H), 1.75 (d, j= 10.8 Hz, 1H), 1.71 (s, 3H), 1.61 (s, 3H), 1.08 (s, 3H), 1.01 (d, J= 13.7 Hz, 1H).

Example 66: Preparation of sodium 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)methyl)-2H-l,2,3-triazole-4-carboxylate

1. Preparation of methyl 2-((l-(tert-butoxycarbonyl)azetidin-3-yl)methyl)-2H-l,2,3- triazole-4-carboxylate

[0402] To a solution of tert-butyl 3-(hydroxymethyl)azetidine-l-carboxylate (936 mg), methyl lH-l,2,3-triazole-4-carboxylate (763 mg) and PPh₃ (2.62 g) in DCM (10 mL) was added diisopropyl azodi carboxyl ate (1.2 g) very slowly at 0 °C. The reaction was then stirred at r.t which monitored by TLC. The reaction mixture was quenched with water and extracted with DCM (3 x 20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (silica gel, 1 % ~ 10 % MeOH in DCM) to give methyl 2-((l-(tert-butoxycarbonyl)azetidin-3-yl)methyl)-2H- l,2,3-triazole-4-carboxylate (mg) as a white solid. LC MS: m/z (M+l)⁺ 297. 1H MR (400 MHz, Chloroform -ί/) d 8.13 (s, 1H), 4.93 (d, J= 7.6 Hz, 2H), 4.03 (dd, J= 9.0, 8.2 Hz, 2H), 3.94 (s, 3H), 3.80 (dd, j= 9.0, 5.2 Hz, 2H), 3.17 (tt, j= 7.9, 5.2 Hz, 1H), 1.43 (s, 9H).

2. Preparation of 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)methyl)-2H-l,2,3-triazole-4-carboxylic acid

[0403] To a solution of methyl 2-((l-(tert-butoxycarbonyl)azetidin-3-yl)methyl)-2H-l,2,3- triazole-4-carboxylate (280 mg) in MeOH/H₂0(v/v, 3: 1, 10 mL) was added Li0H ¾0 (40 mg). The mixture was stirred at r.t for 6 hrs. TLC showed the reaction was complete. The solvent was then removed under vacuum to give lithium 2-((l-(tert-butoxycarbonyl)azetidin-3- yl)methyl)-2H-l,2,3-triazole-4-carboxylate (290 mg) as white solid. LC MS: m/z (M-l) 281. The crude product was dissolved in DCM (2.5 mL), and TFA (2.5 mL) was added. The resulting mixture was stirred at room temperature overnight. The solvent was removed under vacuum. The residue was dissolved in MeCN (10 mL), and DIPEA (0.5 mL) was added dropwise while cooling the reaction in an ice bath. To the resulting solution was added (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl (4-nitrophenyl) carbonate (Intermediate 1, 282 mg), and the reaction was stirred at room temperature for 6 hrs. The reaction mixture was quenched with aqueous NaHC0₃ (1 M) and extracted with EA (3 ^c 20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (silica gel, 1 % ~ 10 % MeOH in DCM) to give 2-((l-((((3R,4S,5S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)methyl)-2H-l,2,3-triazole-4-carboxylic acid (88 mg) as a white solid LC MS: m/z (M+l)⁺ 491.

3. Preparation of sodium 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)methyl)-2H-l,2,3-triazole-4-carboxylate

[0404] To a solution of 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)methyl)-2H-l,2,3-triazole-4-carboxylic acid (88 mg) in MeCN (20 mL) was added aqueous NaOH (0.1 M, 0.9 eq, 0.57 mL) at -20 °C. The solvent was removed by lyophilization to give sodium 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)methyl)-2H-l,2,3- triazole-4-carboxylate (88 mg) as a white solid. LC MS: m/z (M-Na) 489. 1H MR (400 MHz, DMSO-d6) d 7.59 (s, 1H), 5.29 (s, 1H), 5.18 (t, J= 7.6 Hz, 1H), 4.93 (s, 2H), 3.86-4.00 (m, 2H), 3.74 (s, 2H), 3.51 (d, = 10.8 Hz, 1H), 3.27 (s, 3H), 3.14 - 3.01 (m, 2H), 2.84 (d, 7= 4.4 Hz, 1H), 2.57 - 2.58 (m, 1H), 2.55 (d, J= 4.2 Hz, 1H), 2.08-2.23 (m, 2H), 1.99 - 1.87 (m, 3H), 1.65-1.80 (m, 3H), 1.70 (s, 3H), 1.60 (s, 3H), 1.07 (s, 3H), 1.00 (d, J= 13.9 Hz, 1H).

Example 67: Preparation of sodium 6-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-6, 7-dihydro-

5H-pyrrolo [3,4-b] pyridine-3-carboxylate

1. Preparation of dimethyl 5-bromopyridine-2,3-dicarboxylate

[0405] To a solution of 5-bromopyridine-2,3-dicarboxylic acid (5 g, 20.3 mmol) in MeOH (50 mL) was added concentrated H₂S0₄ (2 mL). Then the mixture was heated at reflux for 20 h, and TLC showed the reaction was complete. After cooling to r.t., the reaction was quenched with saturated aqueous NaHC0₃ and concentration in vacuo. The residue was diluted with water (50 mL), and then extracted with DCM (50 mL x 3). The combined organic layers were dried over anhydrous Na₂S0₄, filtered, and concentrated in vacuo to give the title compound as a white solid (4.3 g).LC-MS (ESI) found: 275 [M+H]⁺.

2. Preparation of (5-bromopyridine-2,3-diyl)dimethanol

[0406] NaBLL (1.4 g, 36.5 mmol) was added portion-wise to a solution of 5-bromopyridine- 2,3-dicarboxylate (2 g,7.3 mmol) in EtOH (20 mL, pre-cooled to 0 °C). A solution of CaCl₂ (18.2 mL,2L9 mmol) in ethanol was added slowly at 0 °C, and the reaction mixture was warmed to r.t. and stirred overnight. TLC showed the reaction was complete. Excess sodium borohydride was quenched by slow addition of aqueous 2 N HC1 solution (3 mL), followed by stirring at r.t. for 0.5 h. Saturated aqueous sodium bicarbonate solution was added until a pH of ~ 7 was reached. The mixture was concentrated in vacuo, and the residue was purified by a silica gel column chromatography (DCM/MeOH (v/v) = 10/1) to give (5-bromopyridine-2,3- diyl)dimethanol (lg). LC-MS (ESI) found: 219 [M+l]⁺.

3. Preparation of 5-bromo-2,3-bis(chloromethyl)pyridine

[0407] To a solution of (5-bromopyridine-2,3-diyl)dimethanol (900 mg, 4.13 mmol) in DCM (20 mL) was added SOCl₂ (15 mL) dropwise at 0 °C. After 3 h, TLC showed the reaction was complete. The reaction was concentrated and the residue was used in the next step directly. LC-MS (ESI) found: 256 [M+l]⁺.

4. Preparation of 3-bromo-6-(3,4-dimethylbenzyl)-6,7-dihydro-5H-pyrrolo[3,4- b]pyridine

[0408] To a solution of 5-bromo-2,3-bis(chloromethyl)pyridine (900 mg, 3.53 mmol) in DCM (15 mL) was added DIPEA (3.5 mL). Then (2,4-dimethoxyphenyl)methanamine (0.64 mL, 4.24 mmol) was added. And the mixture was stirred at r.t. overnight. TLC showed the reaction was complete. The reaction was concentrated under vacuum. The residue was purified by a silica gel column chromatography (PE/EA (v/v) = 5/1) to give 3-bromo-6-(3,4- dimethylbenzyl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridine (610 mg). LC-MS (ESI) found:350 [M+l]⁺.

5. Preparation of methyl 6-(3,4-dimethylbenzyl)-6,7-dihydro-5H-pyrrolo[3,4- b] pyridine-3-carboxylate

[0409] To a solution of 3-bromo-6-(3,4-dimethylbenzyl)-6,7-dihydro-5H-pyrrolo[3,4- b]pyridine (610 mg, 1.75 mmol) in MeOH (5.5 mL) and TEA (2 mL) was added Pd(dppf)Cl₂ (100 mg). Then, the reaction was degassed with CO, and the resulting mixture was stirred at 80 °C overnight. TLC showed the reaction was complete. The reaction mixture was concentrated, and purified on a silica gel column eluting with PE/EA (2.5/1) to give the title product (130 mg). LC-MS (ESI) found:329 [M+l]⁺.

6. Preparation of lithium 6-(2,4-dimethoxybenzyl)-6,7-dihydro-5H-pyrrolo[3,4- b] pyridine-3-carboxylate

[0410] To a solution of methyl 6-(3,4-dimethylbenzyl)-6,7-dihydro-5H-pyrrolo[3,4- b]pyridine-3-carboxylate (127 mg, 0.39 mmol) in THF (4.5 mL) and H₂0 (1.5 mL) was added Li0H ¾0 (16.2 mg, 0.39 mmol). After stirring at r.t. overnight, TLC showed the reaction was complete. The solvent was removed and the crude product was used in the next step directly. LC-MS (ESI) found:3 l5 [M+l]⁺. 7. Preparation of 6,7-dihydro-5H-pyrrolo[3,4-b]pyridine-3-carboxylic acid

[0411] A solution of lithium 6-(2,4-dimethoxybenzyl)-6,7-dihydro-5H-pyrrolo[3,4- b]pyridine-3-carboxylate (122 mg, 0.39 mmol) in TFA (5 mL) and DCM (1 mL) was stirred at 65 °C overnight. TLC showed the reaction was complete. The solvent was removed and the crude product was used in the next step directly. LC-MS (ESI) found: 165 [M+l]⁺.

8. Preparation of sodium 6-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-6,7-dihydro- 5H-pyrrolo [3,4-b] pyridine-3-carboxylate

[0412] To a solution of 6,7-dihydro-5H-pyrrolo[3,4-b]pyridine-3-carboxylic acid (64 mg, 0.39 mmol) in MeCN (3 mL) was added DIPEA (0.27 mL, 1.56 mmol) at 0 °C. After 30 min, (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl (4-nitrophenyl) carbonate (Intermediate 1 139 mg, 0.31 mmol) was added in one portion and the reaction was warmed to r.t. under N₂. After 2 hrs, TLC showed the reaction was complete. The reaction was concentrated under vacuum to remove the solvent while keeping the temperature under 50 °C. The ammonium salt product (75 mg) was obtained through the prep-HPLC (Cl 8, 0-90% MeCN in H₂0 with 0.1% NH₃ H₂0) as a white solid. To a solution of ammonium salt product (75 mg) in MeCN (2mL) was added 0.1 N NaOH solution (1.7 mL) dropwise at -60°C with stirring, followed by lyophilization to give sodium 6- ((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridine-3-carboxylate (70 mg) as a white solid. LC-MS (ESI) found: 473 [M+l]⁺. 1H NMR (400 MHz, DMSO-d₆) 5:8.92 (s, 1H), 8.15-8.20 (m, 1H), 5.46 (s, 1H), 5.20 (t, J = 7.2 Hz, 1H), 4.55-4.80 (m, 4H), 3.58 (dt, Ji = 10.8 Hz, J₂ = 2.8 Hz, 1H), 3.32-3.33 (m, 3H), 2.90 (d, J = 4.4 Hz, 1 H), 2.57-2.65 (m, 2H), 2.03-2.21 (m, 3H), 1.75-1.93 (m, 3H), 1.71 (s, 3H), 1.62 (s, 3H), 0.98-1.12 (m, 4H). Example 68: Preparation of sodium 2-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2, 3-dihydro- lH-pyrrolo [3,4-c] pyridine-6-carboxylate

1. Preparation of dimethyl 6-cyanopyridine-3,4-dicarboxylate

cCoOoOrMe COOMe

,COOMe Zn(CN)₂, dppf, Pd₂(dba)₃ ^J^^COOMe

CV^ DMF, 100 °C, 3h

NC N

[0413] A mixture of dimethyl 6-chloropyridine-3,4-dicarboxylate (3.9 g, 17.0 mmol),

Zn(CN)₂ (2.4 g, 20.4 mmol), Pd₂(dba)₃ (1.5 g, 1.7 mmol), and dppf (1.9 g, 3.4 mmol) in DMF (70 mL) was stirred for 4 h at 100 °C under nitrogen. TLC showed the reaction was complete. The residue was diluted with ethyl acetate, washed with brine, dried over anhydrous sodium sulfate, and concentrated under vacuum. The residue was purified by silica gel column eluting with petroleum ether/ethyl acetate (5: 1). The resulting dimethyl 6-cyanopyridine-3,4- dicarboxylate (2.4 g) was obtained as a light yellow solid. LC-MS (ESI) found:22l [M+l]⁺.

2. Preparation of 4,5-bis(hydroxymethyl)picolinonitrile

[0414] 2 M LiBH₄ (10.4 mL) THF solution was added portion-wise to a solution of dimethyl

6-cyanopyridine-3,4-dicarboxylate (2.4 g, 10.4 mmol) in EtOH (40 mL, pre-cooled to 0°C).

The reaction mixture was warmed to r.t. and stirred for 1.5 h. TLC showed the reaction was complete. The mixture was then quenched by slow addition of 5 mL of water. The mixture was concentrated and then purified by silica gel column chromatography (DCM/MeOH (v/v) = 10/1) to give 4,5-bis(hydroxymethyl)picolinonitrile (1.45 g). LC-MS (ESI) found: 165 [M+l]⁺.

3. Preparation of 4,5-bis(chloromethyl)picolinonitrile

[0415] To a solution of 4,5-bis(hydroxymethyl)picolinonitrile (1.45 g, 8.83 mmol) in DCM (50 mL) was added SOCl₂ (50 mL) dropwise at 0 °C. After 3 h, TLC showed the reaction was complete. The volatiles were removed, and the residue was used in the next step without purification. LC-MS (ESI) found: 202 [M+l]⁺.

4. Preparation of 2-(3,4-dimethylbenzyl)-2,3-dihydro-lH-pyrrolo[3,4-c]pyridine-6- carbonitrile

[0416] To a solution of 4,5-bis(chloromethyl)picolinonitrile (1.8 g, 8.8 mmol) in DCM (50 mL) was added DIPEA (7.7 mL, 44 mmol) and then(2,4-dimethoxyphenyl)methanamine (1.6 mL, 10.6 mmol) was added. And the mixture was stirred overnight at r.t. TLC showed the reaction was complete. The reaction was concentrated under vacuum. The residue was purified by silica gel column chromatography (PE/EA (v/v) = 2/1) to give 2-(3,4-dimethylbenzyl)-2,3- dihydro-lH-pyrrolo[3,4-c]pyridine-6-carbonitrile (1.5 g). LC-MS (ESI) found: 296 [M+l]⁺.

5. Preparation of 2-(3,4-dimethylbenzyl)-2,3-dihydro-lH-pyrrolo[3,4-c]pyridine-6- carboxylic acid

[0417] 2 M NaOH (20 mL) solution was added to 2-(3,4-dimethylbenzyl)-2,3-dihydro-lH- pyrrolo[3,4-c]pyridine-6-carbonitrile (550 mg, 1.86 mmol). Then, the mixture was heated to reflux for 18 hours. TLC showed the reaction was complete. The mixture was cooled to 0 °C and acidified to pH 5-6 with concentrated HC1. Then the mixture was concentrated in vacuo to give crude 2-(3,4-dimethylbenzyl)-2,3-dihydro-lH-pyrrolo[3,4-c]pyridine-6-carboxylic acid (619 mg). LC-MS (ESI) found:3 l5 [M+l]⁺.

6. Preparation of 2,3-dihydro-lH-pyrrolo[3,4-c]pyridine-6-carboxylic acid

[0418] A a solution of 2-(3,4-dimethylbenzyl)-2,3-dihydro-lH-pyrrolo[3,4-c]pyridine-6- carboxylic acid (619 mg, 1.97 mmol) in TFA (5 mL) and DCM (2 mL) was stirred at 65 °C overnight. TLC showed the reaction was complete. The solvent was removed and the residue was used in the next step directly. LC-MS (ESI) found: 165 [M+l]⁺.

7. Preparation of sodium 2-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2, 3-dihydro- lH-pyrrolo [3,4-c] pyridine-6-carboxylate

[0419] To a solution of 2,3-dihydro-lH-pyrrolo[3,4-c]pyridine-6-carboxylic acid (320 mg, 1.95 mmol) in MeCN (10 mL) was added DIPEA (1.36 mL, 7.80 mmol) at 0 °C. After 30 min, (3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl (4-nitrophenyl) carbonate (Intermediate 1, 436 mg, 0.97 mmol) was added, and the reaction was warmed to r.t. under N₂. After 2 h, TLC showed the reaction was complete. The reaction was concentrated under vacuum to remove the solvent while keeping the temperature under 50 °C. The ammonium salt product (68 mg) was obtained by prep-HPLC (Cl 8, 0-90% MeCN in H₂0 with 0.1% NH₃Ή₂0) as a white solid. Then to the solution of ammonium salt product (68 mg) in MeCN (2 mL) was added 0.1 N NaOH solution (l .6mL) dropwise at -60°C with stirring, followed by lyophilization to give sodium 2-((((3R,4S,5S,6R)-

5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-

6-yl)oxy)carbonyl)-2,3-dihydro-lH-pyrrolo[3,4-c]pyridine-6-carboxylate (71 mg) as a white solid. LC-MS (ESI) found: 473 [M+l]⁺. 1H NMR (400 MHz, DMSO-d₆) 5:8.47-8.50 (m, 1H), 7.95-7.99 (m, 1H), 5.52 (s, 1H), 5.27 (t, J = 7.2 Hz, 1H), 4.66-4.83 (m, 4H), 3.65 (dt, Ji = 10.8 Hz, J₂ = 2.8 Hz, 1H), 3.38 (s, 3H), 2.95 (d, J = 4.4 Hz, 1 H), 2.64-2.70 (m, 2H), 2.11-2.27 (m, 3H), 1.82-1.98 (m, 3H), 1.78 (s, 3H), 1.68 (s, 3H), 1.16 (s, 3H), 1.10 (d, J = 13.6 Hz, 1H). Example 69: Preparation of sodium l-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)methyl)-lH-l,2,3-triazole-4-carboxylate

C1JSO4

Sodium ascorbate

rnnivi

1. Preparation of tert-butyl 3-(azidomethyl)azetidine-l-carboxylate

[0420] To a solution of tert-butyl 3-(iodomethyl)azetidine-l-carboxylate, 900 mg) in DMF was added NaN₃ (295 mg, 1.5 eq). The reaction mixture was stirred at r.t. and monitored by TLC. After the reaction completed, the reaction was quenched with water and extracted with EA (3 x 20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (silica gel, 10 % ~ 50 % EA in PE) to give tert-butyl 3-(azidomethyl)azetidine-l-carboxylate (500 mg).

2. Preparation of methyl l-((l-(tert-butoxycarbonyl)azetidin-3-yl)methyl)-lH-l,2,3- triazole-4-carboxylate

CuS0₄

Sodium ascorbate

[0421] /ert-Butyl 3-(azidomethyl)azetidine-l-carboxylate (500 mg) and methyl propiolate were suspended in a 1 :1 mixture of water and tert-butyl alcohol (6 mL/6mL). Sodium ascorbate (0.3 mmol, 300 pL of freshly prepared 1 M solution in water) was added, followed by copper(II) sulfate pentahydrate (7.5 mg, 0.03 mmol, in 100 pL of water). The heterogeneous mixture was stirred vigorously overnight, at which point the mixture had become clear, and TLC indicated complete consumption of the reactants. The reaction mixture was diluted with water (50 mL), cooled in ice, and the white precipitate was collected by filtration. After washing the precipitate with cold water (2x25 mL), it was dried under vacuum to afford methyl l-((l-(tert-butoxycarbonyl)azetidin-3-yl)methyl)-lH-l,2,3-triazole-4-carboxylate (500 mg, 94%) as a white solid LC MS: m/z (M+l)⁺ 297. 1H NMR (400 MHz, Chloroform -if) d 8.04 (s, 1H), 4.56 (d, J= 7.6 Hz, 2H), 4.01 (t, J= 8.5 Hz, 2H), 3.89 (s, 3H), 3.68 (dd, J= 9.0, 5.0 Hz, 2H), 3.06 (ddd, j= 10.5, 7.9, 5.0 Hz, 1H), 1.37 (s, 9H).

3. Preparation of sodium l-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)methyl)-lH-l,2,3-triazole-4-carboxylate

1. TFA, DCM MeCN te 1

[0422] To a solution of methyl l-((l-(tert-butoxycarbonyl)azetidin-3-yl)methyl)-lH-l,2,3- triazole-4-carboxylate (500 mg) in MeOH/H₂0 (v/v, 3 : 1, 10 mL) was added LiOHH₂0 (40 mg). The mixture was stirred at r.t for 6 hrs. Then the solvent was removed under vacuum to give lithium 1 -(( 1 -(tert-butoxycarbonyl)azeti din-3 -yl )m ethyl)- 1H- 1 ,2,3 -triazole-4-carboxylate (290 mg) as a white solid without purification. LC MS: m/z (M-l) 281. The residue was dissolved in DCM (2.5 mL), then TFA (2.5 mL) was added. The resulting mixture was stirred at room temperature overnight. The solvent was removed under vacuum. The residue was dissolved in MeCN (10 mL), and DIPEA (0.5 mL) was added dropwise while cooling the reaction in an ice bath. To the resulting solution was added (3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6-yl (4- nitrophenyl) carbonate (Intermediate 1, 282 mg), and the reaction was stirred at room temperature for 6 hrs. The reaction mixture was quenched with aqueous NaHC0₃ (1 M) and extracted with EA (3 x 20 mL). The organic layer was separated, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (silica gel, 1 % ~ 10 % MeOH in DCM) to give l-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)methyl)-lH-l,2,3-triazole-4-carboxylic acid (110 mg) as a white solid. LC MS: m/z (M+l)⁺ 491. To a solution of l-((l-((((3R,4S,5S,6R)-5-methoxy-4- ((2R, 3R)-2-methyl -3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)azetidin-3-yl)methyl)-lH-l,2,3-triazole-4-carboxylic acid (110 mg) in MeCN (20 mL) was added aqueous NaOH (0.1 M) at -20 °C. The solvent was removed after lyophilization to give impure product (115 mg, analyzed by ¹HNMR). The product was re- purified with prep-HPLC (Cl8,0 ~ 90 % acetonitrile in H₂0 with 0.1 % NH₃H₂0) and SFC (AD column-20%EtOH+0.l%NH₃H₂O) to give ammonium l-((l-((((3R,4S,5S,6R)-5-methoxy- 4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)methyl)-lH-l,2,3-triazole-4-carboxylate (30 mg) . To this product was added aqueous NaOH (0.1 M) at -20 °C, and the solvent was removed after lyophilization to give sodium l-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)methyl)-lH-l,2,3-triazole-4-carboxylate (29 mg) as a white solid LC MS: m/z (M-Na) 489. 1H NMR (400 MHz, DMSO-i¾) d 8.02 (d, J= 8.8 Hz, 1H), 5.29 (d, J= 3.7 Hz, 1H), 5.24 - 5.05 (m, 1H), 4.55 (d, J= 7.5 Hz, 2H), 3.91 (dt, J= 20.8, 8.4 Hz, 3H), 3.72 (s, 2H), 3.57 - 3.48 (m, 2H), 3.27 (s, 3H), 3.14 - 3.01 (m, 2H), 2.84 (d, J= 4.4 Hz, 1H), 2.55 (d, J= 4.0 Hz, 2H), 2.16 (q, j= 7.6 Hz, 2H), 1.94 (td, j= 13.4, 4.7 Hz, 1H), l.70(s, 3H), l.65-l.79(m, 3H), 1.60 (s, 3H), 1.07 (s, 3H), 0.99 (d, j= 13.8 Hz, 1H).

Example 70: Preparation of sodium 3-fluoro-l-((((.?/?, S,5»S,6/?)-5-methoxy-4-((2/?,.?/?)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)pyrrolidine-3-carboxylate

1. Preparation of 3-fluoro-l-((((3/?, V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)pyrrolidine-3- carboxylic acid

[0423] To a solution of 3-fluoropyrrolidine-3-carboxylicacid (200 mg, 1.5 mmol) in MeCN (5 mL) was added DIPEA (0.5 mL, 3.1 mmol) dropwise at 0 °C. The mixture was stirred at 0 °C for 10 min, then (5f?,4,S^,,5S^,,6/?)-5-methoxy-4-[(2f?,5f?)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl 4-nitrophenylcarbonate (Intermediate 1 806 mg, 1.8 mmol) was added. The reaction mixture was stirred at r.t. overnight under N₂ atmosphere. TLC showed the reaction was complete. The mixture was concentrated under vacuum to remove the solvent while keeping the temperature below 40 °C. The residue was purified by column chromatography (silica gel, 1 % ~ 10% MeOH in DCM) and prep- HPLC (Cl 8, 0 ~ 90 % acetonitrile in H₂0 with 0.1 % NH₃ H₂0) to give 3-fluoro- 1 -( { [(3/(-/N5.5', 6//)-5-rnethoxy-4- [(2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6- yl]oxy}carbonyl)pyrrolidine-3-carboxylic acid (210 mg) as a colorless oil. LC-MS (ESI) m/z (M+l): 442.

2. Preparation of sodium 3-fluoro- l-((((J/?, V,5,V,6/?)-5-methoxy-4-((2/?,J/?)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)pyrrolidine-3-carboxylate

[0424] To a mixture of 3-fluoro-l-({[(3f?,4ri^,,5ri^,, 6/?)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)pyrrolidine-3- carboxylic acid (160 mg, 0.36 mmol) in MeCN (1 mL) was added aq. 0.1 M NaOH (3.2 mL) at -60 °C with stirring. The reaction mixture was lyophilized to give l-(3R,4S,5S, 6R)-5- methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6- yl 3 -sodium 3-fluoropyrrolidine-l,3-dicarboxylate (130 mg) as a white solid. LC-MS (ESI) m/z (M+l): 442. 1H MR (400 MHz, DMSO-d6) d 5.36 (s, 1H), 5.19 (t, J= 7.0 Hz, 1H), 3.77 - 3.37 (m, 4H), 3.29 (s, 3H), 2.85 (d, J= 4.1 Hz, 1H), 2.57 (dd, 7= 11.1, 6.4 Hz, 2H), 2.42 - 2.08 (m, 3H), 1.98 (dd, 7 = 18.5, 10.1 Hz, 2H), 1.85 - 1.73 (m, 2H), 1.70 (s, 3H), 1.61 (s, 3H), 1.08 (d, 7= 3.7 Hz, 3H), 0.99 (d, J= 12.3 Hz, 1H). Example 71: Preparation of sodium 2-((((J ?, V,5»V,6 ?)-5-methoxy-4-((2 ?,J ?)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-5-oxa-2- azaspiro [3.4] octane-6-carboxylate

1. Preparation of 5-oxa-2-azaspiro[3.4]octane-6-carboxylic acid

[0425] To a mixture of 2-[(tert-butoxy)carbonyl]-5-oxa-2-azaspiro[3.4]octane-6-carboxylic acid (250 mg, 0.97 mmol) in DCM (3 mL) was added TFA (1 mL). The reaction mixture was stirred at room temperature for 2 hrs. TLC showed the reaction was complete. The resulting mixture was concentrated to give crude product, which was used in the next step without further purification. LC-MS (ESI) m/z (M+l): 158.

2. Preparation of 2-((((3/?, V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-5-oxa-2- azaspiro [3.4] octane-6-carboxylic acid

[0426] To a solution of 5-oxa-2-azaspiro[3.4]octane-6-carboxylic acid (150 mg, 0.95 mmol) in MeCN (5 mL) was added DIPEA (0.6 mL, 3.8 mmol) dropwise at 0 °C. The mixture was stirred at 0 °C for 10 min, then (3i?,4ri^,,5ri^,,6/?)-5-methoxy-4-[(2i?,3i?)-2-methyl-3-(3-methylbut- 2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl 4-nitrophenyl carbonate (Intermediate 1, 426 mg, 0.95 mmol) was added. The reaction mixture was stirred at r.t. overnight under N₂ atmosphere. TLC showed the reaction was complete. The mixture was concentrated under vacuum to remove the solvent while keeping the temperature below 40°C. The residue was purified by column chromatography (silica gel, 1 % ~ 10% MeOH in DCM) and prep-HPLC (Cl 8, 0 ~ 90 % acetonitrile in H₂0 with 0.1 % NH₃ H₂0) and SFC (ChiralPak IG

250x21.2mm I.D., 5pm - 30% MeOH + 0.1% NH₃ H₂0) to give 2-({[(3R,4S,5S, 6R)-5- methoxy-4-[((2R,3R) -2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6- yl]oxy}carbonyl)-5-oxa-2-azaspiro[3.4]octane-6-carboxylic acid as 2 separate diasteriomers A and B (102 mg and 70 mg) as colorless oil. LC-MS (ESI) m/z (M+l): 466.

3. Preparation of sodium 2-((((3/?, V,5,V,6/?)-5-methoxy-4-((2/?,3/?)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-5-oxa-2- azaspiro [3.4] octane-6-carboxylate (71)

[0427] To a mixture of 2-({[(3i?, S',5_<S^,,d/?)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut- 2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)-5-oxa-2-azaspiro[3.4]octane- 6-carboxylic acid diastereomer A (102 mg, 0.22 mmol) in MeCN (1 mL) was added aq. 0.1 M NaOH (1.9 mL) at -60 °C with stirring. The reaction mixture was lyophilized to give 2- ( 3R , 4S, 5S, d ?)-5-methoxy-4-[(2R,3R)-2-methyl-3 -(3-methylbut-2-en- 1 -yl)oxiran-2-yl]-l - oxaspiro[2.5]octan-6-yl 6-sodium 5-oxa-2-azaspiro[3.4]octane-2,6-dicarboxylate (71) (94 mg) as a white solid. LC-MS (ESI) m/z (M+l): 466. 1H NMR (400 MHz, DMSO-d6) d 5.30 (s, 1H), 5.19 (t, J= 6.7 Hz, 1H), 4.16 - 4.09 (m, 1H), 4.02 (t, = 6.5 Hz, 1H), 3.86 (dd, J= 16.1, 8.8 Hz, 2H), 3.77 (t, J= 7.6 Hz, 1H), 3.52 (d, J= 10.8 Hz, 1H), 3.28 (s, 3H), 3.23 (d, J= 7.5 Hz, 1H), 2.84 (d, J= 4.3 Hz, 1H), 2.55 (dd, J= 13.0, 5.3 Hz, 2H), 2.17 (s, 2H), 2.07 - 1.88 (m, 4H), 1.76 (dd, J = 14.2, 9.7 Hz, 3H), 1.70 (s, 3H), 1.61 (s, 3H), 1.07 (s, 3H), 1.01 (d, = 11.1 Hz, 1H). Example 72: Preparation of sodium 2-(((( /?, S,5A,6/?)-5-methoxy-4-( 2/?, /?)-2-methyl-3- (3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-5-oxa-2- azaspiro [3.4] octane-6-carboxylate

[0428] To a mixture of 2-({[(3f?, S^,,5S^,,d/?)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut- 2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)-5-oxa-2-azaspiro[3.4]octane- 6-carboxylic acid diastereomer B (75 mg, 0.16 mmol) in MeCN (1 mL) was added aq. 0.1 M NaOH (1.4 mL) at -60 °C with stirring. The reaction mixture was lyophilized to give 2- (3R , 4S, 5S, d/?)-5-methoxy-4-[(2R,3R)-2-methyl-3 -(3-methylbut-2-en- 1 -yl)oxiran-2-yl]-l - oxaspiro[2.5]octan-6-yl 6-sodium 5-oxa-2-azaspiro[3.4]octane-2,6-dicarboxylate (72) (65 mg) as a white solid. LC-MS (ESI) m/z (M+l): 466. 1H NMR (400 MHz, DMSO-d6) d 5.30 (s, 1H), 5.19 (t, 7= 6.7 Hz, 1H), 4.16 - 4.09 (m, 1H), 4.02 (t, 7= 6.5 Hz, 1H), 3.86 (dd, 7= 16.1, 8.8 Hz, 2H), 3.77 (t, 7= 7.6 Hz, 1H), 3.52 (d, 7= 10.8 Hz, 1H), 3.28 (s, 3H), 3.23 (d, 7= 7.5 Hz, 1H), 2.84 (d, 7= 4.3 Hz, 1H), 2.55 (dd, 7= 13.0, 5.3 Hz, 2H), 2.17 (s, 2H), 2.07 - 1.88 (m, 4H), 1.76 (dd, 7 = 14.2, 9.7 Hz, 3H), 1.70 (s, 3H), 1.61 (s, 3H), 1.07 (s, 3H), 1.01 (d, 7= 11.1 Hz, 1H).

Example 73: Preparation of sodium 2-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)ethane-l-sulfonate

1. Preparation of tert- butyl 3-(2-hydroxyethyl)azetidine-l-carboxylate

[0429] To a solution of 2-(l-(tert-butoxycarbonyl)azetidin-3-yl)acetic acid (6.5 g, 30.2 mmol) in THF (80 mL) was added 1 M B¾ THF solution (121 mL) at 0°C under N₂. The mixture was stirred at r.t. overnight. Then, 100 mL of MeOH was added dropwise and the solution was refluxed for 2 h. TLC showed the reaction was complete. The reaction was concentrated and purified by column chromatography (petroleum ether/ethyl acetate, 1 : 1) on silica gel to provide tert- butyl 3-(2-hydroxyethyl)azetidine-l-carboxylate (5 g) as a colorless oil. LC-MS (ESI) found: 202 [M+l]⁺.

2. Preparation of terf-butyl 3-(2-((methylsulfonyl)oxy)ethyl)azetidine-l-carboxylate

[0430] To a solution of /cvV-butyl 3-(2-hydroxyethyl)azetidine-l-carboxylate (1 g, 4.97 mmol) in DCM (10 mL) was added Et₃N (2 mL) and MsCl (0.6 mL) at 0°C. The mixture was stirred for 4 h. TLC showed the reaction was complete. The reaction was concentrated and the residue was purified by column chromatography (petroleum ether/ethyl acetate, 1 :1) on silica gel to afford /cvV-butyl 3-(2-((methylsulfonyl)oxy)ethyl)azetidine-l-carboxylate (1 g) as a colorless oil. LC-MS (ESI) found: 280 [M+l]⁺.

3. Preparation of sodium 2-(l-(tert-butoxycarbonyl)azetidin-3-yl)ethane-l- sulfonate

[0431] To a solution of /cvV-butyl 3-(2-((methylsulfonyl)oxy)ethyl)azetidine-l-carboxylate (400 mg, 1.43 mmol) in EtOH (10 mL) was added Na₂S0₃ (1.8 g, 14.3 mmol). The mixture was heated to reflux overnight. TLC showed the reaction was complete. The reaction was then cooled to r.t., filtered, and concentrated to provide 380 mg crude sodium 2-(l-(tert- butoxycarbonyl)azetidin-3-yl)ethane-l -sulfonate. LC-MS (ESI) found: 266 [M+l]⁺.

4. Preparation of 2-(azetidin-3-yl)ethane-l-sulfonic acid

[0432] To a solution of sodium 2-(l-(tert-butoxycarbonyl)azetidin-3-yl)ethane-l-sulfonate (380 mg, 1.43 mmol) in DCM (4 mL) was added TFA (1.5 mL) at 0 °C. Then, the reaction mixture was slowly warmed to r.t. and stirred for 1.5 h. TLC showed the reaction was complete. The reaction mixture was concentrated, and used directly in the next reaction. LC-MS (ESI) found: 166 [M+l]⁺.

5. Preparation of sodium 2-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-

(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)ethane-l-sulfonate

[0433] To a solution of 2-(azeti din-3 -yl)ethane-l -sulfonic acid (237 mg, 1.43 mmol) in MeCN (5 mL) was added DIPEA (1.0 mL,5.74 mmol) at 0 °C. After 30 min, (3R,4S,5S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl (4-nitrophenyl) carbonate (Intermediate 1, 321 mg, 0.72 mmol) was added in one portion and the reaction was warmed to r.t. under N_2. After 2 h, TLC showed the reaction was complete. Then, the reaction was concentrated under vacuum to remove the solvent and purified by prep-HPLC (Method A, H₂0 (0.1% FA)/ CH₃CN) to give product as ammonium salt (220 mg). Then to a solution of ammonium salt product (150 mg, 0.31 mmol) in MeCN (2 mL) was added 0.1 N NaOH solution (3.1 mL) dropwise at -60°C with stirring, followed by lyophilization to give sodium 2-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)ethane- l-sulfonate (130 mg) as a white solid. LC-MS (ESI) found: 474 [M+l]⁺.¹H NMR (400 MHz, DMSO-d₆) 5:5.29 (s, 1H), 5.19 (t, J = 7.6 Hz, 1H), 3.91 (t, J = 8.0 Hz, 2H), 3.46-3.53 (m, 3H), 3.28 (s, 3H), 2.83 (d, J = 4.0 Hz, 1H), 2.55-2.64 (m, 3H), 2.35 (t, J = 7.6 Hz, 2H), 2.10-2.22 (m, 2H), 1.74-2.03 (m, 6H), 1.71 (s, 3H), 1.61 (s, 3H), 1.08 (s, 3H), 1.01 (d, J = 13.6 Hz, 1H).

Biological Examples:

Methionine Aminopeptidase 2 (MetAP2) Inhibition

[0434] Enzyme inhibition was assessed with Flag tagged Human recombinant MetAP2.

Assay buffer was prepared as a 2x stock containing 100 mM HEPES containing 200 mM NaCl, pH adjusted to 7.5, when required, assay buffer was created by 1 : 1 dilution with double distilled water. 10 mM stock solutions of compounds were prepared in 100% DMSO and further diluted in 100% DMSO required concentration to 1 mM stocks. The stock solutions and DMSO vehicle controls were diluted 1 :20 in assay buffer prior to addition to the test plate (final in well concentration 10 mM). MAS peptide was formulated to a 7.5 mM stock in distilled water and prior to use further diluted 1 :4. Amino acid oxidase was prepared as a stock solution (6.2 mg/ml) and prior to use further diluted 1 :49.6 in distilled water. A 250 pM solution of MnCl₂ was prepared in advance of thawing an aliquot of MetAP2 enzyme. 40 pl of enzyme was mixed with 100 pl of MnCl₂ then further diluted in assay buffer to a final concentration of 16 pg/ml. 5pl of test compound, lOpl of MAS substrate / amino acid oxidase mixture, 10 pl of MetAP2 was added to test wells in a 384 well black plate with blank wells containing no enzyme, replaced with 10 pl of assay buffer. All compounds we tested in duplicate on two occasions on the same day. The final in well concentrations of the assay are 1% DMSO, 0.272 pg/ml MetAP2, 10 pM MnCl₂, 50.0 pg/ml (0.225 U/ml) amino acid oxidase, 0.75 mM MAS.

[0435] The plate was sealed with a TopSeal A cover and mixed briefly on an orbital mixer at 900 rpm. The plate was incubated for a further 25 minutes at 25 °C A 5x stock of Amplex buffer was prepared (0.25 M sodium phosphate, pH 7.4) and stored at 4 °C. When preparing for use the stock was diluted with distilled water. Amplex Ultraread stock solution was prepared at 2.57 mg/ml in 100% DMSO and stored in 50 mΐ aliquots at -20 °C. 20m1 of 505 U/ml Horse radish peroxidase was diluted in 990 ml of Amplex buffer, 100 mΐ of this was combined with 50 mΐ of Amplex Ultrared in 4850 ml of lx Amplex buffer to generate sufficient detection reagent for a 384 well plate. 25 mΐ detection reagent was added to each well of the test plate, which was re-sealed and mixed briefly on an orbital shaker. The plate was transferred to an Envision Multi-label reader and RFU measured corresponding to excitation 531 nm and emission 595 nm. The raw data file was exported as a .csv file for further analysis. At the end of the MetAP2 incubation add 25 mΐ Amplex/HRP mixture per well and read plate on Perkin Elmer Envision.

Human Vein Umbilical Cell (HUVEC) Proliferation

[0436] HUVECs are cells derived from the endothelium of veins from the umbilical cord. EGM-2 cell culture medium was used to grow cells. Cells were passaged by seeding at 3 x 103 cells/mL on T175 flasks and allowed to reach approximately 80% confluence before used in the experiment. After overnight culture cells were placed in fresh EGM-2 complete medium containing the appropriate concentration of compound in 100 pL/well. After a short incubation (1 or 2 hours), cells were washed with DPBS and placed in fresh EGM-2 complete medium without compound. After 72 hours of culture, cell proliferation was evaluated with CyQuant cell proliferation reagent according to manufacturer’s specifications for 60 minutes in a 37 °C, 5% C0₂ incubator. Fluorescence intensity of samples was measured using a fluorescent microplate reader with excitation at 485 nm and emission detection at 530 nm. Fluorescence intensity values were exported from the plate reader and average fluorescence was calculated across samples. Inhibition of cell proliferation was calculated using GraphPad Prism 7

(GraphPad Software, Inc.).

Diet Induced Obese Mouse Experiments

[0437] Taconic Biosciences maintains an inventory of C57BL/6NTac male mice on 60% kcal high fat diet (D12492; Research Diets). Mice are put on the diet at 6 weeks of age. Male DIO C57B1/6 mice at -19-21 weeks of age (13 - 15 weeks on high fat diet) were received from Taconic approximately 2 weeks prior to study start. Animals were singly housed in normal cages and continued on a high fat chow (D 12492; Research Diets) from the day they were received and throughout the study. Test articles were provided by the Sponsor. Compounds were formulated into a 100% DMSO stock (at 9 mg/mL) prior to the start of the study. Daily aliquots were formulated (to the dosing vehicle of 10% DMSO in water) each morning on Days 1-10 prior to dosing. A daily aliquot for each group was removed from the freezer and thawed on Days 1-10 prior to dosing and made up to the desired concentration in 10% DMSO. Animals were randomized into study groups (N=4) based on body weights collected on Day -3. All animals were administered vehicle via oral gavage on Days -3 to -1. For all groups on the mornings of Days 1-10, test compounds or vehicle were administered via oral gavage. Animals were dosed, based on a body weight taken each morning. Starting on Day -3, individual animal body weights were collected daily. Body weights collected throughout the study will be compared to the pre-dose body weight measured on Day -1.

[0438] On Day 11, all mice were euthanized via C0₂ asphyxiation and had blood and tissues collected at approximately 24 hours after the Day 10 dose. Animals were sacrificed by C0₂ and tissues collected and snap frozen in liquid nitrogen. For the liver, 2 aliquots were generated (-200 mg each into 2 mL conical tube). Brain was collected, snap frozen, and placed in a 50 mL conical tube. All tissues were stored on dry ice/at -70 °C.

Tissue N-Terminal Thioredoxin in DIO Mouse

[0439] The tissue samples were homogenized 1 :3 (w/v) in T-PER. The homogenized samples were centrifuged at 10,000 rpm for 10 min. The supernatant (lysate) was transferred to another tube. The total protein concentration of the supernatant was measured by BCA assay. The samples were mixed with 1 mM CaCl₂ in 100 mM Tris buffer, pH 8.0 and then reduced with TCEP at room temperature for 10 min. The samples were denatured at 100 °C for 10 min and cooled to room temperature. The samples were mixed with iodoacetamide (IAM) and alkylated at 300 rpm on the incubation shaker for 30 min in the dark. DTT was added to react with excess IAM at room temperature for 10 min. Samples were mixed (25: 1, protein/enzyme) with endoproteinase Glu-C solution and digested overnight (16 hr) at 37 °C. The reaction was quenched by adding 10% formic acid. After digestion, the samples were centrifuged at 10,000 rpm for 10 min. The supernatant (20 pL) was transferred to a new sample plate and mixed with 10 pL of 0.1% formic acid in 90/10 water/acetonitrile (v/v). IS spiking solution (3 pL) was added. The samples were mixed well by pipetting. The sample plate was cover with heating foil and submitted for LC -MS/MS analysis. The analytes and standards were measured by LC- MS/MS. Injections (25 pL) were made onto an Acquity EIPLC BEH 300 C18 column, using a Shimadzu SIL-30ACMP autosampler and Shimadzu LC-20AD pump. The total running time per sample was 15 min. The analytes were detected with an API Sciex TripleTOF 6600 mass spectrometer. The levels of the N-terminal peptide of thioredoxin (amino acid 1-6 MVKLIE, or THRX) was read off of a standard curve and reported as ng/mg total protein (tp) in tissues or as a ratio of the concentration related to total protein in multiple tissues.

[0440] The results are shown in Table 1 below. Table 1

BQL = Below Quantifiable Limit

INCORPORATION BY REFERENCE

[0441] All publications and patents mentioned herein, including those items listed below, are hereby incorporated by reference in their entirety for all purposes as if each individual publication or patent was specifically and individually incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

EQUIVALENTS

[0442] While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

[0443] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term“about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention.

[0444] What is claimed is:

Claims

1. A pharmaceutically acceptable base salt of a compound represented by:

wherein:

L¹ is -NR^XR²;

R¹ is H or Ci-3alkyl, and R² is selected from the group consisting of: a 4-9 membered monocyclic heterocyclic ring, heteroaryl, -Ci_-2alkylene-heteroaryl, phenyl, -Ci_-2alkylene- phenyl and -Ci_-6alkylene; wherein -Ci_-6alkylene may be straight or branched and may optionally be substituted with one or more substituents each independently selected from R^p; or

R¹ and R² taken together with the nitrogen to which they are attached form a 4-9 membered monocyclic, fused bicyclic, or spirocyclic heterocyclic ring; wherein the 4-9 membered monocyclic, fused bicyclic, or spirocyclic heterocyclic ring is optionally substituted with one or more substituents each independently selected from R^h;

L² is selected from the group consisting of: Ci_-4alkylene, -0-Ci_-6alkylene, -NR^a-Ci. ₄alkylene, -S-Ci_-4alkylene, - S(0)₂-Ci_-4alkylene, -S(0)-Ci_-4alkylene, =CR^a-, -Ci_-2alkylene- heteroaryl, a 4-9 membered monocyclic heterocyclic ring, and a bond; wherein Ci_-4alkylene and -0-Ci_-6alkylene may be straight or branched and may optionally be substituted with one or more substituents each independently selected from R^p;

T is selected from the group consisting of: -C(0)0H, tetrazole, -S0₃H, isoxazol-3-ol, isothiazol-3-ol, l-alkyl-lH-pyrazol-3-ol, l,2,4-oxadiazol-5-ol, l,2,4-thiadiazol-5-ol, 1H- imidazole-2,5-dione, oxazolidine-2,4-dione-5-yl, thiazolidine-2,4-dione-5-yl, 1,2,4- oxadiazolidine-3,5-dione-2-yl, 4-hydroxy-l,5-dihydro-2H-pyrrol-2-one-l-yl, 6-hydroxy-4H- l,3-dioxin-4-one-2-yl, -C(0)NHS(0)₂-Ci_-3alkyl, -NH-S(0)₂-Ci_-3alkyl, -S(0)₂-NH-Ci_-3alkyl, and -P(0)(0H)(0R^b); wherein -C(0)NHS(0)₂-Ci_-3alkyl, -NH-S(0)₂-Ci_-3alkyl and -S(0)₂-NH- C_l-3alkyl may optionally be substituted with one or more substituents each independently selected from R^p;

R^p is independently selected, for each occurrence, from the group consisting of fluorine, hydroxyl, cyano, and -C(0)NR^aR^b; or two R^p are taken together to form =0; R^h is independently selected, for each occurrence, from the group consisting of hydrogen, Ci_-3alkyl, fluoro and hydroxyl; or two R^h are taken together to form =0; and

R^a and R^b are independently selected, for each occurrence, from the group consisting of hydrogen and C_l-3 alkyl.

2. The pharmaceutically acceptable base salt of claim 1, wherein L¹ is selected from the group consisting of:

wherein independently for each occurrence p is 1 or 2, q is 1 or 2, r is 0, 1 or 2, Z is CH₂ or O, and Y is N or CH.

3. The pharmaceutically acceptable base salt of claim 1 or 2, wherein L¹ is selected from the group consisting of:

, ; p y .

5. The pharmaceutically acceptable base salt of any one of claims 1-4, wherein the base salt is an alkali metal salt.

6. The pharmaceutically acceptable base salt of any one of claims 1-5, wherein the alkali metal is selected from the group consisting of sodium, potassium, and lithium.

7. The pharmaceutically acceptable base salt of any one of claims 1-6, wherein T is selected from the group consisting of:

8. The pharmaceutically acceptable base salt of any one of claims 1-7, selected from the group consisting of: sodium 3-((S)-2-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)-3-methylbutyl)-5-oxo-l,2,4-oxadiazol-4-ide; sodium 2-(l- ((((3R,4S, 5 S,6R)-5-methoxy-4-((2R,3R)-2 -methyl -3-(3-methylbut-2-en- l-yl)oxiran-2- yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)acetate; sodium 1- ((((3R,4S, 5 S,6R)-5-methoxy-4-((2R,3R)-2 -methyl -3-(3-methylbut-2-en-l-yl)oxiran-2- yl)-l -oxaspiro[2.5 ] octan-6-yl)oxy)carbonyl)piperi dine-4-carboxylate; sodium 5 -(( 1 - ((((3R,4S, 5 S,6R)-5-methoxy-4-((2R,3R)-2 -methyl -3-(3-methylbut-2-en- l-yl)oxiran-2- yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)methyl)tetrazol-2-ide; sodium 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)oxy)acetate; sodium 3-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)propanoate;

sodium 5-(2-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2- en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)ethyl)tetrazol-2-ide; sodium (2-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)acetyl)(methylsulfonyl)amide; sodium ethyl(2-(l- ((((3R,4S, 5 S,6R)-5-methoxy-4-((2R,3R)-2 -methyl -3-(3-methylbut-2-en- l-yl)oxiran-2- yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)ethyl)phosphonate; sodium 2- ((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-

2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)oxy)-2-methylpropanoate; sodium 3-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-2,2- dimethylpropanoate; sodium (3-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-

3 -(3 -methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6- yl)oxy)carbonyl)azetidin-3-yl)propanoyl)(methylsulfonyl)amide; sodium 5-((l-

((((3R,4S, 5 S,6R)-5-methoxy-4-((2R,3R)-2 -methyl -3-(3-methylbut-2-en- l-yl)oxiran-2- yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)methyl)isoxazol-3-olate; sodium 5-(2-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2- en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)ethyl)isoxazol-3-olate; sodium 3-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)oxy)propanoate; sodium 2-(((S)-l-((((3R,4S,5S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)pyrrolidin-3-yl)oxy)acetate; sodium 2-((l-

((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2- yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)amino)acetate; sodium 2-

(((R)-l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)pyrrolidin-3-yl)oxy)acetate; sodium 2-(3-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-

1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)azetidin- 1 -yl)acetate; sodium 4-((((((3R,4S,5S,6R)-5-methoxy-4-((2S,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)methyl)benzoate;

sodium 4-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)benzoate; sodium 6-

((((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2- yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)methyl)nicotinate; sodium 1-

((((3R,4S, 5 S,6R)-5-methoxy-4-((2R,3R)-2 -methyl -3-(3-methylbut-2-en- l-yl)oxiran-2- yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidine-3-carboxylate; sodium 2-((R)-3-

(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2- yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)pyrrolidin-l-yl)acetate; sodium 2-

((S)-3-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)pyrrolidin-l-yl)acetate; sodium 2-(4-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en- l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)piperidin-l-yl)acetate; sodium ethyl (l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2- en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)phosphonate; sodium 3 -(3 -(((((3R,4S, 5 S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3 -(3 - methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6- yl)oxy)carbonyl)amino)azetidin-l-yl)propanoate; sodium 2-(4-((((3R,4S,5S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)piperazin-l-yl)acetate; sodium 2-(4- ((((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2- yl)- 1 -oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)methyl)- 1H- 1,2,3 -triazol- 1 - yl)acetate; sodium 2-(4-((((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5]octan-6- yl)oxy)carbonyl)(methyl)amino)methyl)-lH-l,2,3-triazol-l-yl)acetate; sodium (3R,5R)- 3,5-dihydroxy-7-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2- en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)heptanoate; sodium 3-(3-(((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)azetidin-l-yl)-2,2- dimethylpropanoate; sodium (3R, 5R)-3 , 5 -dihydroxy-7 -(( 1 -((((3R,4S, 5 S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)oxy)heptanoate; sodium 2-(6- ((((3R,4S, 5 S,6R)-5-methoxy-4-((2R,3R)-2 -methyl -3-(3-methylbut-2-en-l-yl)oxiran-2- yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2,6-diazaspiro[3.3]heptan-2-yl)acetate; sodium 2-(4-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2-oxopiperazin-l-yl)acetate; sodium 2-((2-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-

1-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2-azaspiro[3.3]heptan-6- yl)oxy)acetate; sodium 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-3- methylazetidin-3-yl)oxy)acetate; sodium 2-(4-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-

2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)piperazin-l-yl)acetate; sodium l-(l-((((3R,4S,5S,6R)-5-methoxy-4- ((2R,3R)-2 -methyl -3-(3-methylbut-2-en- l-yl)oxiran-2 -yl)-l -oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)-lH-pyrazole-4-carboxylate; sodium 5- ((((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2- yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)amino)methyl)picolinate; sodium l-(l- ((((3R,4S, 5 S,6R)-5-methoxy-4-((2R,3R)-2 -methyl -3-(3-methylbut-2-en- l-yl)oxiran-2- yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-lH-pyrazole-3-carboxylate; sodium 6-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)nicotinate;

sodium 5-(3-hydroxy-l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)picolinate; sodium 6-(3-hydroxy-l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)nicotinate; sodium 3-fluoro-l-((((3R,4S,5S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidine-3-carboxylate; sodium 6-(3-fluoro-l- ((((3R,4S, 5 S,6R)-5-methoxy-4-((2R,3R)-2 -methyl -3-(3-methylbut-2-en- l-yl)oxiran-2- yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)nicotinate; sodium 5-(l- ((((3R,4S, 5 S,6R)-5-methoxy-4-((2R,3R)-2 -methyl -3-(3-methylbut-2-en- l-yl)oxiran-2- yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl) picolinate; sodium l-[l- ({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2- yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3-yl]-lH-imidazole-4-carboxylate; sodium 5-(3-fluoro-l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)picolinate; sodium 2-(l-((((3R,4S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- ylidene)acetate; sodium 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)thio)acetate; sodium 2-((l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)sulfonyl)acetate; sodium 2-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en- 1 -yl)oxiran-2-yl)- 1 -oxaspiro[2.5 ] octan-6-yl)oxy)carbonyl)-2- azaspiro[3.3]heptane-6-carboxylate; sodium 2,2-difluoro-3-(l-((((3R,4S,5S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)propanoate; sodium 2-((l- ((((3R,4S, 5 S,6R)-5-methoxy-4-((2R,3R)-2 -methyl -3-(3-methylbut-2-en- l-yl)oxiran-2- yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)sulfmyl)acetate; sodium l-(l- ((((3R,4S, 5 S,6R)-5-methoxy-4-((2R,3R)-2 -methyl -3-(3-methylbut-2-en- l-yl)oxiran-2- yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-6-oxo-l,6-dihydropyridine-3- carboxylate; sodium l-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)-2-oxo-l,2-dihydropyridine-4-carboxylate; sodium l-(l-((((3R,4S,5S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-2-oxo-l,2-dihydropyridine-3- carboxylate; sodium 4-[l-({[(3R,4S,5S,6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl]-l-oxaspiro[2.5]octan-6-yl]oxy}carbonyl)azetidin-3- yl]-l,3-thiazole-2-carboxylate; sodium 3-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)-2-oxopropanoate; sodium l-(l-((((3R,4S,5S,6R)-5- methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l- oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3-yl)-4-oxo-l,4-dihydropyridine-3- carboxylate; sodium 2-hydroxy-3-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2- methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)propanoate; sodium 2-((l-((((3R,4S,5S,6R)-5-methoxy- 4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)methyl)-2H-l,2,3-triazole-4-carboxylate; sodium 6- ((((3R,4S, 5 S,6R)-5-methoxy-4-((2R,3R)-2 -methyl -3-(3-methylbut-2-en-l-yl)oxiran-2- yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-6,7-dihydro-5H-pyrrolo[3,4-b]pyridine-3- carboxylate; sodium 2-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-2,3-dihydro- lH-pyrrolo[3,4-c]pyridine-6-carboxylate; sodium l-((l-((((3R,4S,5S,6R)-5-methoxy-4- ((2R,3R)-2-methyl-3-(3-methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6- yl)oxy)carbonyl)azetidin-3-yl)methyl)-lH-l,2,3-triazole-4-carboxylate; sodium 3- fluoro-l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)pyrrolidine-3-carboxylate; sodium 2-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3-methylbut-2-en-l- yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)-5-oxa-2-azaspiro[3.4]octane-6- carboxylate ; and sodium 2-(l-((((3R,4S,5S,6R)-5-methoxy-4-((2R,3R)-2-methyl-3-(3- methylbut-2-en-l-yl)oxiran-2-yl)-l-oxaspiro[2.5]octan-6-yl)oxy)carbonyl)azetidin-3- yl)ethane-l -sulfonate, and a pharmaceutically acceptable salt or stereoisomer thereof.

9. A method of treating or mitigating a liver, intestine, and/or kidney disorder in a patient in need thereof, comprising administering an effective amount of a compound of any one of claims 1-8 to the patient.

10. The method of claim 9, wherein the liver disorder is one or more of: non-alcoholic steato- hepatitis, alcoholic steato-hepatitis, hepato-cellular carcinoma, liver cirrhosis, and hepatitis B.

11. The method of claim 9, wherein the kidney disorder is one or more of chronic kidney

disease, glomerular disease, IGA nephropathy, lupus nephritis, and polycystic kidney disease.

12. A method of treating hepatocellular carcinoma in a patient in need thereof, comprising adminsetering an effective amount of a compound of any one of claims 1-8 to the patient.

13. The method of any one of claims 9-11, wherein the patient suffers from type 2 diabetes, hepatitis B, cirrhosis, alpha-l antitrypsin deficiency, and/or is obese.

14. A method of treating, or preventing the development of, hepatic preneoplastic lesions in patient in need thereof, comprising administering an effective amount of a compound of any one of claims 1-8.

15. The method of claim 14, wherein the patient is at risk for but not yet suffering from NASH.

16. The method of claim 14, wherein the patient has at least one of: minimal liver fibrosis, minimal hepatic steatosis, lobular inflammation, and/or balloon degeneration.

17. The method of any one of claims 1-16, wherein the patient suffers from type 2 diabetes and/or is obese.